Bone marrow‐derived cells in palatal wound healing

Oral Diseases (2010) 16 , 788–794 Objective: Myofibroblasts are responsible for contraction and scarring after cleft palate repair. This leads to growth disturbances in the upper jaw. We hypothesized that cells from the bone marrow are recruited to palatal wounds and differentiate into myofibroblasts. Methods: We transplanted bone marrow from green fluorescent protein (GFP)‐transgenic rats into lethally irradiated wild‐type rats. After recovery, experimental wounds were made in the palatal mucoperiosteum, and harvested 2 weeks later. GFP‐expressing cells were identified using immunostaining. Myofibroblasts, activated fibroblasts, endothelial cells, and myeloid cells were quantified with specific markers. Results: After transplantation, 89 ± 8.9% of mononuclear cells in the blood expressed the GFP and about 50% of adherent cells in the bone marrow. Tissue obtained during initial wounding contained only minor numbers of GFP‐positive cells, like adjacent control tissue. Following wound healing, 8.1 ± 5.1% of all cells in the wound area were positive, and 5.0 ± 4.0% of the myofibroblasts, which was significantly higher than in adjacent tissue. Similar percentages were found for activated fibroblasts and endothelial cells, but for myeloid cells it was considerably higher (22 ± 9%). Conclusions: Bone marrow‐derived cells contribute to palatal wound healing, but are not the main source of myofibroblasts. In small wounds, the local precursor cells are probably sufficient to replenish the defect.     

Local injection of IFN-gamma reduces the number of myofibroblasts and the collagen content in palatal wounds

Wound contraction and scar formation after cleft palate surgery impair maxillary growth and dentoalveolar development. Since myofibroblast numbers and scar formation are reduced by interferon-gamma (IFN-gamma) in the healing of dermal wounds, the hypothesis was tested that local administration of IFN-gamma reduces the numbers of myofibroblasts and the elevated amount of collagen during palatal mucoperiosteal wound healing. Standardized mucoperiosteal excision wounds were made in the palatal mucoperiosteum of young rats. Either IFN-gamma or vehicle alone (sham group) was repeatedly injected into the wound site between 4 and 29 days post-wounding. The results were compared with unmanipulated control wounds. Samples of wound tissue were prepared for biochemical and microscopic analysis. The hydroxyproline, sulfated glycosaminoglycan and DNA contents of the wound tissues were analyzed biochemically. The degree of re-epithelialization, tissue thickness, the numbers of myofibroblasts, and the amounts of elastin and collagen types I and III were evaluated on histological sections. Injection of vehicle alone affected almost all healing parameters, compared with the controls, and delayed the wound-healing process. IFN-gamma stimulated re-epithelialization and decreased the numbers of myofibroblasts when compared with vehicle-treated wounds. It also decreased the hydroxyproline and glycosaminoglycan contents of 60-day-old wound tissue, but the histological characteristics of scar tissue persisted. Therefore, IFN-gamma is able to reduce the numbers of myofibroblasts and the collagen content of scar tissue after palatal wound healing. It may be a promising pharmaceutical agent for the reduction of wound contraction and scarring after cleft palate surgery.     

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.     

Myofibroblasts in healing laser wounds of rat tongue mucosa

The distribution of myofibroblasts was studied in healing laser incisions compared with scalpel-incision and excision wounds in dorsal tongue mucosa and excision wounds in back skin. Myofibroblasts (m-f-b) were visualized by staining with NBD-phallacidin, a fluorescent probe specific for F-actin, and by electron microscopy. Few, randomly-orientated m-f-b were found in laser wounds over 28 days. Neither m-f-b nor contraction were seen in the scalpel-incisions. No contraction was observed in the laser wounds whereas an organized network of m-f-b with substantial contraction occurred in excision wounds. It is suggested that lack of contraction in laser wounds is partially due to the fewness of m-f-b. The residual connective tissue matrix resisting the laser treatment also seems to play a role in preventing the wound contraction.     

Evidence for apoptosis induction in myofibroblasts during palatal mucoperiosteal repair.

