Self-Assembled Collagen Microparticles by Aerosol as a Versatile Platform for Injectable Anisotropic Materials.

Extracellular matrices (ECM) rich in type I collagen exhibit characteristic anisotropic ultrastructures. Nevertheless, working in vitro with this biomacromolecule remains challenging. When processed, denaturation of the collagen molecule is easily induced in vitro avoiding proper fibril self-assembly and further hierarchical order. Here, an innovative approach enables the production of highly concentrated injectable collagen microparticles, based on collagen molecules self-assembly, thanks to the use of spray-drying process. The versatility of the process is shown by performing encapsulation of secretion products of gingival mesenchymal stem cells (gMSCs), which are chosen as a bioactive therapeutic product for their potential efficiency in stimulating the regeneration of a damaged ECM. The injection of collagen microparticles in a cell culture medium results in a locally organized fibrillar matrix. The efficiency of this approach for making easily handleable collagen microparticles for encapsulation and injection opens perspectives in active tissue regeneration and 3D bioprinted scaffolds.

IL-1ß-Primed Mesenchymal Stromal Cells Improve Epidermal Substitute Engraftment and Wound Healing via Matrix Metalloproteinases and Transforming Growth Factor-ß1.

Since the 1980s, deep and extensive skin wounds and burns are treated with autologous split-thickness skin grafts, or cultured epidermal autografts, when donor sites are limited. However, the clinical use of cultured epidermal autografts often remains unsatisfactory because of poor engraftment rates, altered wound healing, and reduced skin functionality. In the past few decades, mesenchymal stromal cells (MSCs) have raised much attention because of their anti-inflammatory, protrophic, and pro-remodeling capacities. More specifically, gingival MSCs have been shown to possess enhanced wound healing properties compared with other tissue sources. Growing evidence also indicates that MSC priming could potentiate therapeutic effects in diverse in vitro and in vivo models of skin trauma. In this study, we found that IL-1ß-primed gingival MSCs promoted cell migration, dermal-epidermal junction formation, and inflammation reduction in vitro, as well as improved epidermal substitute engraftment in vivo. IL-1ß-primed gingival MSCs had different secretory profiles from naive gingival MSCs, characterized by an overexpression of transforming growth factor-ß and matrix metalloproteinase (MMP) pathway agonists. Eventually, MMP-1, MMP-9, and transforming growth factor-ß1 appeared to be critically involved in IL-1ß-primed gingival MSC mechanisms of action.

Therapeutic potential of gingival fibroblasts for cutaneous radiation syndrome: comparison to bone marrow-mesenchymal stem cell grafts.

Mesenchymal stem cell (MSC) therapy has recently been investigated as a potential treatment for cutaneous radiation burns. We tested the hypothesis that injection of local gingival fibroblasts (GFs) would promote healing of radiation burn lesions and compared results with those for MSC transplantation. Human clinical- grade GFs or bone marrow-derived MSCs were intradermally injected into mice 21 days after local leg irradiation. Immunostaining and real-time PCR analysis were used to assess the effects of each treatment on extracellular matrix remodeling and inflammation in skin on days 28 and 50 postirradiation. GFs induced the early development of thick, fully regenerated epidermis, skin appendages, and hair follicles, earlier than MSCs did. The acceleration of wound healing by GFs involved rearrangement of the deposited collagen, modification of the Col/MMP/TIMP balance, and modulation of the expression and localization of tenascin-C and of the expression of growth factors (VEGF, EGF, and FGF7). As MSC treatment did, GF injection decreased the irradiation-induced inflammatory response and switched the differentiation of macrophages toward an M2-like phenotype, characterized by CD163(+) macrophage infiltration and strong expression of arginase-1. These findings indicate that GFs are an attractive target for regenerative medicine, for easier to collect, can grow in culture, and promote cutaneous wound healing in irradiation burn lesions.

Inhibition of the differentiation of monocyte-derived dendritic cells by human gingival fibroblasts.

