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.

Biotechnological Management of Skin Burn Injuries: Challenges and Perspectives in Wound Healing and Sensory Recovery.

Many wound management protocols have been developed to improve wound healing after burn with the primordial aim to restore the barrier function of the skin and also provide a better esthetic outcome. Autologous skin grafts remain the gold standard in the treatment of skin burn, but this treatment has its limitation especially for patients presenting limited donor sites due to extensive burn areas. Deep burn injuries also alter the integrity of skin-sensitive innervation and have an impact on patient's quality of life by compromising perceptions of touch, temperature, and pain. Thus, patients can suffer from long-term disabilities ranging from cutaneous sensibility loss to chronic pain. The cellular mechanisms involved in skin reinnervation following injury are not elucidated yet. Depending on the depth of the burn, nerve sprouting can occur from the wound bed or the surrounding healthy tissue, but somehow this process fails to provide correct reinnervation of the wound during scarring. In addition, several clinical observations indicate that damage to the peripheral nervous system influences wound healing, resulting in delayed wound healing or chronic wounds, underlining the role of innervation and neuromediators for normal cutaneous tissue repair development. Promising tissue engineering strategies, including the use of biomaterials, skin substitutes, and stem cells, could provide novel alternative treatments in wound healing and help in improving patient's sensory recovery.

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.

The 23rd Annual Meeting of the European Tissue Repair Society (ETRS) in Reims, France.

The 23rd Annual Meeting of the European Tissue Repair Society, Reims, France, October 23 to 25, 2013 focused on tissue repair and regenerative medicine covering topics such as stem cells, biomaterials, tissue engineering, and burns.

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.

The myofibroblast, multiple origins for major roles in normal and pathological tissue repair.

Myofibroblasts differentiate, invade and repair injured tissues by secreting and organizing the extracellular matrix and by developing contractile forces. When tissues are damaged, tissue homeostasis must be re-established, and repair mechanisms have to rapidly provide harmonious mechanical tissue organization, a process essentially supported by (myo)fibroblasts. Under physiological conditions, the secretory and contractile activities of myofibroblasts are terminated when the repair is complete (scar formation) but the functionality of the tissue is only rarely perfectly restored. At the end of the normal repair process, myofibroblasts disappear by apoptosis but in pathological situations, myofibroblasts likely remain leading to excessive scarring. Myofibroblasts originate from different precursor cells, the major contribution being from local recruitment of connective tissue fibroblasts. However, local mesenchymal stem cells, bone marrow-derived mesenchymal stem cells and cells derived from an epithelial-mesenchymal transition process, may represent alternative sources of myofibroblasts when local fibroblasts are not able to satisfy the requirement for these cells during repair. These diverse cell types probably contribute to the appearance of myofibroblast subpopulations which show specific biological properties and which are important to understand in order to develop new therapeutic strategies for treatment of fibrotic and scarring diseases.

Endoluminal gingival fibroblast transfer reduces the size of rabbit carotid aneurisms via elastin repair.

OBJECTIVE: Matrix metalloproteinase-9 is considered to play a pivotal role in aneurismal formation. We showed that gingival fibroblasts (GF) in vitro reduced matrix metalloproteinase-9 activity via increased secretion of tissue inhibitor of metalloproteinase 1. We aimed to evaluate in vivo the efficacy of GF transplantation to reduce aneurism development in a rabbit model. METHODS AND RESULTS: Seventy rabbit carotid aneurisms were induced by elastase infusion. Four weeks later, GF, dermal fibroblast, or culture medium (DMEM) were infused into established aneurisms. Viable GF were abundantly detected in the transplanted arteries 3 months after seeding. GF engraftment resulted in a significant reduction of carotid aneurisms (decrease of 23.3% [P<0.001] and 17.6% [P=0.01] of vessel diameter in GF-treated arteries, 1 and 3 months after cell therapy, respectively), whereas vessel diameter of control DMEM and dermal fibroblast-treated arteries increased. GF inhibited matrix metalloproteinase-9 activity by tissue inhibitor of metalloproteinase 1 overexpression and matrix metalloproteinase-9/tissue inhibitor of metalloproteinase 1 complex formation, induced elastin repair, and increased elastin density in the media compared with DMEM-treated arteries (38.2 versus 18.0%; P=0.02). Elastin network GF-induced repair was inhibited by tissue inhibitor of metalloproteinase 1 blocking peptide. CONCLUSIONS: Our results demonstrate that GF transplantation results in significant aneurism reduction and elastin repair. This strategy may be attractive because GF are accessible and remain viable within the grafted tissue.

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.

Phenotypic study of human gingival fibroblasts in a medium enriched with platelet lysate.

