Palmar Fibroblasts Research Articles

Blue light irradiation and its beneficial effect on Dupuytren's fibroblasts.

Dupuytren’s contracture is a fibroproliferative disorder affecting the palmar fascia of the hand. Most affected are the ring fingers, and little fingers of middle-aged men. Symptomatic for this disease is the increased proliferation and differentiation of fibroblasts to myofibroblasts, which is accompanied by an elevated α-SMA expression. The present study evaluated the therapeutic benefit of blue light (λ = 453 nm, 38 mW/cm2, continuous radiance, spot size 10–12 cm2) as well as the molecular mechanism mediating this effect. It could be determined that blue light significantly diminished the induced α-SMA protein expression in both normal palmar fibroblasts and Duypuytren’s fibroblasts. The beneficial effect mediated by this irradiance, radiant exposure and wavelength was associated with an elevated reactive oxygen species generation. Furthermore, the data underlines the potential usefulness of blue light irradiation as a promising therapy option for Dupuytren’s disease, especially for relapse prevention, and may represent a useful strategy to treat further fibrotic diseases, such as keloids, hypertrophic scarring, and scleroderma.

The expression pattern of keratin 24 in tissue-engineered dermo-epidermal human skin substitutes in an in vivo model.

The use of autologous tissue-engineered skin substitutes is a promisingapproach to cover large skin defects in patients. Preclinical investigation is pivotal to test and improve the quality of these bio-engineered substitutes. In the skin, the epidermis, formed mainly by keratinocytes, provides the first physical barrier protecting from the environment. Proper keratinocyte differentiation and, thus, formation of astratified epidermis is essential for this function. Keratins, the main structural support of keratinocytes, play a vital role regarding differentiation of keratinocytes. Here, we examined the expression pattern of a recently described keratinocyte differentiation marker, namely Keratin 24, in our skin substitutes. Human epidermal keratinocytes, melanocytes, dermal fibroblasts, palmar fibroblasts or sweat gland cells were used to prepare skin substitutes. Fibroblast-containing collagen hydrogels were prepared, and keratinocytes or sweat gland cells and melanocytes were seeded onto the hydrogels. The generated tissue-engineered dermo-epidermal skin analogs were transplanted onto full-thickness skin wounds created on the back of immuno-incompetent rats. The skin substitutes were excised at different time points and histologically examined with regard to Keratin 24 expression. We observed the expression of Keratin 24 in keratinocytes of the upper stratum spinosum of the epidermis. In particular, we observed an intensified expression of Keratin 24 13weeks after transplantation compared to 4weeks after transplantation. Importantly, we noticed a markedly higher presence of Keratin 24 in more spinous layers if we used palmar fibroblasts or sweat gland cells in our skin substitutes compared non-palmar fibroblasts or epidermal keratinocytes. Our observations prove that the keratinocyte differentiation marker Keratin 24 is expressed in our dermo-epidermal skin substitutes in a normal pattern. This highlights that our bio-engineered skinanalogs mature and reach homeostasis in an in vivo assay. These findings harbor favorable implications regarding future clinical application.

The influence of stromal cells on the pigmentation of tissue-engineered dermo-epidermal skin grafts.

It has been shown in vitro that melanocyte proliferation and function in palmoplantar skin is regulated by mesenchymal factors derived from fibroblasts. In this study, we investigated in vivo the influence of mesenchymal-epithelial interactions in human tissue-engineered skin substitutes reconstructed from palmar- and nonpalmoplantar-derived fibroblasts. Tissue-engineered dermo-epidermal analogs based on collagen type I hydrogels were populated with either human palmar or nonpalmoplantar fibroblasts and seeded with human nonpalmoplantar-derived melanocytes and keratinocytes. These skin substitutes were transplanted onto full-thickness skin wounds of immunoincompetent rats. Four weeks after transplantation the development of skin color was measured and grafts were excised and analyzed with regard to epidermal characteristics, in particular melanocyte number and function. Skin substitutes containing palmar-derived fibroblasts in comparison to nonpalmoplantar-derived fibroblasts showed (a) a significantly lighter pigmentation; (b) a reduced amount of epidermal melanin granules; and (c) a distinct melanosome expression. However, the number of melanocytes in the basal layer remained similar in both transplantation groups. These findings demonstrate that human palmar fibroblasts regulate the function of melanocytes in human pigmented dermo-epidermal skin substitutes after transplantation, whereas the number of melanocytes remains constant. This underscores the influence of site-specific stromal cells and their importance when constructing skin substitutes for clinical application.

Myofibroblast phenotype and apoptosis in keloid and palmar fibroblasts in vitro.

Keloid formation is a wound healing response, which fails to resolve and leads to formation of a raised collagen mass extending beyond the original wound margins. Keloids are typically excluded from palms and soles. Therefore we compared keloid and palmar fibroblasts in vitro using fibroblasts from nonaffected individuals as controls. Collagen I, alpha-smooth muscle actin and thrombospondin-1 were found at higher levels in keloid than in palmar fibroblasts. These differences were ameliorated by addition of TGFbeta1. The potential for resolution of the wound healing response was estimated analyzing apoptosis during serum starvation. Annexin V and TUNEL assays showed that palmar fibroblasts underwent faster apoptosis, than did the keloid fibroblasts, and started detaching. Addition of TGFbeta1 counteracted this effect. The weak expression of the myofibroblast phenotype and the advanced apoptosis of palmar fibroblasts suggest mechanisms for the exclusion of keloids from palmar sites.