We investigated whether gingival fibroblasts (GFs) can modulate the differentiation and/or maturation of monocyte-derived dendritic cells (DCs) and analyzed soluble factors that may be involved in this immune modulation. Experiments were performed using human monocytes in co-culture with human GFs in Transwell® chambers or using monocyte cultures treated with conditioned media (CM) from GFs of four donors. The four CM and supernatants from cell culture were assayed by ELISA for cytokines involved in the differentiation of dendritic cells, such as IL-6, VEGF, TGFß1, IL-13 and IL-10. The maturation of monocyte-derived DCs induced by LPS in presence of CM was also studied. Cell surface phenotype markers were analyzed by flow cytometry. In co-cultures, GFs inhibited the differentiation of monocyte-derived DCs and the strength of this blockade correlated with the GF/monocyte ratio. Conditioned media from GFs showed similar effects, suggesting the involvement of soluble factors produced by GFs. This inhibition was associated with a lower stimulatory activity in MLR of DCs generated with GFs or its CM. Neutralizing antibodies against IL-6 and VEGF significantly (P<0.05) inhibited the inhibitory effect of CM on the differentiation of monocytes-derived DCs and in a dose dependent manner. Our data suggest that IL-6 is the main factor responsible for the inhibition of DCs differentiation mediated by GFs but that VEGF is also involved and constitutes an additional mechanism.

Mechanisms of pathological scarring: role of myofibroblasts and current developments.

Myofibroblasts play a key role in the wound-healing process, promoting wound closure and matrix deposition. These cells normally disappear from granulation tissue by apoptosis after wound closure, but under some circumstances, they persist and may contribute to pathological scar formation. Myofibroblast differentiation and apoptosis are both modulated by cytokines, mechanical stress, and, more generally, cell-cell and cell-matrix interactions. Tissue repair allows tissues and organs to recover, at least partially, functional properties that have been lost through trauma or disease. Embryonic skin wounds are repaired without scarring or fibrosis, whereas skin wound repair in adults always leads to scar formation, which may have functional or esthetic consequences, as in the case of hypertrophic scars, for example. Skin wound repair involves a precise remodeling process, particularly in the dermal compartment, during which fibroblasts/myofibroblasts play a central role. This article reviews the origins of myofibroblasts and their role in normal and pathological skin wound healing. This article focuses on traumatic skin wound healing, but largely, the same mechanisms apply in other physiological and pathological settings. Tissue healing in other organs is examined by comparison, as well as the stromal reaction associated with cancer. New approaches to wound/scar therapy are discussed.

Impact of photodynamic therapy on inflammatory cells during human chronic periodontitis.

The aim of this study was to evaluate the effects of the photodynamic therapy (PDT) on the inflammatory infiltrate and on the collagen network organization in human advanced chronic periodontitis. Two different drug delivery systems (DDS) were tested (liposomes and nanoemulsions) to determine if the effects of PDT could differ according to the DDS used. Sixteen patients presenting two teeth with chronic advanced periodontitis and important tooth mobility with clinical indication of extraction were included in the group liposomes (group L, n=8) or in the group nanoemulsions (group N, n=8) in order to compare the effects of each DDS. Seven days before extractions one tooth of each patient was treated with PDT using phthalocyanine derivatives as photosensitizers and the contralateral tooth was taken as control. In group L the density of gingival collagen fibers (66±19%) was significantly increased (p<0.02) when compared to controls (35±21%). Concerning the antigen-presenting cells, PDT had differential effects depending on the drug delivery system; the number of macrophages was significantly decreased (p<0.05) in group L while the number of Langerhans cells was significantly decreased in group N (p<0.02). These findings demonstrate that PDT presents an impact on gingival inflammatory phenomenon during chronic periodontitis and leads to a specific decrease of antigen-presenting cells populations according to the drug delivery system used.

Fusiform aneurysm model in rabbit carotid artery.

