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ABSTRACT: Background: Children with a cleft in the soft palate have difficulties with speech, swallowing, and sucking. Despite successful surgical repositioning of the muscles, optimal function is often not achieved. Scar formation and defective regeneration may hamper the functional recovery of the muscles after cleft palate repair. Therefore, the aim of this study is to investigate the anatomy and histology of the soft palate in rats, and to establish an in vivo model for muscle regeneration after surgical injury. Methods: Fourteen adult male Sprague Dawley rats were divided into four groups. Groups 1 (n = 4) and 2 (n = 2) were used to investigate the anatomy and histology of the soft palate, respectively. Group 3 (n = 6) was used for surgical wounding of the soft palate, and group 4 (n = 2) was used as unwounded control group. The wounds (1 mm) were evaluated by (immuno)histochemistry (AZAN staining, Pax7, MyoD, MyoG, MyHC, and ASMA) after 7 days. Results: The present study shows that the anatomy and histology of the soft palate muscles of the rat is largely comparable with that in humans. All wounds showed clinical evidence of healing after 7 days. AZAN staining demonstrated extensive collagen deposition in the wound area, and initial regeneration of muscle fibers and salivary glands. Proliferating and differentiating satellite cells were identified in the wound area by antibody staining. Conclusions: This model is the first, suitable for studying muscle regeneration in the rat soft palate, and allows the development of novel adjuvant strategies to promote muscle regeneration after cleft palate surgery.
PLoS ONE 03/2013; 8(3):e59193. · 4.09 Impact Factor
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ABSTRACT: Children with a cleft in the soft palate have difficulties with speech, swallowing, and sucking. Despite successful surgical repositioning of the muscles, optimal function is often not achieved. Scar formation and defective regeneration may hamper the functional recovery of the muscles after cleft palate repair. Therefore, the aim of this study is to investigate the anatomy and histology of the soft palate in rats, and to establish an in vivo model for muscle regeneration after surgical injury.
Fourteen adult male Sprague Dawley rats were divided into four groups. Groups 1 (n = 4) and 2 (n = 2) were used to investigate the anatomy and histology of the soft palate, respectively. Group 3 (n = 6) was used for surgical wounding of the soft palate, and group 4 (n = 2) was used as unwounded control group. The wounds (1 mm) were evaluated by (immuno)histochemistry (AZAN staining, Pax7, MyoD, MyoG, MyHC, and ASMA) after 7 days.
The present study shows that the anatomy and histology of the soft palate muscles of the rat is largely comparable with that in humans. All wounds showed clinical evidence of healing after 7 days. AZAN staining demonstrated extensive collagen deposition in the wound area, and initial regeneration of muscle fibers and salivary glands. Proliferating and differentiating satellite cells were identified in the wound area by antibody staining.
This model is the first, suitable for studying muscle regeneration in the rat soft palate, and allows the development of novel adjuvant strategies to promote muscle regeneration after cleft palate surgery.
PLoS ONE 01/2013; 8(3):e59193. · 4.09 Impact Factor
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ABSTRACT: The biomechanical character of the periodontal ligament (PDL) is crucial in its response to functional and orthodontic forces. Collagen has been the primary subject of investigations in this field. Several studies, however, indicate that oxytalan fibres, which belong to the elastic fibre family, also contribute to the biomechanical character and behaviour of the PDL. In order to elucidate this, we have evaluated the available literature on the oxytalan fibre network within the PDL and supra-alveolar tissues with respect to development, morphology and distribution, and response to mechanical stimulation. To this end, we have combined the classical histological studies with more recent in vitro studies. Oxytalan fibres develop simultaneously with the root and the vascular system within the PDL. A close association between oxytalan fibres and the vascular system also remains later in life, suggesting a role in vascular support. Mechanical loading of the PDL, through orthodontic force application, appears to induce an increase in the number, size, and length of oxytalan fibres. In line with this, in vitro stretching of PDL fibroblasts (PDLFs) results in an increased production of fibrillin, a major structural component of the microfibrils that make up oxytalan fibres. The available data suggest a mechanical function for oxytalan, but to date experimental data are limited. Further research is required to clarify their exact mechanical function and possible role in orthodontic tooth movement.
