Regulation of fibroblast-induced collagen gel contraction by interleukin-1β

Fibroblasts incorporated within collagen gels induce a cell-mediated contraction of the gel to form a three-dimensional, tissue-like structure by a mechanism thought to mimic wound contraction in vivo . In this study a gel contraction model was used to investigate the ability of fibroblasts derived from adult gingiva, adult skin and fetal skin to organise a collagen matrix. In addition the effects of interleukin-1β (IL-1β) on the contraction process was also investigated. Over the concentration range 5-50 U/ml, IL-1β induced a statistically significant inhibition of gel contraction in all fibroblast cell types ( P <0.05), although fetal fibroblasts appeared least responsive and gingival fibroblasts most responsive to the inhibitory effects of this cytokine. Comparison of gel contraction by the different fibroblast strains indicated that fetal and gingival fibroblasts shared similar contraction kinetics. For the adult skin fibroblasts, three of five strains studied showed significantly diminished levels of gel contraction compared to fetal and gingival cells. This apparent difference in fibroblast phenotype may, at least in part, explain the fetal-like wound healing pattern seen in the oral mucosa.     

Fibronectins in healing incision, excision and laser wounds

The distribution of extracellular matrix (ECM) glycoproteins, fibronectins (FNs), was studied in healing scalpel incision, excision and laser wounds of rat tongue dorsal mucosa over a healing period of 42 days by indirect immunofluorescence microscopy using mono- and polyclonal antibodies. A monoclonal antibody (Mab) DH1 was used to detect extradomain-A containing cellular fibronectin (ED-AcFN), and a polyclonal antiserum was utilized to recognize all forms of FNs. In normal tissue ED-AcFN was confined only to the endothelia of larger blood vessels whereas in healing wounds abundant immunoreactive deposits were found in regenerating connective tissue and endothelia of capillaries. The increased content of FNs revealed with both antibodies subsided later on during healing. The results suggest that the locally produced ED-AcFN is essential for tissue regeneration and plays a distinct functional role during wound healing. Laser treatment did not affect the ability of wound fibroblasts to synthesize and deposit cFN. The results provide further evidence that certain embryonic characteristics are seen in regenerating tissue.     

Wound healing effects of gingival fibroblasts cultured in animal‐free medium

Oral Diseases (2010) 16 , 438–444 Objective: The purpose of this study was to develop a graft material made of gingival fibroblasts cultured in animal‐free medium (HFDM1). Methods: We examined the effects of human serum (HS) on cell growth and wound healing capability, demonstrated by cytokine production, of gingival fibroblasts cultured in HFDM1. Subsequently, the capability of fibroblasts cultured in HFDM1 with 2% HS to promote the healing of skin defects was evaluated using nude mice. Results: The proliferation of human gingival fibroblasts was increased when HS at a concentration of 0.5–2% was added to HFDM1. Wound healing cytokines, including transforming growth factor‐β, keratinocyte growth factor, hepatocyte growth factor, vascular endothelial growth factor, and IL‐6 produced by gingival fibroblasts were increased by adding 2% HS to HFDM1. In addition, gingival fibroblasts cultured in HFDM1 with 2% HS improved wound healing of mouse skin defects as well as those cultured in Dulbecco’s modified Eagle’s medium with 10% fetal calf serum. Conclusion: Gingival fibroblasts cultured in HFDM1 with 2% HS may be useful as a graft material for reconstruction.     

Multiple functions of gingival and mucoperiosteal fibroblasts in oral wound healing and repair