AIMS: To develop a reproducible and accessible model of elastase-induced fusiform aneurysm in carotid rabbit arteries. METHODS: Elastase, at a concentration of 1-30 U, was incubated into the lumen of carotid rabbit arteries. Four weeks later, angiography, histomorphometry, immunohistochemistry and zymography were performed. RESULTS: The optimal concentration of elastase in this model was 3 U according to the balance between mortality and thrombosis rates. Indeed, at 3 U, external carotid diameter increased from 1.9 +/- 0.1 to 3.1 +/- 0.4 mm (p < 0.0001) associated with degradation of elastic fibers, matrix metalloproteinase-9 secretion, apoptosis and macrophage infiltration. CONCLUSIONS: Our study underlines that abdominal aortic aneurysm can be reliably duplicated in an elastase-induced aneurysm in carotid artery, a much more accessible vessel.

Gingival fibroblast inhibits MMP-7: evaluation in an ex vivo aorta model.

Matrix metalloproteinases (MMP) play a deleterious role in numerous vascular diseases. In contrast, gingival matrix remodelling is adequately regulated by the gingival fibroblast (GF). Here, we aimed to evaluate the GF activity on MMP-7 expression and secretion in coculture with aorta rings. We evaluated MMP-7 transcription and secretion in rabbit aorta rings cultured or not with gingival fibroblasts in collagen gels. GF induced an increase of TIMP-1 transcription and secretion, followed, similarly to other MMPs, by the formation of TIMP-1/MMP-7 complexes. There was also a decrease of MMP-7 mRNA by RT-PCR in aorta rings cocultured with gingival fibroblasts. Interestingly, in contrast with other MMPs (which were not influenced at a transcription level), GF stimulated the release of TGF-beta1, which in turn inhibited the transcription and synthesis of MMP-7, as shown by neutralizing MMP-7 inhibition due to gingival fibroblast by overexpressing decorin (a TGF beta 1 inhibitor) or by silencing TGF beta 1 using siRNA. We showed that healing properties of the GF could be transposed to another organ, i.e., ex vivo aneurism model, implicating a down-regulation of MMP-7.

Human bone marrow-derived cells: an attractive source to populate dermal substitutes.

We have previously shown the importance of dermal fibroblasts within skin substitutes for promoting the emergence of a functional neodermis after grafting in humans. However, the use of fibroblasts from sources other than the dermis needs to be evaluated for patients with extensive skin loss. Here we examined the capacity of human bone marrow-derived cells (BMDCs), selected for their ability to adhere to plastic culture dishes, to behave like human dermal fibroblasts when incorporated within a 3D in vitro reconstructed tissue that promotes dermal fibroblast differentiation. Like dermal fibroblasts, BMDCs contracted a collagen matrix and were growth regulated by the matrix environment. They had the same shape and their nuclei had the same form factor as dermal fibroblasts. In addition, both cell types expressed desmin and vimentin but not alpha-smooth muscle actin. BMDCs deposited collagen types I and III, and fibrillin-1 with similar efficiency to dermal fibroblasts. In addition, BMDCs have the potential to regulate this deposition, as they produced metalloproteinases (MMP1, MMP2, and MMP9) and metalloproteinase inhibitors (TIMP1) very similarly to dermal fibroblasts. BMDCs can thus be induced to express functions resembling those of dermal fibroblasts, including those involved in the wound healing process.

Infrared radiation induces the p53 signaling pathway: role in infrared prevention of ultraviolet B toxicity.

We have previously observed that preirradiation with naturally occurring doses of near-infrared (IR) protects normal human dermal fibroblasts from ultraviolet (UV) cytotoxicity in vitro. This effect was observed in temperature-controlled conditions, without heat shock protein (Hsp72-70) induction. Moreover, IR inhibited UVB-induced apoptosis by modulating the Bcl2/Bax balance, pointing to a role of p53. Here, we show for the first time that p53-deficient SaOs cells are not protected from UVB cytotoxicity by IR preirradiation, suggesting that the response to IR is p53-dependent. Thus, we investigated the effect of IR on the p53 signaling pathway. Normal human dermal fibroblasts exposed in vitro to IR accumulated p53 protein, involving p53 stabilization and phosphorylation of serine 15 (Ser15) and Ser20. IR-induced p53 accumulation correlated with increased expression of p21 and GADD45, showing that IR also stimulates p53 transcriptional activity. By modulating UVB-induced targets of the p53 signaling pathway, IR irradiation appears to anticipate the UVB response and to prepare cells to better resist subsequent UV-induced stress. This is reinforced by the fact that IR preirradiation reduces the formation of UVB-induced thymine dimers.