The effect of interferon-alpha2b on an in vitro model Dupuytren's contracture.

The effects of interferon-alpha2b (IFN-alpha2b) on Dupuytren's and control palmar fibroblasts were evaluated using the fibroblast-populated collagen lattice model. Three paired strains of Dupuytren's and control fibroblasts were exposed to IFN-alpha2b for 96 hours before incorporation into triplicate collagen lattices. Contraction of the lattices was recorded and Northern blot analysis of cytoskeletal mRNA was performed. Comparisons of Dupuytren's and control fibroblasts revealed significantly increased contractility of the Dupuytren's fibroblasts in 2 of the 3 strains. Treatment with IFN-alpha2b significantly inhibited contraction in both Dupuytren's and control fibroblasts. In Dupuytren's fibroblasts, treatment with IFN-alpha2b significantly down-regulated mRNA expression for cytoplasmic beta-actin and gamma-actin.

Pharmacologic regulation of Dupuytren's fibroblast contraction in vitro

Dupuytren's disease is associated with contraction of specialized fibroblasts present in the diseased palmar fascia. Pharmacologic agents were evaluated for their ability to promote or inhibit contraction of Dupuytren's fibroblasts in vitro using a collagen lattice contraction assay. In the first part of the study, lysophosphatidic acid (LPA), serotonin, angiotensin II, and prostaglandin F2 alpha were tested for their ability to promote Dupuytren's fibroblast contraction. Lysophosphatidic acid was found to significantly promote Dupuytren's fibroblast contraction as compared with controls. This response to LPA is dose dependent, with a half-maximal response of 0.07 microM. Angiotensin II, serotonin, and prostaglandin F2 alpha at 1 mM, induced a significant amount of contraction as compared to controls, but the amount of contraction was at least six times less than that observed for LPA. In the second part of the study, prostaglandins E1 and E2 or the calcium blockers nifedipine and verapamil were tested for their ability to inhibit LPA-promoted contraction. It was found that both types of inhibitors partially block LPA-promoted contraction of Dupuytren's fibroblasts. The effect of the various pharmacologic agents on normal palmar fibroblasts was not evaluated. The focus of this study was to examine the regulation of contraction of Dupuytren's fibroblasts. This study demonstrates that LPA is a potent agonist of Dupuytren's fibroblast contraction and that this contraction can be inhibited by specific pharmacologic agents. These findings provide a rational basis for investigating further the clinical use of the calcium channel blockers nifedipine or verapamil and prostaglandins E1 and E2 to control Dupuytren's disease and possibly other fibrotic conditions.

Correlation of α-smooth muscle actin expression and contraction in Dupuytren's disease fibroblasts

We studied 11 nodules from patients with Dupuytren's contracture to determine whether alpha-smooth muscle actin expression in Dupuytren's fibroblasts is related to the generation of contractile force. Tissue was placed into explant culture and fibroblast strains were obtained. The mean percent of cultured Dupuytren's fibroblasts expressing alpha-smooth muscle actin, as determined by immunofluorescence, was 14 +/- 8 and ranged from 1% to 26%. The ability of Dupuytren's fibroblasts to generate contractile force was determined by using a previously described collagen lattice contraction assay. We observed a significant positive correlation between the expression of alpha-smooth muscle actin and the generation of contractile force in cell strains of Dupuytren's fibroblasts. In addition, six fibroblast strains from palmar fascia of individuals undergoing carpal tunnel release were examined. In six strains of palmar fibroblasts the mean percent of cells expressing alpha-smooth muscle actin was 5 +/- 3 and ranged from 1% to 9%. Six Dupuytren's fibroblast strains, in which more than 15% of the cells expressed alpha-smooth muscle actin, were significantly more contractile than the palmar fibroblasts. These results suggest that Dupuytren's fibroblasts can acquire smooth muscle characteristics and that the acquisition of a smooth muscle-like phenotype correlates with increased contractility.

Generation of contractile force by cultured Dupuytren's disease and normal palmar fibroblasts

Contractile fibroblasts are believed to be responsible for palmar fascia contracture in Dupuytren's Disease. An in vitro collagen lattice model was used to examine the contractile properties of Dupuytren's fibroblasts from 10 patients undergoing partial fasciectomy, and palmar fascia fibroblasts from 6 patients undergoing carpel tunnel release. Dupuytren's and palmar fascia fibroblasts cultured within a stabilized collagen lattice acquired morphological characteristics similar to those of ‘myofibroblasts’ in Dupuytren's diseased fascia. Both types of fibroblasts generated contractile forces that resulted in rapid collagen lattice contraction after release of the lattice from points of stabilization. Generation of contractile force by the fibroblasts was inhibited by disruption of the actin cytoskeleton, lack of cells, or serum removal. Afferent neuropeptides (substance P, galanin and neurokinin A) did not promote lattice contraction. These results demonstrate that normal palmar fascia fibroblasts can modulate into Dupuytren's-like fibroblasts and that cultured fibroblasts, from either Dupuytren's diseased or normal palmar fascia, can generate contractile forces that are transmitted to extracellular matrix. In addition, fibroblast contraction is an actin based process which requires specific factor(s) present in serum. It is suggested that in Dupuytren's disease extracellular cues trigger the modulation of fibroblasts to Dupuytren's fibroblasts and the promotion of contractile forces responsible for palmar fascia contracture.