Archives of oral biology 07/2012; 57(8):1003-11. · 1.65 Impact Factor
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ABSTRACT: The restoration of muscles in the soft palate of patients with cleft lip and/or palate is accompanied by fibrosis, which leads to speech and feeding problems. Treatment strategies that improve muscle regeneration have only been tested in limb muscles. Therefore, in the present study the myogenic potential of muscle progenitor cells (MPCs) isolated from head muscles was compared with that of limb muscles. Muscle progenitor cells were isolated from the head muscles and limb muscles of rats and cultured. The proliferation of MPCs was analysed by DNA quantification. The differentiation capacity was analysed by quantifying the numbers of fused cells, and by measuring the mRNA levels of differentiation markers. Muscle progenitor cells were stained to quantify the expression of paired box protein Pax 7 (Pax-7), myoblast determination protein 1 (MyoD), and myogenin. Proliferation was similar in the head MPCs and the limb MPCs. Differentiating head and limb MPCs showed a comparable number of fused cells and mRNA expression levels of myosin-1 (Myh1), myosin-3 (Myh3), and myosin-4 (Myh4). During proliferation and differentiation, the number of Pax-7(+), MyoD(+), and myogenin(+) cells in head and limb MPCs was equal. It was concluded that head and limb MPCs show similar myogenic capacities in vitro. Therefore, in vivo myogenic differences between those muscles might rely on the local microenvironment. Thus, regenerative strategies for limb muscles might also be used for head muscles.
European Journal Of Oral Sciences 02/2012; 120(1):38-45. · 1.88 Impact Factor
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ABSTRACT: Nutritional factors such as vitamin intake contribute to the etiology of cleft palate. Vitamin A is a regulator of embryonic development. Excess vitamin A can cause congenital malformations such as spina bifida and cleft palate. Therefore, preventive nutritional strategies are required. This review identifies putative biological mechanisms underlying the association between maternal vitamin A intake and cleft palate. Excessive vitamin A may disturb all three stages of palatogenesis: 1) during shelf outgrowth, it may decrease cell proliferation and thus prevent tissue development; 2) it may prevent shelf elevation by affecting extracellular matrix composition and hydration; and 3) during shelf fusion, it may affect epithelial differentiation and apoptosis, which precludes the formation of a continuous palate. In general, high doses of vitamin A affect palatogenesis through interference with cell proliferation and growth factors such as transforming growth factor β and platelet-derived growth factor. The effects of lower doses of vitamin A need to be investigated in greater depth in order to improve public health recommendations.
Nutrition Reviews 10/2011; 69(10):613 - 624. · 4.47 Impact Factor
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ABSTRACT: Nutritional factors such as vitamin intake contribute to the etiology of cleft palate. Vitamin A is a regulator of embryonic development. Excess vitamin A can cause congenital malformations such as spina bifida and cleft palate. Therefore, preventive nutritional strategies are required. This review identifies putative biological mechanisms underlying the association between maternal vitamin A intake and cleft palate. Excessive vitamin A may disturb all three stages of palatogenesis: 1) during shelf outgrowth, it may decrease cell proliferation and thus prevent tissue development; 2) it may prevent shelf elevation by affecting extracellular matrix composition and hydration; and 3) during shelf fusion, it may affect epithelial differentiation and apoptosis, which precludes the formation of a continuous palate. In general, high doses of vitamin A affect palatogenesis through interference with cell proliferation and growth factors such as transforming growth factor β and platelet-derived growth factor. The effects of lower doses of vitamin A need to be investigated in greater depth in order to improve public health recommendations.
Nutrition Reviews 10/2011; 69(10):613-24. · 4.47 Impact Factor
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ABSTRACT: Wounded skin recruits progenitor cells, which repair the tissue defect. These cells are derived from stem cells in several niches in the skin. In addition, bone marrow-derived cells (BMDCs) are recruited and contribute to wound repair. We hypothesized that larger wounds recruit more cells from the bone marrow. Wild-type rats were lethally irradiated and transplanted with bone marrow cells from green fluorescent protein (GFP)-transgenic rats. Seven weeks later, 4, 10, and 20 mm wounds were created. The wound tissue was harvested after 14 days. The density of GFP-positive cells in the wounds and the adjacent tissues was determined, as well as in normal skin from the flank. Bone marrow-derived myofibroblasts, activated fibroblasts, and macrophages were also quantified. After correction for cell density, the recruitment of BMDCs (23±11%) was found to be independent of wound size. Similar fractions of GFP-positive cells were also detected in nonwounded adjacent tissue (29±11%), and in normal skin (26±19%). The data indicate that BMDCs are not preferentially recruited to skin wounds. Furthermore, wound size does not seem to affect the recruitment of BMDCs.