Infrared radiation affects the mitochondrial pathway of apoptosis in human fibroblasts.

We have previously observed that near-infrared (IR) pre-irradiation protects normal human dermal fibroblasts from ultraviolet (UV) cytotoxicity in vitro. Here, we show that IR pre-irradiation of human fibroblasts inhibited UVB activation of caspase-9 and -3, leading us to study early events in the mitochondrial apoptotic pathway after IR irradiation. IR irradiation led to a partial release of cytochrome c and Smac/Diablo but not apoptosis-inducing factor (AIF). This was accompanied by a slight but transient decrease in the mitochondrial membrane potential (Deltapsim) and by the insertion of Bax into mitochondrial membrane. Early apoptotic events in the mitochondrial pathway thus occurred after IR irradiation despite a lack of caspase-9 and -3 activation. This could be explained by the induction by IR of the expression of heat shock protein Hsp27, which is known to prevent apoptosome assembly. Furthermore, the balance between pro-apoptotic (i.e., Bax) and anti-apoptotic (i.e., Bcl-2 or Bcl-xL) proteins, which was rather pro-apoptotic after IR exposure, became anti-apoptotic 24 h later, suggesting a protective effect. Together, these actions could also contribute to prepare the cell to resist UVB-triggered apoptosis. Finally, isolated rat liver mitochondria-released cytochrome c in response to IR, demonstrating that mitochondria were a primary target of IR radiation.

ERK activation by mechanical strain is regulated by the small G proteins rac-1 and rhoA.

Physical forces play an important role in regulating cell functions. We applied mechanical strain to human fibroblasts by magnetic attraction of superparamagnetic arginine-glycine-aspartic acid (RGD)-coated beads. We confirmed that the MAP kinases Erk and p38 are activated by mechanical strain, and went further by demonstrating the activation of Elk-1 by mechanical strain, mainly through a MEK-Erk pathway. Transfection of a dominant negative form of the G protein rac-1 (rac T17N), and inhibition of PI3K, an effector of rac-1, efficiently prevented Elk-1 activation by mechanical forces. Transfection with C3 transferase, known to inhibit rhoA, and inhibition of rock (a downstream effector of rhoA), gave similar results. However, contrary to the active form of rhoA (rho G14V), transfection of the active form of rac-1 (rac G12V) induced Elk activation and mimicked the effects of mechanical strain. These results point out that the two small G proteins rhoA and rac-1 participate in cell sensitivity to mechanical strain and lead to the modulation of the Erk pathway.

p38 mitogen-activated protein kinase activation by ultraviolet A radiation in human dermal fibroblasts.

UVA radiation penetrates deeply into the skin reaching both the epidermis and the dermis. We thus investigated the effects of naturally occurring doses of UVA radiation on mitogen-activated protein kinase (MAPK) activities in human dermal fibroblasts. We demonstrated that UVA selectively activates p38 MAPK with no effect on extracellular-regulated kinases (ERK1-ERK2) or JNK-SAPK (cJun NH2-terminal kinase-stress-activated protein kinase) activities. We then investigated the signaling pathway used by UVA to activate p38 MAPK. L-Histidine and sodium azide had an inhibitory effect on UVA activation of p38 MAPK, pointing to a role of singlet oxygen in transduction of the UVA effect. Afterward, using prolonged cell treatments with growth factors to desensitize their signaling pathways or suramin to block growth factor receptors, we demonstrated that UVA signaling pathways shared elements with growth factor signaling pathways. In addition, using emetine (a translation inhibitor altering ribosome functioning) we detected the involvement of ribotoxic stress in p38 MAPK activation by UVA. Our observations suggest that p38 activation by UVA in dermal fibroblasts involves singlet oxygen-dependent activation of ligand-receptor signaling pathways or ribotoxic stress mechanism (or both). Despite the activation of these two distinct signaling mechanisms, the selective activation of p38 MAPK suggests a critical role of this kinase in the effects of UVA radiation.