Fibroblasts are cells of mesenchymal origin. They are responsible for the production of most extracellular matrix in connective tissues and are essential for wound healing and repair. In recent years, it has become clear that fibroblasts from different tissues have various distinct traits. Moreover, wounds in the oral cavity heal under very special environmental conditions compared with skin wounds. Here, we reviewed the current literature on the various interconnected functions of gingival and mucoperiosteal fibroblasts during the repair of oral wounds. The MEDLINE database was searched with the following terms: (gingival OR mucoperiosteal) AND fibroblast AND (wound healing OR repair). The data gathered were used to compare oral fibroblasts with fibroblasts from other tissues in terms of their regulation and function during wound healing. Specifically, we sought answers to the following questions: (i) what is the role of oral fibroblasts in the inflammatory response in acute wounds; (ii) how do growth factors control the function of oral fibroblasts during wound healing; (iii) how do oral fibroblasts produce, remodel and interact with extracellular matrix in healing wounds; (iv) how do oral fibroblasts respond to mechanical stress; and (v) how does aging affect the fetal‐like responses and functions of oral fibroblasts? The current state of research indicates that oral fibroblasts possess unique characteristics and tightly controlled specific functions in wound healing and repair. This information is essential for developing new strategies to control the intraoral wound‐healing processes of the individual patient.     

Distinct patterns of angiogenesis in oral and skin wounds

Clinical observation suggests that oral mucosal wounds heal faster than skin; however, little is known about the site-specific differences. Since fetal skin wounds heal rapidly, but are less vascular than adult wounds, we hypothesized that less robust wound angiogenesis might be observed in healing oral mucosa. This study investigated angiogenesis in equivalent-size oral and skin murine wounds. Change in wound bed vascularity was significantly lower in oral wounds than in skin. Also, vascular endothelial growth factor (VEGF) levels were less in oral than cutaneous wounds. Because keratinocytes are a prominent source of VEGF in wounds, we compared VEGF production by oral and epidermal keratinocytes in vitro. Significantly higher levels of VEGF protein and mRNA were observed in epidermal keratinocytes than in oral keratinocytes after 18 hrs of hypoxia. This study demonstrates distinct angiogenesis patterns in oral and skin wounds and intrinsic site-specific differences in VEGF production by keratinocytes.     

'Young' oral fibroblasts are geno/phenotypically distinct

Wound healing within the oral mucosa results in minimal scar formation compared with wounds within the skin. We have recently demonstrated distinct differences in the aging profiles of cells (oral mucosal and patient-matched skin fibroblasts) isolated from these tissues. We hypothesized that the increased replicative potential of oral mucosal fibroblasts may confer upon them preferential wound-healing capacities. Passage-matched early cultures of oral mucosal fibroblasts and skin fibroblasts demonstrated distinct gene expression profiles, with several genes linked to wound healing/tissue repair. This was related to an increased ability of the 'replicatively younger' oral mucosal fibroblasts to repopulate a wound space and reorganize their surrounding extracellular matrix environment, key activities during the wound-healing process. We conclude that oral mucosal fibroblasts exhibit a preferential healing response in vivo, due to their 'replicatively younger' phenotype when compared with that of patient-matched skin fibroblasts.     

Histatins,wound healing,and cell migration

Wounds in the oral mucosa heal faster and more efficiently than those in the skin, although the mechanisms underlying these differences are not completely clear. In the last 10 years, a group of salivary peptides, the histatins, has gained attention on behalf of their ability to improve several phases of the wound‐healing process. In addition to their roles as anti‐microbial agents and in enamel maintenance, histatins elicit other biological effects, namely by promoting the migration of different cell types contained in the oral mucosa and in non‐oral tissues. Histatins, and specifically histatin‐1, promote cell adhesion and migration in oral keratinocytes, gingival and dermal fibroblasts, non‐oral epithelial cells, and endothelial cells. This is particularly relevant, as histatin‐1 promotes the re‐epithelialization phase and the angiogenic responses by increasing epithelial and endothelial cell migration. Although the molecular mechanisms associated with histatin‐dependent cell migration remain poorly understood, recent studies have pointed to the control of signaling endosomes and the balance of small GTPases. This review aimed to update the literature on the effects of histatins in cell migration, with a focus on wound healing. We will also discuss the consequences that this increasing field will have in disease and therapy design.     

