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.     

Differential Expression of HIF-1α in Skin and Mucosal Wounds

Despite accelerated epithelial closure, oral mucosal wounds exhibit lower levels of VEGF and a more refined angiogenic response than do skin wounds. The specific differences in angiogenesis suggest that skin and oral mucosal wounds may experience dissimilar levels of hypoxia and HIF-1α. Using a model of comparable wounds on murine dorsal skin and tongue, we determined levels of hypoxia and HIF-1α. Skin wounds were found to be significantly more hypoxic and had higher levels of HIF-1α than mucosal wounds. Furthermore, under stressed conditions, skin wounds, but not mucosal wounds, exhibited a further elevation of HIF-1α beyond that of non-stressed levels. To determine if manipulation of oxygen levels might equalize the repair response of each tissue, we exposed mice to hyperbaric oxygen treatment (HBOT) following wounding. HBOT did not significantly change HIF-1α or VEGF expression in either skin or mucosal wounds, nor did it alter wound bed vascularity. These studies suggest that skin wounds have higher levels of hypoxia than do mucosal wounds, along with a differential expression of HIF-1α. Interestingly, modulation of oxygen by HBOT does not ameliorate this difference. These results suggest that differential responses to hypoxia may underlie the distinctive wound-healing phenotypes seen in skin and oral mucosa.     

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.     

'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.     

Extracellular matrix components of oral mucosa differ from skin and resemble that of foetal skin

Objective

Wounds of both the oral mucosa and early-to-mid gestation foetuses have a propensity to heal scarless. Repair of skin wounds in adults, however, regularly results in scar formation. The extracellular matrix (ECM) plays an important role in the process of healing. The fate of scarless or scar forming healing may already be defined by the ECM composition, prior to wounding. In this study, the presence of several ECM components in oral mucosa (palatum) and skin was investigated.

Design

Immunohistochemical stainings of different ECM components were performed on skin, obtained from abdominal dermolipectomy surgery, and oral mucosa, derived after pharynx reconstruction.

Results

Expression of fibronectin, its splice variant ED-A, and chondroitin sulphate was elevated in oral tissue, whereas elastin expression was higher in skin. Tenascin-C, hyaluronic acid, biglycan, decorin, and syndecan-1 were expressed at similar levels in both tissues. Oral mucosa contained more blood vessels than skin samples. Finally, oral keratinocytes proliferated more, while dermal keratinocytes demonstrated higher differentiation.

Conclusions

Comparing ECM components of the skin and oral mucosa coincides with differences earlier observed between foetal and adult skin, and this might indicate that some ECM components are involved in the mode of repair.     

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.     

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.     

Preferential recruitment of bone marrow-derived cells to rat palatal wounds but not to skin wounds

ObjectiveTo investigate the contribution of bone marrow-derived cells to oral mucosa wounds and skin wounds.BackgroundBone marrow-derived cells are known to contribute to wound healing, and are able to differentiate in many different tissue-specific cell types. As wound healing in oral mucosa generally proceeds faster and with less scarring than in skin, we compared the bone marrow contribution in these two tissues.DesignBone marrow cells from GFP-transgenic rats were transplanted to irradiated wild-type rats. After recovery, 4-mm wounds were made in the mucoperiosteum or the skin. Two weeks later, wound tissue with adjacent normal tissue was stained for GFP-positive cells, myofibroblasts (a-smooth muscle actin), activated fibroblasts (HSP47), and myeloid cells (CD68).ResultsThe fraction of GFP-positive cells in unwounded skin (19%) was larger than in unwounded mucoperiosteum (0.7%). Upon wounding, the fraction of GFP-positive cells in mucoperiosteum increased (8.1%), whilst it was unchanged in skin. About 7% of the myofibroblasts in both wounds were GFP-positive, 10% of the activated fibroblasts, and 25% of the myeloid cells.ConclusionsThe results indicate that bone marrow-derived cells are preferentially recruited to wounded oral mucosa but not to wounded skin. This might be related to the larger healing potential of oral mucosa.     