Apoptosis is thought to be a requisite event for maintaining kinetic homeostasis within continually renewing tissues such as the oral mucosa and skin. However, no systematic study of the apoptotic process in fibroblasts in the oral mucosa following injury has been performed. In this study, we have assessed the expression of transforming growth factor-beta1 (TGF-beta1) and basic fibroblast growth factor (bFGF), which are among the most important modulators of wound repair, during wound healing following mucoperiosteal injury in the rat palate. In addition, we have investigated fibroblast differentiation and apoptosis by immunohistochemical analysis for alpha-smooth-muscle (alpha-SM) actin or DNA strand breaks, respectively, to clarify the mechanisms of the wound healing process. TGF-beta1-positive cells were noted in the subepithelium from Day 2 to Day 14 after injury, by which time the wounds were completely reepithelialized. Strong expression of bFGF was observed, mainly in macrophages and monocytes at the injured site, from Day 10 to Day 14 after injury. TGF-beta1 and bFGF-immunostaining was significantly lower during the later phase of wound healing. In addition, the number of myofibroblasts expressing alpha-SM actin increased (peak at Day 14), and thereafter gradually decreased. In parallel, the apoptosis in myofibroblasts was prominent on Day 14. These results suggest that TGF-beta1 and bFGF may be potential stimulators of apoptosis in myofibroblasts after re-epithelialization in the palatal wound healing process. The regulation of apoptotic phenomena during wound healing may be important in scar establishment and development of pathological scarring.     

Quantitative evaluation of myofibroblast apoptosis during wound healing in rat palate after post-operative administration of basic fibroblast growth factor (bFGF)

Abstract

Objective. Excessive wound contraction apparently inhibits maxillary growth; thus, myofibroblast apoptosis needs to be accelerated in mucoperiosteal denudation after palatoplasty. The aim of this study was to evaluate myofibroblast apoptosis during wound healing in mucoperiosteal denudation of rat palates immediately after post-operative administration of basic fibroblast growth factor (bFGF). Materials and methods. A total of 100 male Wistar rats aged 20 days were divided into control, scar, sham and bFGF groups (n = 25 each). In the scar, sham and bFGF groups, mucoperiosteum was removed from the palate and fibrin glue was applied to the exposed bone surface immediately after surgery. In the bFGF group, 10 μL of 2 μg/μL bFGF solution was injected into the operated area beneath the fibrin glue. At 2, 5, 7, 14 and 28 days post-operatively, myofibroblast apoptosis during the wound healing process was investigated by double immunofluorescence staining. The apoptotic area of myofibroblasts was measured using image software. Results. In the bFGF group, at 2 days, apoptosis of myofibroblasts in the lamina propria and submucosa was marked, as compared with the other three groups and apoptosis of myofibroblasts was scarcely seen at 5 days. At 5 and 7 days, the apoptotic area of myofibroblasts in the bFGF group was statistically significantly smaller when compared to the scar and sham groups. Conclusion. The results confirmed that bFGF injection immediately after surgery accelerated apoptosis of myofibroblasts in mucoperiosteal denudation of rats. This may reduce maxillary growth retardation due to excessive wound contraction.     

Effects of locally injected interferon-β on palatal mucoperiosteal wound healing

BACKGROUND: Wound contraction and scar formation in the palatal mucoperiosteum after cleft palate surgery impair maxillary growth. The aim of this study was to determine the effects of IFN-beta on palatal mucoperiosteal wound healing in growing rats. METHODS: Standardized wounds were made in the palatal mucoperiosteum of young rats. Either IFN-beta or vehicle were injected at the wound site between 4 and 29 days after wounding. The results were compared with control wounds. Tissue samples were collected at 8, 15, 30, and 60 days PW for biochemical and microscopic analysis. RESULTS: IFN-beta stimulated re-epithelialization but did not reduce the number of myofibroblasts or scar tissue formation. Surprisingly, the injection of vehicle alone delayed the healing process. CONCLUSION: IFN-beta might be suitable to stimulate re-epithelialization but it does not reduce scar tissue formation in rat palatal wound healing. The injection of agents into palatal wounds might severely impair the healing process.     

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.     

Differential injury responses in oral mucosal and cutaneous wounds

Oral mucosa heals faster than does skin, yet few studies have compared the repair at oral mucosal and cutaneous sites. To determine whether the privileged healing of oral injuries involves a differential inflammatory phase, we compared the inflammatory cell infiltrate and cytokine production in wounds of equivalent size in oral mucosa and skin. Significantly lower levels of macrophage, neutrophil, and T-cell infiltration were observed in oral vs. dermal wounds. RT-PCR analysis of inflammatory cytokine production demonstrated that oral wounds contained significantly less IL-6 and KC than did skin wounds. Similarly, the level of the pro-fibrotic cytokine TGF-b1 was lower in mucosal than in skin wounds. No significant differences between skin and mucosal wounds were observed for the expression of the anti-inflammatory cytokine IL-10 and the TGF-beta1 modulators, fibromodulin and LTBP-1. These findings demonstrate that diminished inflammation is a key feature of the privileged repair of oral mucosa.     