Laser burn wound healing in naso-labial region of fetal and neonatal mice

OBJECTIVE: To investigate the characteristics of wound healing in the mouse naso-labial region in both the fetal and neonatal stages, histological and immunohistochemical analyses were performed using a newly established laser burn wound healing system. MATERIALS AND METHODS: Fetal mice at embryonic day 14 (E 14) were wounded as a model of fetal wound healing. To compare it, neonatal mice at day 5 after birth (d 5) were adopted as a model of neonatal wound healing. The healing process was examined by van Gieson staining and immunohistochemistry for fibronectin and tenascin. RESULTS: Relatively large damage remained after wound healing even in fetal mice. In both types of wound healing, rapid regeneration of muscle tissues were observed. Fibronectin and tenascin immunostaining was detected not only in wound healing region, but also in the endomysium of regenerating muscle tissues. Especially, tenascin showed a restricted expression pattern. CONCLUSIONS: Rapid regeneration of muscle tissues in the naso-labial region in both the fetal and neonatal mice seemed to leave relatively large damage even in the fetal wound healing. Contracted force exerted by muscle tissues may be a reason for this phenomenon. Fibronectin and tenascin were closely related to the wound healing process including muscle regeneration in this region.     

Dynamic protein expression patterns during intraoral wound healing in the rat

Wound healing after cleft palate surgery is often associated with impairment of maxillary growth and dento-alveolar development. Wound contraction and scar tissue formation contribute strongly to these effects. In vitro studies have revealed that fibroblasts isolated during different phases of palatal wound healing show phenotypical differences. They change from a quiescent to an activated state and then partly back to a quiescent state. In this study, we evaluated the existence of fibroblast phenotypes at several time-points during palatal wound healing in the rat. Based on cytoskeletal changes (alpha-sma, vimentin, vinculin), integrin expression (alpha1, alpha2, alpha(v) and beta1) and changes in cellularity, we conclude that phenotypically different fibroblast populations are also present during in vivo wound healing. Alpha-sma and the integrin subunits alpha1 and alpha(v) were significantly up-regulated, and vinculin was significantly down-regulated, at early time-points compared to late time-points in wound healing. These changes point to an activated fibroblast state early in wound healing. Later in wound healing, these activated fibroblasts return only partially to the unwounded situation. These results strongly support the idea that different fibroblast populations with specific phenotypes occur in the course of palatal wound healing.     

Growth factor modulation of fibroblasts in simulated wound healing

Growth factors are potent bioactive molecules responsible for the co-ordination of many cell functions and interactions. Of these agents PDGF and IGF have shown particular promise as agents which may be used to stimulate periodontal regeneration. In order to further understand the mechanisms by which growth factors may work, a simple model of in vitro wound healing has been utilized to assess the effects of PDGF on human periodontal ligament fibroblasts and its potential to stimulate wound healing. Human periodontal ligament fibroblasts were plated into 24-well plates and upon reaching confluence were wounded by creating uniform discoid lesions stripped of cells. The influence of various concentrations of PDGF on cell proliferation, cell migration and extracellular matrix synthesis was monitored. The results of this study indicated that in the presence of 10 ng/ml PDGF and 0.2% fetal calf serum, both cell proliferation and cell migration were significantly stimulated. In the wounded cultures, PDGF appeared to cause a moderate stimulation of proteoglycan synthesis compared to unwounded cultures. In conclusion, the model system tested appears to be useful for studying fundamental cellular and biochemical events associated with wound healing. The effects of PDGF in this system confirm that it is capable of modulating fibroblasts in a manner compatible with the events associated with wound repair.     

Extraction wound healing in desalivated rats

Wound licking has been shown to promote extraoral wound healing among animals. Although the oral mucosa is bathed in saliva. Little information about the role of saliva in oral wound healing is found. The present study evaluates the healing of extraction wounds in desalivated rats. Experimental rats underwent sialadenectomy of the submandibular and sublingual glands and ligation of the parotid ducts. Maxillary left first molars were extracted. Rats were killed at 0, 1, 3, 5, 7, 10, 14, and 21 days after surgery and maxillae were prepared for light microscopy examination. Generally, a delay in socket healing in the desalivated rats was found. No differences were observed in blood clot formation. Replacement of the clot by granulation tissue was relatively slow concomitant with a longer inflammatory process. Bone formation kinetics were slower among the experimental rats.     