Wound Repair and Regeneration 03/2011; 19(2):260-7. · 2.91 Impact Factor
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ABSTRACT: Wound contraction and scar formation after cleft palate repair lead to growth impairment of the maxilla and midface. Myofibroblasts play a key role in these processes. The application of an interferon-γ (IFN-γ)-loaded collagen scaffold after surgery might reduce the differentiation of myofibroblasts. In this study, the tissue response to IFN-γ-loaded collagen scaffolds was evaluated after implantation in the palate of rats. Scaffolds, with or without IFN-γ, were implanted submucoperiosteally in the palate of two groups of 25 five-week-old male Wistar rats. Groups of five rats were sacrificed at 1, 2, 4, 8, and 16 weeks post-implantation and processed for histological analyses. On haematoxylin and eosin-stained sections, the cell density and number of giant cells within the scaffolds were determined. Blood vessels, inflammatory cells, and myofibroblasts were detected by immunohistochemistry. The data for cell density, blood vessels, and giant cells were compared with a two-way analysis of variance. The scores for myofibroblasts and inflammation were compared by a rank sum test. A mild and rapidly subsiding inflammatory and foreign body response was found in both groups. Angiogenesis had already begun after 1 week, showed a peak after 4 weeks, and declined thereafter. IFN-γ induced a faster influx of host cells and a major reduction in myofibroblast numbers. The scaffolds might be suitable for future applications in oral surgery.
The European Journal of Orthodontics 02/2011; 33(1):1-8. · 0.89 Impact Factor
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ABSTRACT: Wound healing is an intricate process requiring the concerted action of keratinocytes, fibroblasts, endothelial cells, and macrophages. Here, we review the literature on normal wound healing and the pathological forms of wound healing, such as hypertrophic or excessive scar formation, with special emphasis on the heme-heme oxygenase (HO) system and the versatile effector molecules that are formed after HO-mediated heme degradation. Excessive scar formation following wounding is thought to relate to prolonged oxidative and inflammatory stress in the skin. Evidence is accumulating that the heme-HO system forms a novel and important target in the control of wound healing. Heme-protein derived heme can act as a potent oxidative and inflammatory stress inducer, and excess levels of heme may thus contribute to delayed resolution of oxidative and inflammatory insults in the skin. This emphasizes the need for a timely reduction of the levels of heme. Heme-binding proteins, heme transporters, and the heme degrading protein, HO, form therefore a necessary defense. Deficiencies in these defense proteins or a disturbed redox status, as in diabetic patients, may render individuals more prone to heme-induced deleterious effects. A better understanding of the heme-heme oxygenase system as target during wound healing may result in novel strategies to reduce scar formation.
Current drug targets 12/2010; 11(12):1571-85. · 3.93 Impact Factor
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ABSTRACT: To develop a model for muscle fibrosis based on full-thickness muscle defects, and to evaluate the effects of implanted stromal-derived factor (SDF)-1α-loaded collagen scaffolds.
Full-thickness defects 2 mm in diameter were made in the musculus soleus of 48 rats and either left alone or filled with SDF-1α-loaded collagen scaffolds. At 3, 10, 28 and 56 days postsurgery, muscles were analyzed for collagen deposition, satellite cells, myofibroblasts and macrophages.
A significant amount of collagen-rich fibrotic tissue was formed, which persisted over time. Increased numbers of satellite cells were present around, but not within, the wounds. Satellite cells were further upregulated in regenerating tissue when SDF-1α-loaded collagen scaffolds were implanted. The scaffolds also attracted macrophages, but collagen deposition and myofibroblast numbers were not affected.