BACKGROUND: The modulation abilities of gingival fibroblasts open new therapeutic strategies for the treatment of vascular diseases (e.g., aneurism) and irradiation burns. Culture media are classically supplemented with animal sera to provide nutriments. Unfortunately, because of their potential for interspecies transmission of microorganisms, these media are not used for cells destined for human transplantation. This preliminary phenotypic study aims to test a serum-free (SF) culture medium for human gingival fibroblasts (hGF) supplemented with human platelet lysates (PLs) for rapid cell expansion. METHODS: An SF medium was first elaborated to compete with hGF proliferation in a reference medium containing 10% fetal bovine serum (BSmedium). Adhesion, proliferation, and doubling kinetics were run in the presence of PLs (SF+PL). Cytoskeletal proteins were analyzed and chromosomal abnormalities were evaluated by karyotype analyses. The SF+PL influence on secretion of molecules implied in tissue remodeling (i.e., matrix metalloproteinases [MMPs], their tissue inhibitors [TIMPs], and several growth factors) was studied. RESULTS: SF+PL increased the proliferation rate 1.5-fold in a week compared to BSmedium. Cytoskeleton protein expression was similar in BSmedium and in SF+PL. Chromosomal abnormalities were rare in SF+PL. MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, TIMP-1, and the growth factors interleukin-1ß and -4 and transforming growth factor-ß1 secretions were stable during the experiment. TIMP-2 and interleukin-6 were slightly decreased in SF+PL compared to BSmedium. CONCLUSION: While waiting confirmation from a proteomic approach, this SF culture medium could allow a secured faster hGF proliferation adapted for human cell transplant therapy.

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.

Multipotent progenitor cells in gingival connective tissue.

The gum has an exceptional capacity for healing. To examine the basis for this property and explore the potential of conferring it to organs with inferior healing capacity, we sought the presence of progenitor cells in gingival connective tissue. Colony-forming units of fibroblast-enriched cells from gingival fibroblast cultures were assessed for expression of membrane markers of mesenchymal stem cells; capacity to differentiate into osteoblasts, chondroblasts, and adipocytes; and engraftment efficiency after in vivo transfer. On the basis of their ability to differentiate into several lineages, proliferate from single cells, induce calcium deposits, and secrete collagen in vivo after transfer on hydroxyapatite carriers, we suggest that this population represents gingival multipotent progenitor cells. The discovery of progenitor cells in gingival connective tissue may help improve our understanding of how the wounded gum is capable of almost perfect healing and opens the prospect of cellular therapy for wound healing using readily available cells at limited risk to the patient.

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.

Increase of gingival matured dendritic cells number in elderly patients with chronic periodontitis.

OBJECTIVE: The purpose of this study was to evaluate the inflammatory cell subset proportions in the upper gingival connective tissue, including mature dendritic cells (DC) in elderly and younger patients with generalized chronic periodontitis in order to further understand the effect of aging on gingival inflammatory phenomenon. METHODS: Gingival tissue specimens presenting chronic periodontitis from 8 elderly patients aged >75 (test group, group T) and from 8 younger patients aged 50-60 (considered as controls, group C) were analysed by immunohistochemistry using monoclonal antibodies against CD45RB, CD4, CD8, CD19, CD68, DC-SIGN, DC-LAMP molecules. The number of each immunolabelled cells subset was counted using image analysis. RESULTS: The difference in the number of CD45RB+leucocytes in the upper gingival connective tissue between groups was not significant permitting to use it as reference. As compared to group C, the lymphocyte subsets/CD45RB+leucocytes ratios tended to decrease in group T but the decrease was significant only for CD4+T lymphocytes/CD45RB+cells ratio (p<0.03). On the opposite, the ratios of antigen-presenting cells DC-SIGN+cells/CD45RB+cells and DC-LAMP+cells/CD45RB+cells were significantly increased (p<0.03 and <0.0001, respectively) in group T. Moreover, in group T the DC-LAMP+cells/DC-SIGN+cells ratio was significantly increased (p<0.05) showing an increased number of matured dendritic cells. CONCLUSION: During chronic periodontitis in elderly patients, our results show a decrease in the ratio of gingival CD4+lymphocyte subset associated with an increase in the ratios of antigen-presenting cells subsets and more particularly maturated DC-LAMP+dendritic cells.

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.

Gingival fibroblasts inhibit MMP-1 and MMP-3 activities in an ex-vivo artery model.