Effect of cultured autologous oral keratinocyte suspension in fibrin glue on oral wound healing in rabbits

The effect of cultured autologous oral keratinocyte suspension in fibrin glue on the healing of surgically produced oral mucosal wounds was assessed in the rabbit model. Using the light microscope and a digital image analysis system, the epithelization parameters (marginal epithelization and percentage of wound re-epithelization) were measured in haematoxylin-eosin stained sections of the wound area and compared with those of wounds treated with fibrin glue alone and untreated ones. The epithelization was significantly higher in keratinocytes plus fibrin glue-treated wounds on postoperative days 3 and 7. No significant differences were observed on postoperative day 1, when the healing process had just begun, and on postoperative day 14, when re-epithelization was completed or nearly completed in all groups. The inflammatory infiltration of the wounded mucosa was weakest in keratinocyte-treated wounds and strongest in untreated wounds. In conclusion, suspension of cultured autologous oral keratinocytes in fibrin glue significantly accelerates oral wound healing in the rabbit model and could be beneficial in the treatment of oral wounds in patients.     

Effects of He-Ne laser irradiation on fibroblasts derived from scar tissue of rat palatal mucosa

This study was carried out to clarify the mechanism of stimulating effects of low power He-Ne laser irradiation on wound healing process of rat palatal mucosa. Experimental wounds were made in 8-week-old male SD rats by surgically excising mucoperiosteum in hard palate. Fibroblasts were cultured from the scar tissue one month after the excision. Cells were investigated ultrascopically and the effects of He-Ne laser irradiation were examined by measuring DNA and collagen synthesis. Thermal change of the medium was also monitored. Cultured cells from the palatal wounds exhibited features of myofibroblasts. They exhibited well-developed bundles of microfilaments in the cytoplasm and had nuclei with foldings. Single irradiation of He-Ne laser at 28J/cm2 stimulated uptake of [3H] Thymidine into myofibroblasts and the stimulating effect decreased time dependently. There was no significant change in collagen synthesis. No thermal change was detected during irradiation. Ultrascopically, dilatation of rER was observed immediately after irradiation at 28J/cm2. But there was no modification in other cytoplasmic structures. The author concludes that He-Ne laser irradiation stimulates DNA synthesis of myofibroblasts without any degenerative changes in the organelle.     

TGF-β1-induced connexin43 promotes scar formation via the Erk/MMP-1/collagen III pathway

Wound healing can be divided into different phases, and timely initiation and cessation of these stages is key to successful wound healing; otherwise, scar tissue forms in the wounded area. Connexins (Cxs) were confirmed to influence scar formation, and Cx43, an indispensable member of the Cx family, was shown to be involved in this process. Our study investigated the regulatory role of Cx43 in scar formation and the possible cell signalling pathways. We established oral mucosa and skin wound healing models in C57BL/6J mice. RT-PCR, western blotting, immunohistochemistry and immunofluorescence were used to examine the expression of ECM components and key proteins in cell signalling pathways (TGF-β1, Smad2/3, Cx43, Erk1/2 MMP-1 and collagen III). After injury, buccal mucosa wounds healed with no scar, whereas skin wounds healed with an evident scar. Nevertheless, TGF-β1 expression gradually increased by the 5th day after injury; Cx43 expression showed a similar response, with a progressive increase in the skin and a peak on day 14. In contrast, TGF-β1 and Cx43 expression in the oral mucosa remained low. The high level of TGF-β1 increased p-Smad2/3 levels and then induced Cx43, whereas increased expression of Cx43 antagonised the phosphorylation of Erk1/2, a protein downstream of Cx43, which affected MMP-1 synthesis. MMP-1 deficiency led to collagen III accumulation and facilitated scar formation. We demonstrated that TGF-β1-induced Cx43 promotes scar formation via the Erk/MMP-1/collagen III pathway.     