脱细胞异体真皮基质修复口腔黏膜缺损的临床研究

目的评价脱细胞异体真皮基质口腔黏膜补片修复口腔黏膜缺损的临床效果。方法采用脱细胞异体真皮修复42例浅表的口腔黏膜缺损,缺损部位为前庭沟、舌、口底、颊部、腭部、牙龈及唇部,缺损面积2.25～12.00cm2。将脱细胞真皮基质基底膜面向外,分别用缝线固定、反包扎、基托固定的方式,覆盖于黏膜缺损区。术后随访3个月～1年。结果完全成活40例,大部成活2例,无失败病例。脱细胞异体真皮存在两种成活形式,其中上皮化愈着成活12例,附着成活30例。术后收缩发生在2～4周,4周内收缩率为3.7%±1.1%。3个月后基本稳定。无瘢痕挛缩。结论脱细胞异体真皮可以作为自体皮片的替代品,取得满意的口腔黏膜修复效果。     

Subpopulations of fetal-like gingival fibroblasts: characterisation and potential significance for wound healing and the progression of periodontal disease

Wound healing in the adult is commonly compromised by excessive scar formation. In contrast, fetal wound healing is a regenerative process characterised by the conspicuous absence of scarring. Available evidence suggests that phenotypic differences between fetal and adult fibroblasts are important determinants of these distinct modes of tissue repair. In this context, a number of groups (including our own) have documented differences between fetal and adult fibroblasts with respect to such potentially relevant characteristics as migratory activity, motogenic response to cytokines and the synthesis of motility factors, cytokines and matrix macromolecules. The oral mucosa appears to be a privileged site in the adult in that it continues to display a fetal-like mode of wound healing. Data are presented in this review indicating that a subpopulation of gingival fibroblasts expresses several 'fetal-like' phenotypic characteristics. These observations are discussed in terms of both the continued expression of a fetal-like mode of wound healing in the oral mucosa and the possible differential involvement of distinct fibroblast subpopulations in the progression of periodontal disease.     

口腔癌成纤维细胞对口腔癌细胞MMP-2、9表达的影响

目的:研究口腔癌成纤维细胞对口腔癌细胞MMP-2、9表达的影响.方法:采用明胶酶谱法和免疫细胞化学法分析MMP-2、MMP-9在口腔癌细胞与口腔癌成纤维细胞(CAFs)中共培养前后表达的变化.结果:①单独培养的Tca-8113细胞中未检测到MMP-2、9表达,单独培养的CAFs中只检测到微弱活性的MMP-2,未检测到活性形式的MMP-9.直接共培养后,上清液中MMP-9的活性随CAFs细胞数量的增加而增加,各条带的平均积分灰度值分别为82.15±0.45、96.40±0.32、103.87±0.47,各组间比较有显著性差异(P<0.05).而MMP-2活性略有升高.间接共培养后,MMP-2的活性随CAFs细胞数量的增加而增加,各条带的平均积分灰度值分别为84.15±0.25、94.40±0.38、105.87±0.57,各组间比较有显著性差异(P<0.05).未检测到活性形式的MMP-9.②免疫组化结果表明直接共培养(1∶3比例)后,Tca-8113表达MMP-9较对照组增强,尤其在被CAFs环绕的Tca-8113表达更强.间接共培养(1∶3比例)后,Tca-8113表达MMP-2也较对照组增强.结论:MMP-2表达增加可能受到源自口腔癌相关成纤维细胞可溶性因子的旁分泌介导,而MMP-9表达可能需要口腔癌细胞与癌相关成纤维细胞的直接接触.     

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.     

Matrix mechanics and regulation of the fibroblast phenotype

The oral cavity hosts a variety of different fibroblast populations that are generally responsible for maintaining homeostasis of the soft connective tissue. In addition to regulating the turnover and structural arrangement of collagen and other proteins of the extracellular matrix, fibroblasts perform a number of specialized functions. Certain fibroblast subpopulations in the gingiva, oral mucosa and periodontal ligament serve as progenitor cells with multilineage differentiation and self‐renewal characteristics. In the periodontal ligament, fibroblasts further appear to function as mechanosensing entities that regulate collagen‐secretory and collagen‐remodeling activities according to the level of strain in the ligament. Mechanical challenge also plays an important role during the activation of periodontal fibroblasts in response to injury. Dysregulation of this activation process can lead either to poor healing and chronic wounds or to overly healed wounds with fibrosis. This review will elaborate on the roles of mechanical factors and mechanoperception in fibroblast activation, the molecular features of activated fibroblasts and the regulation mechanisms of fibroblast contraction. Pharmacological interference at each level is currently being pursued to improve the outcome of healing of injured periodontal tissue.     

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.     

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.     

Healing pattern of experimental soft tissue lacerations after application of novel topical anesthetic agents - an experimental study in rabbits.