Tumor Necrosis Factor‐α and Interleukin (IL)‐1β, IL‐6, and IL‐8 Impair In Vitro Migration and Induce Apoptosis of Gingival Fibroblasts and Epithelial Cells,Delaying Wound Healing

Background: Multiple factors affect oral mucosal healing, such as the persistence of an inflammatory reaction. The present study evaluates effects of tumor necrosis factor (TNF)‐α and interleukin (IL)‐1β, IL‐6, and IL‐8 on epithelial cells (ECs) and human gingival fibroblasts (GFs) in vitro. Methods: GFs and ECs were seeded in 96‐well plates (1 × 10⁴ cells/well) in plain culture medium (Dulbecco’s modified Eagle’s medium [DMEM]) containing 1% antibiotic/antimycotic solution and 10% fetal bovine serum, and incubated for 24 hours. Both cell lines were exposed for 24 hours to the following cytokines: 1) TNF‐α (100 ng/mL); 2) IL‐1β (1 ng/mL); 3) IL‐6 (10 ng/mL); and 4) IL‐8 (10 ng/mL). All cytokines were diluted in serum‐free DMEM. Control cultures were exposed only to serum‐free DMEM. Effects of exposure to inflammatory cytokines were determined by means of: 1) apoptosis (anexin V); 2) cell migration (wound healing assay); 3) inflammatory cytokine synthesis (enzyme‐linked immunosorbent assay). Data were analyzed by Kruskal–Wallis and Mann–Whitney U tests (α = 0.05). Results: Increased apoptosis rates were noted when cells were exposed to inflammatory cytokines, except ECs exposed to IL‐1β. Cell migration was negatively affected by all inflammatory cytokines for both cell lines. ECs and GFs exposed to IL‐6 and IL‐8 significantly increased synthesis of TNF‐α and IL‐1β. Conclusions: Demonstrated results indicate negative effects of tested inflammatory cytokines on ECs and GFs, inducing apoptosis and impairing cell migration. These results can justify delayed oral mucosa healing in the presence of inflammatory reaction.     

Matrix metalloproteinase-3 differences in oral and skin fibroblasts

While skin wounds heal by scarring, wounds of oral mucosa show privileged healing with minimal scar formation. Our hypothesis was that phenotypic differences between oral and skin fibroblasts underlie these differences in healing. The aims of this study were to compare MMP-3 expression by oral and skin fibroblasts and investigate a role for MMP-3 in mediating collagen gel contraction. Oral fibroblasts induced significantly greater gel contraction than did paired skin cells. Inhibition of MMP activity significantly inhibited gel contraction by both cell types. Specific inhibition of MMP-3 activity reduced gel contraction by oral, but not skin, fibroblasts. Oral fibroblasts produced significantly higher levels of MMP-3 than did skin fibroblasts at all levels studied. TGF-beta1 and -beta3 isoforms stimulated MMP-3 expression at mRNA, protein, and activity levels by both fibroblast populations. Results suggest that increased MMP-3 production by oral fibroblasts may underlie the differences in wound-healing outcome seen in skin and oral mucosa.     

Basic fibroblast growth factor and epidermal growth factor reverse impaired ulcer healing of the rabbit oral mucosa

BACKGROUND: The therapies for refractory ulcers on the oral mucosa are symptomatic and very unsatisfactory. We hypothesized that application of growth factors might be able to achieve successful remission of the lesion. We evaluated the effects of systemic administration and topical application of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) on impaired wound healing of ulcers in the rabbit gingiva. METHODS: Almost uniform round ulcers could be created on the gingiva of the rabbits by chemical injury with acetic acid. When the submandibular glands were removed or i.v. injection of cisplatin (CDDP) and peplomycin sulfate was performed before ulcer formation, healing of the ulcers took longer than in untreated rabbits. To ascertain whether or not human EGF and bFGF affect rabbit cells, we first examined the effects of EGF and bFGF on the proliferation of the cells derived from rabbit gingiva. We then applied EGF or bFGF in these impaired healing models. RESULTS: EGF and bFGF promoted proliferation of the fibroblasts, and EGF also promoted proliferation of the keratinocytes isolated from gingival tissue of rabbits in vitro. Systemic injections of EGF and bFGF in rabbits, which had their submandibular glands removed, and topical application of bFGF accelerated healing of ulcers created in rabbits injected with CDDP and peplomycin sulfate. The ability of bFGF to promote the healing of ulcers was much greater than that of EGF. CONCLUSION: Basic FGF may be effective for refractory oral mucosal lesions.     