A comparative study of wound healing following incision with a scalpel,diode laser or Er,Cr:YSGG laser in guinea pig oral mucosa: A histological and immunohistochemical analysis

Abstract

Objective. This study compared wound healing following incisions with either a scalpel, a diode laser or an Er,Cr:YSGG laser in guinea pig oral mucosa. Material and methods. Three types of wound were made randomly with either a stainless-steel scalpel, a diode laser or an Er,Cr:YSGG laser in the buccal mucosa of 24 guinea pigs. Five guinea pigs were sacrificed on each of Days 1, 3, 5 and 7 post-surgery. Four guinea pigs were sacrificed on Day 14 post-surgery. Biopsy samples from each oral mucosa wound were examined using light microscopy and the expression of tumor necrosis factor (TNF)-α and transforming growth factor (TGF)-β1 was determined by immunohistochemical staining. The expression of TNF-α and TGF-β1 was evaluated by calculating the percentage of positively stained cells and immunostaining intensity was evaluated using a scale ranging from 0 to 3. Results. Infiltration of inflammatory cells decreased rapidly at Day 5 post-surgery in all three groups of animals. The highest level of TNF-α expression was found at Day 1 post-surgery for the diode laser wounds. The intensity of TNF-α immunostaining was highest at Day 3 post-surgery and lowest at Day 7 post-surgery for all three groups of animals. For the scalpel wounds, a lower level of TGF-β1 expression was seen until Day 3 post-surgery and a higher level from Day 7 post-surgery compared to laser wounds. The intensity of TGF-β1 immunostaining was highest at Day 1 post-surgery for the diode laser wounds. Conclusions. The diode laser is considered a good cutting device for oral mucosa; however, more tissue damage occurs than with the use of a scalpel or an Er,Cr:YSGG laser. Larger studies will be needed before fully endorsing the widespread use of the diode laser.     

A comparative histological and immunohistochemical study of wound healing following incision with a scalpel,CO2 laser or Er,Cr:YSGG laser in the Guinea pig oral mucosa

Abstract

Purpose. This study was undertaken to compare wound healing following incisions with either a scalpel, CO₂ laser or Er,Cr:YSGG laser in Guinea pig oral mucosa. Materials and methods. Three types of wounds were randomly introduced with either a stainless steel scalpel, CO₂ laser or Er,Cr:YSGG laser in the buccal mucosa of each of 22 Guinea pigs. Four Guinea pigs were sacrificed on day 1, day 3 and day 5 post-surgery. Five Guinea pigs were sacrificed on day 7 and day 14 post-surgery. Biopsy samples from each oral mucosa wound were examined and the expression of TNF-α and TGF-ß1 was determined by immunohistochemical staining. Results. At day 3 post-surgery, the histological pattern of the healing process was similar in the scalpel and Er,Cr:YSGG laser wounds and there were more ulcerations present in the CO₂ laser wounds than in the scalpel and Er,Cr:YSGG laser wounds. The level of TNF-α expression was twice in the laser wounds that in the scalpel wounds. A higher level of TGF-β1 expression was seen at day 7 post-surgery and a lower level at day 14 post-surgery in the CO₂ laser wounds than in the scalpel and Er,Cr:YSGG laser wounds. Conclusions. The Er,Cr:YSGG laser has many advantages for oral surgery due to a low inflammatory response and minimal damage of the tissue. Although a CO₂ laser has better hemostatic ability, its use causes greater tissue damage than a scalpel and Er,Cr:YSGG laser. However, further larger studies would be needed before fully endorsing its widespread use.     

Collagen implants do not preserve periodontal ligament homeostasis in periodontal wounds

An improved understanding of the differentiation of periodontal ligament cells could facilitate the development of new treatment approaches for overcoming the loss of specialized cell types caused by periodontitis. To study healing of wounded periodontal tissues and the differentiation of mineralizing connective tissue cells in periodontal ligament, we have examined the influence of wound size and collagen implantation on the regeneration of periodontium and on immunohistochemical staining for osteopontin and bone sialoprotein. Four groups of Wistar rats were wounded by drilling through the alveolar bone and by extirpation of the periodontal ligament. Wounds were 0.6 or 1.8 mm in diameter and defects were either implanted with collagen gels or were treated without implants. Rats were killed at 1 wk or 2 months after wounding and tissue sections were stained with monoclonal antibodies against rat osteopontin and bone sialoprotein. Collagen implants strongly increased staining for osteopontin and bone sialoprotein in defects at 1 wk. By 2 months alveolar bone healed completely regardless of the wound size but in large defects, periodontal ligament width was significantly reduced with or without implants. In large wounds at 2 months, collagen implants inhibited bone regeneration and there was stronger staining for osteopontin and bone sialoprotein in the bone replacing the implant, indicating that collagen prolonged bone remodelling. We conclude that implantation of exogenous collagen affects alveolar bone healing but does not preserve the width of the regenerated periodontal ligament. Therefore collagen does not appear to contribute to homeostasis in the periodontium following wounding.     

