Colin Wiebe

Wound healing in oral mucosa results in reduced scar formation as compared with skin : Evidence from the red Duroc pig model and humans

Scar formation is a common, unwanted result of wound healing in skin, but the mechanisms that regulate it are still largely unknown. Interestingly, wound healing in the oral mucosa proceeds faster than in skin and clinical observations have suggested that mucosal wounds rarely scar. To test this concept, we created identical experimental wounds in the oral mucosa and skin in red Duroc pigs and compared wound healing and scar development over time. We also compared the pig oral mucosal wound healing to similar experimental wounds created in human subjects. The findings showed significantly reduced scar formation at both clinical and histological level in the pig oral mucosa as compared with skin 49 days after wounding. Additionally, the skin scars contained a significantly increased number of type I procollagen immunopositive cells and an increased fibronectin content, while the oral mucosal wounds demonstrated a prolonged accumulation of tenascin‐C. Furthermore, the pig oral mucosal wounds showed similar molecular composition and clinical and histological scar scores to human oral mucosal wounds. Thus, the reduced scar formation in the pig oral mucosa provides a model to study the biological processes that regulate scarless wound healing to find novel approaches to prevent scar formation in skin.

Scarless healing of oral mucosa is characterized by faster resolution of inflammation and control of myofibroblast action compared to skin wounds in the red Duroc pig model

BACKGROUND Scar formation following skin trauma can have devastating consequences causing physiological and psychosocial concerns. Currently, there are no accepted predictable treatments to prevent scarring which emphasizes a need for a better understanding of the wound healing and scar formation process. OBJECTIVES Previously it was shown that healing of small experimental wounds in the oral mucosa of red Duroc pigs results in significantly reduced scar formation as compared with equivalent full-thickness skin wounds. In the present study, scar formation was assessed in 17 times larger experimental wounds in both oral mucosa and skin of the red Duroc pigs. METHODS Equivalent experimental wounds were created in the oral mucosa and dorsal skin of red Duroc pigs, and scar formation, localization and abundance of key wound healing cells, transforming growth factor-beta (TGF-beta) and phosphorylated Smad3 (pSmad3) were assessed. RESULTS Oral mucosal wounds displayed significantly less clinical and histological scar formation than did the corresponding skin wounds. The number of macrophages, mast cells, TGF-beta and pSmad3 immunopositive cells was significantly reduced in the oral mucosal wounds as compared with skin wounds during the maturation stage of the healing process. Although the number of myofibroblasts was significantly elevated, the oral mucosal wounds showed significantly less contraction than did the skin wounds over time. CONCLUSIONS Earlier resolution of the inflammatory reaction and reduced wound contraction may promote scarless oral mucosal wound healing. In addition, scar formation likely depends not only on the number of myofibroblasts but also on the extracellular environment which regulates their function.

Expression of Integrin αvβ6 and TGF-β in Scarless vs Scar-forming Wound Healing

Oral mucosal wounds heal with reduced scar formation compared with skin. The epithelial integrin αvβ6 is induced during wound healing, and it can activate fibrogenic transforming growth factor β1 (TGF-β1) and anti-fibrogenic TGF-β3 that play key roles in scar formation. In this study, expression of β6 integrin and members of the TGF-β pathway were studied in experimental wounds of human gingiva and both gingiva and skin of red Duroc pigs using real-time PCR, gene microarrays, and immunostaining. Similar to human wounds, the expression of β6 integrin was induced in the pig wounds 7 days after wounding and remained upregulated >49 days. The αvβ6 integrin was colocalized with both TGF-β isoforms in the wound epithelium. Significantly higher expression levels of β6 integrin and TGF-β1 were observed in the pig gingival wounds compared with skin. Early gingival wounds also expressed higher levels of TGF-β3 compared with skin. The spatio-temporal colocalization of αvβ6 integrin with TGF-β1 and TGF-β3 in the wound epithelium suggests that αvβ6 integrin may activate both isoforms during wound healing. Prolonged expression of αvβ6 integrin along with TGF-β3 in the gingival wound epithelium may be important in protection of gingiva from scar formation.