Topical anesthetics based on a combination of 2.5% lidocaine and 2.5% prilocaine are efficient in eliminating pain from needle stick when placed on skin and oral mucosa. This suggests their application in soft tissue lacerations before suturing to enable pain-free exploration and suturing of traumatic lacerations without prior injection needle stick. The aim of the present study was to study the healing of experimental oral lacerations after topical anesthetic substances were placed in the lacerations. Thirty-six standardized incisions were made bilaterally in the lower and the upper labial mucosa of nine white New Zealand rabbits. All wounds were intentionally contaminated with saliva to simulate laceration wounds in trauma situation. EMLA cream and Oraqix thermosetting gel were applied into 30 lacerations and six lacerations were left untreated as control. In some lacerations the topical anesthetic agent was left in the wound, while in others they were rinsed off by saline before suturing the laceration wound. The rabbits were then killed after 3 days, 2 weeks and 4 weeks of healing and the lips were processed for histological evaluation. Similar normal histological healing patterns were seen in wounds in which EMLA and Oraqix were applied compared with control lacerations at all stages of healing. No adverse tissue or foreign body reactions were seen in any of the lacerations. We conclude that EMLA and Oraqix can be used in oral mucosal lacerations prior to suturing without the risk of adverse tissue reaction.     

应用组织工程口腔黏膜固有层修复颊黏膜缺损的实验研究

目的：评价组织工程口腔黏膜固有层修复颊黏膜缺损的效果。方法：以壳多糖-胶原凝胶为网架,与体外培养的Wistar大鼠口腔黏膜成纤维细胞构建壳多糖-胶原凝胶口腔黏膜固有层（FPCCL）,用5-BrdU标记其中的成纤维细胞后．移植修复同种异体大鼠口腔颊黏膜圆形缺损（用直径8mm的圆形刀制成）;21只Wistar大鼠随机分为FPCCL组（12只）及对照组（9只）。术后行大体观察、创面直径测量、组织学及免疫组织化学观察;所有数据用SPSSll．5统计软件包进行t检验。结果：术后大鼠创面无感染,术后7d时,2组创面均被浅黄色膜覆盖,术后14、21d时,2组创面完全被新生黏膜覆盖。术后7d时,对照组创面比FPCCL组创面显著为小（P〈0．05）。组织学观察：术后7d,2组创面未完全上皮化。术后14、21d时,2组创面完全上皮化,有钉突;上皮复层,表面有角化物;新生固有层中细胞数量较多。含毛细血管。免疫组织化学染色,FPCCL组术后7、14、21d时阳性成纤维细胞出现在肉芽组织的细胞密集处和新生固有层中．与新生组织共同参与了颊黏膜缺损的修复重建。未出现免疫排斥现象。结论：FPCCL在修复重建颊黏膜缺损、限制创口收缩方面明显优于对照组。FPCCL作为颊黏膜缺损区永久性固有层替代物是可行和有效的。     

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.     

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.     

Matrix metalloproteinase inhibitors reduce collagen gel contraction and alpha-smooth muscle actin expression by periodontal ligament cells

Background and Objective:  Orthodontic tooth movement requires remodeling of the periodontal tissues. The matrix metalloproteinases (MMPs) degrade the extracellular matrix components of the periodontal ligament, while the tissue inhibitors of metalloproteinases (TIMPs) control their activity. Synthetic MMP inhibitors have been developed to inhibit MMP activity. In this study, periodontal ligament cells in contracting collagen gels served as a model for enhanced periodontal remodeling. The effect of MMP inhibitors on gel contraction and on MMP and TIMP expression was analyzed.
Material and Methods:  Human periodontal ligament cells were cultured in three-dimensional collagen gels and incubated with the MMP inhibitors BB94, CMT-3, doxycycline and Ilomastat. Gel contraction was determined using consecutive photographs. The relative amounts of MMPs and TIMPs were analyzed using substrate zymography and mRNA expression using quantitative polyermase chain reaction.
Results:  All MMP inhibitors reduced MMP activity to about 20% of the control activity. They all reduced contraction, but CMT-3 and doxycycline had the strongest effect. These inhibitors also reduced MMP-2, MMP-3 and α-smooth muscle actin mRNA expression. The expression of MMP-1 mRNA seemed to be increased by CMT-3. No effects were found on the amounts of MMPs and TIMPs.
Conclusion:  Synthetic MMP inhibitors strongly reduced gel contraction by periodontal ligament cells. This was primarily caused by an inhibitory effect on MMP activity, which reduces matrix remodeling. In addition, α-smooth muscle actin expression was reduced by CMT-3 and doxycycline, which limits the contractile activity of the fibroblasts.