The main arterial pathologies can be associated with a deregulation of remodeling involving matrix metalloproteinases (MMPs), whereas gingival healing is characterized by an absence of fibrosis or irreversible elastin/collagen degradation. The aim of our study was to evaluate the effect of gingival fibroblasts on MMP-1 and MMP-3 secretion in an organotypic artery culture. MMP-1 and MMP-3 secretions and activities (dot blots, zymography, ELISA) were evaluated in coculture of rabbit artery in the presence or not of gingival fibroblasts. MMP-1/TIMP-1 and MMP-3/TIMP-1 complexes forms were measured by ELISA. Complementary studies were performed using human aortic smooth muscle cells cocultured with adventitial, dermal, or gingival fibroblasts. Our results indicated that MMP-1 and MMP-3 free-forms activities were significantly reduced in coculture. This inhibition was linked to a significant increase of TIMP-1 leading to formation of TIMP-1/MMPs complexes. Due to the presence of gingival fibroblasts, the decrease in MMP-1 and MMP-3 efficiency thus contributes to diminish the degradation of artery. This cellular therapy strategy could be promising in artery pathologies treatment.

Preservation of rabbit aorta elastin from degradation by gingival fibroblasts in an ex vivo model.

OBJECTIVE: Embryo-like gingival healing properties are attributed to the gingival fibroblast (GF) and could be used as a model for other types of healing dysfunctions. Abdominal aortic aneurysm (AAA) formation is associated with elastin degradation and increase in matrix metalloproteinase (MMP)-9 activity. We aimed to validate the concept of using GF healing properties in arteries. METHODS AND RESULTS: We evaluated MMP-9 and its tissue inhibitor (TIMP-1) in rabbit aortic rings cultured in collagen gels with or without GFs and observed throughout 21 days. We also performed cocultures of human smooth muscle cells (hSMCs) with either gingival, dermal, or adventitial fibroblasts, and alone (control). In control arteries, elastic fibers became spontaneously sparse. In presence of GFs, elastic fibers were preserved. There was a dramatically reduced protein level of MMP-9 in coculture of aorta and GFs, in contrast with control aorta. MMP-9 expression was unaffected by GFs. MMP-9 inhibition was related to increased TIMP-1 secretion, TIMP-1 forming a complex with MMP-9. Cell cocultures of hSMC with GFs showed similar results. Dermal and adventitial fibroblasts did not affect MMP-9. CONCLUSIONS: Elastic fiber degradation was specifically preserved by GFs via reduction of MMP-9 protein level by increasing TIMP-1 synthesis. Vascular transfer of gingival fibroblasts could be a promising approach to treat AAA.

Do Langerhans cells behave similarly in elderly and younger patients with chronic periodontitis?

OBJECTIVE: The purpose of this study was to compare the number, the distribution and the expression of markers of maturation of Langerhans cells (LC) in elderly and younger patients with chronic periodontitis in order to evidence the effect of aging on LC in inflammatory gingival tissue. METHODS: Gingival tissue specimens presenting chronic periodontitis from 8 elderly patients aged >75 (group E) and from 8 younger patients aged 50-60 (considered as controls, group C) were used for immunohistochemistry with monoclonal antibodies against CD45RB (leucocytes), CD1a (LC), markers of LC maturation (DC-LAMP, CD83) and number of immunolabelled cell subsets was evaluated using image analysis. RESULTS: The difference in the number of CD45RB+ leucocytes in the upper connective tissue between groups was not significant. In group E, the number of CD1a+ LC was significantly decreased (P<0.002) in the epithelium and significantly increased (P<0.0004) in the upper connective tissue. Furthermore, in group E, intraepithelial CD1a+ LC are more often observed in the upper epithelium and their dendritic processes were shorter and less numerous. Concerning the expression of markers of maturation, the numbers of intraepithelial DC-LAMP+ cells and CD83+ cells were significantly increased (P<0.0007 and P<0.02, respectively) in group E. CONCLUSION: During chronic periodontitis in elderly patients, the decrease in the number of intraepithelial LC and the alteration of dendritic processes could be balanced by a cellular distribution often observed in the upper epithelium associated with changes in cell maturation in response to bacterial elements.

Comparison of ultraviolet B-induced imbalance of antioxidant status in foreskin- and abdominal skin-derived human fibroblasts.

Ultraviolet B radiation (UVB) is involved in the development of deleterious cutaneous damage. Several changes could be attributed to UVB-induced reactive oxygen species attacks in fibroblasts. However dermal cells from young and adult skin could respond differently to oxidative stress. So antioxidant status and its consequences on cytotoxicity and apoptosis were compared in child foreskin fibroblasts (FF) and adult abdominal skin fibroblasts (AF) in response to UVB. Basal levels of lipid peroxidation tended to be higher in AF than in FF, which could be related to a reshaping of antioxidant defences (higher catalase and lower superoxide dismutase activities). AF and FF appeared to react similarly to high UVB doses as regards cytotoxicity and apoptosis which increased significantly 24h after exposure. The enhancement of cell death could be due to the inherent oxidative stress: glutathione appeared significantly decreased in both cell populations. As a consequence AF, but not FF, presented significantly increased levels of lipid peroxidation, which could be explained by the pre-cited differences of basal antioxidant defences. These results suggest that AF and FF do not respond to UVB by the same pathway.