Abnormal deposition of type VII collagen in Kindler syndrome

Abstract Kindler syndrome is an extremely rare genetic disorder with features of epidermolysis bullosa and poikiloderma congenitale. Approximately 70 cases have been documented in the past 50 years, but only a few investigations of the basement membrane components have been done on these patients. The aim of this study was to examine the components of the basement membrane zone in search of the pathobiological defect(s) responsible for the clinical findings from a female 16-year-old patient diagnosed with Kindler syndrome. This patient also suffered from advanced early-onset periodontal disease. Biopsies were taken from inflamed gingiva and noninflamed oral mucosa as part of periodontal treatment. The basement membrane zone was examined using immunofluorescence microscopy to bullous pemphigoid antigens 1 and 2, collagen types IV and VII, laminins-1 and -5, and integrins ·3‚1 and ·6‚4. The biopsies studied revealed blistering with trauma above the level of lamina densa based on distribution of type IV collagen and laminin-1 at the blister floor. In the noninflamed mucosa, discontinuous areas of the basement membrane zone were found. Expression of the basement membrane zone components and the integrins studied appeared otherwise normal with the exception of type VII collagen which was found in abnormal locations deep in the connective tissue stroma. Our results suggest that Kindler syndrome is associated with abnormalities in the construction of the basement membrane, especially in the expression of type VII collagen. These alterations are likely to play a role as etiological factors leading to blister formation and early onset periodontal disease.

Initial torque stability of a new bone condensing dental implant. A cohort study of 140 consecutively placed implants.

The aim of this paper was to determine the torque resistance of this new implant during placement in different types of bone, immediate placement into sockets, and in grafted bone. The torque at time of placement serves as an indication of initial stability, which is accepted as an important factor for implant osseointegration and immediate loading. Within a 13-month period, 140 NobelActive implants in 84 consecutive patients were placed into types I-IV bone in fresh sockets, and into grafted bone (both in maxillary sinuses and on the facial alveolar surfaces where bone had been lost). The final torque was measured with a manual torque control wrench as manufactured by Nobel Biocare for clinical use with this type of implant. One hundred forty implants with 3.5 to 5 mm diameters and 10 to 15 mm lengths were placed in different types of bone, either as delayed or immediate implants into fresh extraction sockets. These implants demonstrated a mean torque stability value of 50.8 Ncm. The average insertion torque for delayed implants was 49.7 Ncm. For immediate implants the average torque was 52.6 Ncm. Placement into soft bone was also favorable at an average of 47.9 Ncm. Typical straight walled and tapered implants generally exhibit 10 to 35 Ncm insertion torques. The NobelActive implant consistently reaches higher torque levels. This may indicate they are more favorably suited to early provisionalization and loading. Soft bone (type IV) did not seem to decrease significantly the torque of insertion of these implants. Further longer term studies are needed to investigate whether this indeed makes these implants more suited for early provisionalization and loading than traditional root form. Long term studies are also needed to investigate maintenance of bone levels surrounding these implants.

Critical role for αvβ6 integrin in enamel biomineralization

Summary Tooth enamel has the highest degree of biomineralization of all vertebrate hard tissues. During the secretory stage of enamel formation, ameloblasts deposit an extracellular matrix that is in direct contact with the ameloblast plasma membrane. Although it is known that integrins mediate cell–matrix adhesion and regulate cell signaling in most cell types, the receptors that regulate ameloblast adhesion and matrix production are not well characterized. We hypothesized that &agr;v&bgr;6 integrin is expressed in ameloblasts where it regulates biomineralization of enamel. Human and mouse ameloblasts were found to express both &bgr;6 integrin mRNA and protein. The maxillary incisors of Itgb6−/− mice lacked yellow pigment and their mandibular incisors appeared chalky and rounded. Molars of Itgb6−/− mice showed signs of reduced mineralization and severe attrition. The mineral-to-protein ratio in the incisors was significantly reduced in Itgb6−/− enamel, mimicking hypomineralized amelogenesis imperfecta. Interestingly, amelogenin-rich extracellular matrix abnormally accumulated between the ameloblast layer of Itgb6−/− mouse incisors and the forming enamel surface, and also between ameloblasts. This accumulation was related to increased synthesis of amelogenin, rather than to reduced removal of the matrix proteins. This was confirmed in cultured ameloblast-like cells, in which &agr;v&bgr;6 integrin was not an endocytosis receptor for amelogenins, although it participated in cell adhesion on this matrix indirectly via endogenously produced matrix proteins. In summary, integrin &agr;v&bgr;6 is expressed by ameloblasts and it plays a crucial role in regulating amelogenin deposition and/or turnover and subsequent enamel biomineralization.

Localization and potential function of kindlin-1 in periodontal tissues

Kindlin-1 is an intracellular focal adhesion protein that regulates the actin cytoskeleton. Patients suffering from Kindler syndrome have a homologous mutation of the kindlin-1 gene and develop skin blisters, periodontal disease, and intestinal complications because of deficient adhesion of the basal epithelial cells. We investigated kindlin-1 localization in periodontal tissue and its functions in cultured keratinocytes and showed that kindlin-1 co-localizes with migfilin and paxillin in the basal epithelial cells of oral mucosa and in cultured keratinocytes. The kindlin-1-deficient oral mucosal tissue from a patient with Kindler syndrome showed a complete lack of paxillin and reduced migfilin immunostaining in the basal keratinocytes. Co-immunoprecipitation showed that migfilin directly interacted with kindlin-1. RNA interference-induced kindlin-1 deficiency in keratinocytes led to an altered distribution of migfilin-containing focal adhesions, reduced cell spreading, decreased cell proliferation, and decelerated cell migration. Disruption of microtubules in the kindlin-1-deficient cells further reduced cell spreading, suggesting that microtubules can partially compensate for kindlin-1 deficiency. Kindlin-1 supported mature cell-extracellular matrix adhesions of keratinocytes, as downregulation of kindlin-1 expression significantly reduced the cell-adhesion strength. In summary, kindlin-1 interacts with migfilin and plays a crucial role in actin-dependent keratinocyte cell adhesion essential for epidermal and periodontal health.