Persistent muscle fibrosis is induced by full-thickness defects 2 mm in diameter. SDF-1α-loaded collagen scaffolds accelerated muscle regeneration around the wounds, but did not reduce muscle fibrosis.
Regenerative Medicine 09/2010; 5(5):737-47. · 3.72 Impact Factor
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ABSTRACT: Our aim was to compare the dentoalveolar development in beagle dogs after palatal repair according to the Von Langenbeck technique with and without implantation of a dermal substitute.
Nineteen beagles (age, 12 weeks) were assigned to 2 experimental groups and an untreated control group. Palatal surgery was performed with the Von Langenbeck technique in the 2 experimental groups. The dermal substitute Integra (Plainsboro, NJ, USA) was implanted in 1 experimental group, and the other served as sham group. Dental casts were made before surgery and at several times in all groups to measure dentoalveolar development. Transversal distances, arch depth, tipping, and rotation were determined. Histologic evaluations were performed at 3, 7, and 15 weeks after surgery. The degrees of reepithelialization and tissue organization were evaluated microscopically.
All wounds healed without complications. Scar tissue attached to the bone was found in both experimental groups. Deposition of bone in the Integra occurred after implantation, indicating its osteoconductivity. Transversal dentoalveolar development was similar in both experimental groups, but it was significantly less than in the control group.
Implantation of Integra after the Von Langenbeck procedure for palatal repair does not improve dentoalveolar development.
American journal of orthodontics and dentofacial orthopedics: official publication of the American Association of Orthodontists, its constituent societies, and the American Board of Orthodontics 07/2010; 138(1):58-66. · 1.33 Impact Factor
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ABSTRACT: Skeletal muscle regeneration is a complex process, which is not yet completely understood. Satellite cells, the skeletal muscle stem cells, become activated after trauma, proliferate, and migrate to the site of injury. Depending on the severity of the myotrauma, activated satellite cells form new multinucleated myofibers or fuse to damaged myofibers. The specific microenvironment of the satellite cells, the niche, controls their behavior. The niche contains several components that maintain satellite cells quiescence until they are activated. In addition, a great diversity of stimulatory and inhibitory growth factors such as IGF-1 and TGF-beta1 regulate their activity. Donor-derived satellite cells are able to improve muscle regeneration, but their migration through the muscle tissue and across endothelial layers is limited. Less than 1% of their progeny, the myoblasts, survive the first days upon intra-muscular injection. However, a range of other multipotent muscle- and non-muscle-derived stem cells are involved in skeletal muscle regeneration. These stem cells can occupy the satellite cell niche and show great potential for the treatment of skeletal muscle injuries and diseases. The aim of this review is to discuss the niche factors, growth factors, and other stem cells, which are involved in skeletal muscle regeneration. Knowledge about the factors regulating satellite cell activity and skeletal muscle regeneration can be used to improve the treatment of muscle injuries and diseases.
Journal of Cellular Physiology 03/2010; 224(1):7-16. · 3.87 Impact Factor
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ABSTRACT: Wound contraction and scar formation after cleft palate repair impair the growth of the maxilla. The implantation of a growth factor-loaded scaffold might solve these problems.
The tissue response to fibroblast growth factor (FGF)-2 loaded collagen scaffolds was evaluated after implantation in the palate of rats. Scaffolds, with and without FGF-2, were implanted submucoperiosteally in the palate of 25 rats and evaluated after up to 16 weeks. On hematoxylin and eosin (H&E)-stained sections, the cell density and the number of giant cells within the scaffolds were quantified. Infiltration of inflammatory cells, myofibroblasts, and the number of blood vessels were quantified after immunohistochemistry.
The cell density was significantly higher in the FGF-2 group up to 4 weeks after implantation (102% at 2 weeks, P < 0.001). The number of blood vessels was also significantly higher in the FGF-2 group at 1 and 2 weeks (316% at 1 week, P = 0.003), but the myofibroblast score was lower (100% at 2 weeks, P = 0.008). A comparable mild and rapidly subsiding inflammatory response and foreign body reaction were found in both groups.
FGF-2-loaded scaffolds displayed a faster influx of host cells, an increased rate of vascularization, and a reduced differentiation of myofibroblasts. These scaffolds might therefore be highly suitable for intra-oral reconstructions, such as cleft palate repair.