Distribution of tenascin in healing incision, excision and laser wounds

The distribution of the extracellular matrix glycoprotein, tenascin, was studied in normal mucosa and during healing of scalpel incised or excised and CO, laser-wounded rat tongue dorsal mucosa in 51 male Sprague-Dawley rats over a period of 21 days. A polyclonal antibody specific for tenascin was applied in indirect immunofluorescence microscopy. In normal mucosa tenascin was sparsely distributed in a discontinuous manner at the tips of the connective tissue papillae in association with the basement membrane (BM) and in the walls of the capillaries. In all the healing wounds there was a marked increase in the distribution of tenascin, particularly close to the BMs at the wound edges beneath the proliferating and migrating epithelium, and later on during healing in the regenerating connective tissue (CT) area. This expression subsided later on during healing. Laser surgery did not alter the ability of fibroblasts to synthesize tenascin. The transient expression of tenascin in the BMs and CT of the healing wounds suggests that this protein could play an important role in providing ideal conditions for cell movement, and in the deposition and organization of other extracellular matrix (ECM) glycoproteins during tissue repair.     

Myofibroblasts and matrix components in healing palatal wounds in the rat

In order to identify wound contraction and scar formation during palatal mucoperiosteal wound healing in growing rats, the temporal and spatial distribution of myofibroblasts and matrix components were determined immunohistochemically. Myofibroblasts were found in the mucosal part of the palatal wound tissue between 4 and 22 days, with the highest density at 8 days post-wounding. The number of collagen type I and type III fibers gradually increased until about 8 days postwounding, and thereafter the staining intensity of collagen type III decreased. At 60 days post-wounding there were more transversely oriented collagen type I fibers and less type III fibers and elastin present in the submucosa than in normal tissue. The results suggest that in this model wound contraction mainly takes place in the mucosa between 4 and 22 days postwounding. Furthermore, palatal wounds made in young rats heal with distinct scar tissue formation. Therefore, this model is useful to test the effects of therapies that aim to reduce wound contraction and scarring after cleft palate surgery.     

IL-22 mediates the oral mucosal wound healing via STAT3 in keratinocytes

ObjectiveWounds are common in the oral cavity. During wound healing, several cytokines are released, which are probably helpful in providing wound debridement, removal of damaged tissues and microbes. Most of the target cells of IL-22 are epithelial cells, which play an important role in mucosa immunity.DesignThe function of IL-22 in oral diseases is not well understood. We investigated the expression level of IL-22, collagen I and p-stat3 (Tyr705) via a mice tongue wound model in vivo and detected the effect of IL-22 on the expression of MMP-1, type I collagen and p-stat3 in keratinocytes.ResultsIL-22 and p-stat3 were associated with wound healing, and STAT3 was activated when the keratinocytes or the tongue tissue were stimulated by IL-22. In addition, IL-22 could mediate gene expression involved in wounds involving keratinocytes, such as type I collagen and MMP-1, which may contribute to scarless healing.ConclusionOur study suggests that IL-22 mediates wound healing via STAT3 in keratinocytes. This study reveals a new role for IL-22 in mediating wound healing.     

Enamel Matrix Derivative Promotes Healing of a Surgical Wound in the Rat Oral Mucosa