'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.     

Construction and characterization of human oral mucosa equivalent using hyper-dry amniotic membrane as a matrix

BackgroundHuman amniotic membrane(HAM) as a graft material has been used in various fields. Hyper-dry amniotic membrane (HD-AM) is a novel dried amniotic membrane that is easy to handle and can be preserved at room temperature without time limitation. The purpose of this study was to investigate the useful properties of HD-AM in reconstruction of the oral mucosa.MethodsHuman oral keratinocytes were isolated and seeded on HD-AM in serum-free culture system. Oral mucosa equivalent (OME) was developed and transplanted onto full-thickness wound on athymic mice. The wound healing was analyzed and the OME both before and after transplantation was analyzed with hematoxylin-eosin staining and immunohistochemicial staining for Cytokines 10 (CK10), Cytokines 16 (CK16), and Ivolucrin (IVL).ResultsOral keratinocytes spread and proliferated well on HD-AM. Two weeks after air-lifting, OME had formed with good differentiation and morphology. We confirmed immunohistochemically that the expression of CK10 was positive in all suprabasal layers, as was CK16 in the upper layers, while IVL was present in all cell layers. Three weeks after transplantation to athymic mice, the newly generated tissue had survived well with the smallest contraction. The epithelial cells of newly generated tissue expressed CK10 throughout in all suprabasal layers, IVL was mainly in the granular layer, and CK16 positive cells were observed in all spinous layer and granular layer but were not expressed in the mouse skin, all of which were similar to native gingival mucosa.ConclusionsThe OME with HD-AM as a matrix revealed a good morphology and stable wound healing. This study demonstrates that HD-AM is a useful and feasible biomaterial for oral mucosa reconstruction.     

Lysophosphatidic acid induces expression of genes in human oral keratinocytes involved in wound healing

ObjectiveEpithelial cells participate in wound healing by covering wounds, but also as important mediators of wound healing processes. Topical application of the phospholipid growth factor lysophosphatidic acid (LPA) accelerates dermal wound healing and we hypothesized that LPA can play a role in human oral wound healing through its effects on human oral keratinocytes (HOK).DesignHOK were isolated from gingival biopsies and exposed to LPA. The LPA receptor profile, signal transduction pathways, gene expression and secretion of selected cytokines were analyzed.ResultsHOK expressed the receptors LPA₁, LPA₅ and LPA₆ and LPA activated the ERK1/2, JNK and p38 intracellular pathways, substantiated by secretion of IL-6 and IL-8. The early (2 h) and intermediate (6 h) gene expression profiles of HOK after LPA treatment showed a wide array of regulated genes. The majority of the strongest upregulated genes were related to chemotaxis and inflammation, and became downregulated after 6 h. At 6 h, genes coding for factors involved in extracellular matrix remodeling and re-epithelialization became highly expressed. IL-36γ, not earlier known to be regulated by LPA, was strongly transcribed and translated but not secreted.ConclusionsAfter stimulation with LPA, HOK responded by regulating factors and genes that are essential in wound healing processes. As LPA is found in saliva and is released by activated cells after wounding, our results indicate that LPA has a favorable physiological role in oral wound healing. This may further point towards a beneficial role for application of LPA on oral surgical or chronic wounds.     

Palatal mucoperiosteal wound healing in the rat

The objective of this study was to analyze the changes in tissue architecture and matrix composition during healing of palatal wounds of immature rats, and to compare this with rats of the same age that did not receive mucoperiosteal wounds. Wounds were made in the mucoperiosteum of the palate of 35-d-old rats. Samples were evaluated histologically at numerous points in time after wounding. The DNA, hydroxyproline and sulphated glycosaminoglycan contents were determined at 8, 15, 30, and 60 d post-wounding. Eight-d-old granulation tissue contained 43% less hydroxyproline, and 100% more glycosaminoglycans and cells than unwounded palatal tissue of 43-d-old rats. Sixty-d-old wounds contained 100% more DNA and 39% more hydroxyproline than unwounded tissue of 95-d-old rats. At the same time, densely packed and transversely aligned collagen fibres were present. It is concluded that palatal mucoperiosteal wounds made in 35-d-old rats heal with distinct scar tissue formation. The scar contains more collagen than non-wounded palatal tissue of rats of the same age. Therefore, this model may be of use for the development of therapies aiming to reduce palatal scarring.