Elevated CD26 Expression by Skin Fibroblasts Distinguishes a Profibrotic Phenotype Involved in Scar Formation Compared to Gingival Fibroblasts

Compared to skin, wound healing in oral mucosa is faster and produces less scarring, but the mechanisms involved are incompletely understood. Studies in mice have linked high expression of CD26 to a profibrotic fibroblast phenotype, but this has not been tested in models more relevant for humans. We hypothesized that CD26 is highly expressed by human skin fibroblasts (SFBLs), and this associates with a profibrotic phenotype distinct from gingival fibroblasts (GFBLs). We compared CD26 expression in human gingiva and skin and in gingival and hypertrophic-like scar-forming skin wound healing in a pig model, and used three-dimensional cultures of human GFBLs and SFBLs. In both humans and pigs, nonwounded skin contained abundantly CD26-positive fibroblasts, whereas in gingiva they were rare. During skin wound healing, CD26-positive cells accumulated over time and persisted in forming hypertrophic-like scars, whereas few CD26-positive cells were present in the regenerated gingival wounds. Cultured human SFBLs displayed significantly higher levels of CD26 than GFBLs. This was associated with an increased expression of profibrotic genes and transforming growth factor-β signaling in SFBLs. The profibrotic phenotype of SFBLs partially depended on expression of CD26, but was independent of its catalytic activity. Thus, a CD26-positive fibroblast population that is abundant in human skin but not in gingiva may drive the profibrotic response leading to excessive scarring.

Connexin 43 regulates the expression of wound healing-related genes in human gingival and skin fibroblasts

ABSTRACT Fibroblasts are the most abundant connective tissue cells and play an important role in wound healing. It is possible that faster and scarless wound healing in oral mucosal gingiva relative to skin may relate to the distinct phenotype of the fibroblasts residing in these tissues. Connexin 43 (Cx43) is the most ubiquitous Cx in skin (SFBLs) and gingival fibroblasts (GFBLs), and assembles into hemichannels (HCs) and gap junctions (GJs) on the cell membrane. We hypothesized that SFBLs and GFBLs display distinct expression or function of Cx43, and that this may partly underlie the different wound healing outcomes in skin and gingiva. Here we show that Cx43 distinctly formed Cx43 GJs and HCs in human skin and gingiva in vivo. However, in SFBLs, in contrast to GFBLs, only a small proportion of total Cx43 assembled into HC plaques. Using an in vivo‐like 3D culture model, we further show that the GJ, HC, and channel‐independent functions of Cx43 distinctly regulated wound healing‐related gene expression in GFBLs and SFBLs. Therefore, the distinct wound healing outcomes in skin and gingiva may partly relate to the inherently different assembly and function of Cx43 in the resident fibroblasts. HIGHLIGHTSCx43 distinctly forms gap junctions and hemichannels (HCs) in human skin and gingiva.Compared to human gingival fibroblasts, skin fibroblasts contained few Cx43 HCs.Cx43 distinctly regulates gene expression in human gingival and skin fibroblasts.

Intra-operative application of chlorhexidine gel reduces bacterial counts in internal implant cavity.

A prospective clinical trial was conducted to assess the bacterial-inhibitory potential of 1% chlorhexidine (CHX) gel in the internal cavity of implant screw holes, when utilized at the time of implant placement. A total of 40 Straumann (S) and Nobel Biocare (N) implants were divided into test (ST or NT; implant + CHX gel) and control (SC or NC; implant only) groups. Total numbers of colony-forming units (CFUs ml(-1) ) were assessed at a minimum of 3 months postsurgery by aerobic and anaerobic culture. A set of specimens was stained with Gram stain. The mean sample-collection time was 110 d for the test population and 98 d for the controls. The use of 1% CHX gel significantly reduced bacterial counts in both the ST and NT samples by over three logs compared with controls. No statistical differences in the numbers of CFUs ml(-1) were evident between aerobic and anaerobic cultures. Differences in the numbers of CFUs ml(-1) between ST and NT groups were not statistically significant. Microscopic analysis showed mainly Gram-positive coccoid species in most samples.