Background: Enamel matrix proteins (EMPs) play a role in enamel formation and the development of the periodontium. Sporadic clinical observations of periodontal regeneration treatments with enamel matrix derivative (EMD), a commercial formulation of EMPs, suggest that it also promotes post‐surgical healing of soft tissues. In vitro studies showed that EMD stimulates various cellular effects, which could potentially enhance wound healing. This study examines the in vivo effects of EMD on healing of an oral mucosa surgical wound in rats. Methods: A bilateral oral mucosa wound was created via a crestal incision in the anterior edentulous maxilla of Sprague‐Dawley rats. Full‐thickness flaps were raised, and, after suturing, EMD was injected underneath the soft tissues on one side, whereas the EMD vehicle was injected in the contralateral side. Animals were sacrificed after 5 or 9 days, and the wound area was subjected to histologic and immunohistochemical analysis of the epithelial gap, number of macrophages, blood vessels, proliferating cells, and collagen content in the connective tissue (CT). Gene expression analysis was also conducted 2 days post‐surgery. Results: EMD had no effect on the epithelial gap of the wound. On both days 5 and 9, EMD treatment increased significantly the number of blood vessels and the collagen content. EMD also enhanced (by 20% to 40%) the expression of transforming growth factors β1 and β2, vascular endothelial growth factor, interleukin‐1β, matrix metalloproteinase‐1, versican, and fibronectin. Conclusion: EMD improves oral mucosa incisional wound healing by promoting formation of blood vessels and collagen fibers in CT.     

Serum of patients with oral pemphigus vulgaris impairs keratinocyte wound repair in vitro: a time-lapse study on the efficacy of methylprednisolone and pyridostigmine bromide

Objectives:  Pemphigus vulgaris (PV) is an autoimmune blistering disease affecting primarily oral mucosa and skin. Among the drugs used for the therapy of pemphigus, both methylprednisolone (MP) and pyridostigmine bromide (PBr) can prevent acantholysis in vitro . However, their putative therapeutic properties in regenerating PV-like lesions and promoting the healing process still remain to be demonstrated. To address this issue, here we have developed a model for studying the process of epithelial cleft regeneration in PV by artificially wounding keratinocyte monolayers.
Materials and methods:  The experimental model was established by scratching confluent monolayers to simulate the epithelial cleft; then, wound regeneration in the presence of submaximal concentrations of PV sera was studied by time-lapse microscopy, with or without the addition of MP and PBr in the culture medium.
Results:  Pemphigus vulgaris serum inhibited epithelial cleft repair of wounded monolayers. Indeed, in the presence of 10% (v/v) PV serum, keratinocytes reached only 2% confluence within 72 h vs an almost complete healing of controls. When administered together with PV sera, MP significantly ( P  < 0.01) enhanced wound fill by 30% after 72 h. PV-associated wound repair was significantly ( P  < 0.05) ameliorated by PBr by 24 h and keratinocytes reached 20% confluence after 72 h. Interestingly, neither MP nor PBr could accelerate wound healing when compared with untreated control monolayers.
Conclusions:  In PV, MP and PBr exert their curative effects in part by enhancing the regeneration properties of keratinocytes. Indeed, our data suggest that both drugs can specifically counterbalance the detrimental effects of PV serum on keratinocyte wound healing. These findings provide an explanation for the efficacy of MP and PBr in the treatment of PV lesions in human skin and oral mucosa.     

Tissue regeneration in dentistry: Can salamanders provide insight?

The ability to regenerate damaged tissues would be of tremendous benefit for medicine and dentistry. Unfortunately, humans are unable to regenerate tissues such as teeth and fingers or to repair injured spinal cord. With an aging population, health problems are more prominent and dentistry is no exception as loss of bone tissue in the orofacial sphere from periodontal disease is on the rise. Humans can repair oral soft tissues exceptionally well; however, hard tissues, such as bone and teeth, are devoid of the ability to repair well or at all. Fortunately, Mother Nature has solved nearly every problem that we would like to solve for our own benefit and tissue regeneration is no exception. By studying animals that can regenerate, like Axolotls (Mexican salamander), we hope to find ways to stimulate regeneration in humans. We will discuss the role of the transforming growth factor beta cytokines as they are central to wound healing in humans and regeneration in Axolotls. We will also compare wound healing in humans (skin and oral mucosa) to Axolotl skin wound healing and limb regeneration. Finally, we will address the problem of bone regeneration and present results in salamanders which indicate that in order to regenerate bone you need to recruit non‐bone cells. Fundamental research, such as the work being performed in animals that can regenerate, offers insight to help understand why some treatments are successful while others fail when it comes to specific tissues such as bones.