Journal of Oral Pathology and Medicine 04/2009; 38(8):630-8. · 1.63 Impact Factor
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Journal of cranio-maxillo-facial surgery: official publication of the European Association for Cranio-Maxillo-Facial Surgery 01/2009; 37(3):140-4. · 1.25 Impact Factor
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ABSTRACT: Periodontal tissues remodel rapidly, which enables quick adaptation to mechanical changes. Matrix metalloproteinases (MMPs) are involved in these remodeling processes under control of tissue inhibitor of metalloproteinases (TIMPs). In periodontitis, overactivity of MMPs results in pathologic tissue degradation. The aim of this study was to analyze MMPs and TIMPs in healthy and diseased gingiva, periodontal ligament (PDL), and gingival crevicular fluid (GCF).
Samples of gingiva, PDL, and GCF were obtained from healthy controls (gingiva: n = 18; PDL: n = 15; GCF: n = 8) and subjects with periodontitis (gingiva: n = 11; PDL: n = 18; GCF: n = 12). MMPs and TIMPs were analyzed by gelatin-, collagen-, and reverse zymography and by Western blotting. Total MMP activity was analyzed using a fluorogenic substrate.
TIMP-1 and -2, active and pro-MMP-2 and -9, and active MMP-1 and -8 were present in all samples. Large amounts of active MMP-2 complexes and collagenolytic fragments were also found. Their levels were higher in PDL and GCF from subjects with periodontitis. In general, TIMP levels were lower in diseased periodontal tissues. Especially diseased GCF contained more MMPs. Surprisingly, some MMPs were more abundant in healthy gingiva and PDL than in diseased tissue.
Unexpected variations in MMP and TIMP levels in gingiva, PDL, and GCF may result from differences in subject characteristics and disease activity. The levels of active MMP-2 complexes and collagenolytic fragments are higher in the periodontium of subjects with periodontitis and might contribute significantly to periodontal destruction.
Journal of Periodontology 10/2008; 79(9):1704-11. · 2.60 Impact Factor
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ABSTRACT: Hereditary gingival fibromatosis (HGF) is a rare benign disorder characterized by progressive fibrous overgrowth of the gingiva. The proliferation and expression of growth factors of HGF keratinocytes are abnormal. However, the exact role of keratinocytes in HGF pathogenesis is still unknown. The present study aimed to clarify the interactions between HGF keratinocytes and underlying fibroblasts in the pathogenesis of HGF.
Gingival tissues, fibroblasts and keratinocytes from three Chinese HGF patients and three healthy subjects were collected. Histological analyses were performed by histochemical and immunohistochemical staining (Ki-67). Gingival fibroblasts were cocultured with gingival keratinocytes in an in vitro coculture system. The mRNA levels of type I collagen, MMP-1, MMP-3, and TIMP-1 were analysed in the cocultured gingival fibroblasts by reverse-transcription polymerase chain reaction (RT-PCR). The production of type I collagen and TIMP-1 was examined by ELISA.
The number of Ki-67-positive keratinocytes in tissue sections from patients was higher than in those from controls. HGF fibroblasts cocultured with HGF keratinocytes showed an increased expression of type I collagen and TIMP-1. Transmission electron microscopy showed increased rough endoplasmic reticulum and ribosomes in cocultured HGF fibroblasts.
These results suggest that HGF keratinocytes have an important role in HGF pathogenesis by inducing extracellular matrix (ECM) accumulation by fibroblasts.
Archives of oral biology 07/2008; 53(11):1050-7. · 1.65 Impact Factor
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ABSTRACT: Scar formation after repair of the cleft palate leads to growth impairment of the upper jaw and midface. The implantation of a suitable scaffold during surgery may reduce this adverse effect. However, little is known about tissue reactions to scaffolds implanted in the oral cavity. Our goal was to analyze the tissue reactions to cross-linked type I collagen scaffolds after submucoperiosteal implantation in the palate of rats. Collagen type I scaffolds were implanted in the palate of 25 male Wistar rats. Groups of 5 rats were killed consecutively after 1, 2, 4, 8, and 16 weeks and were processed for histologic and immunohistochemical analyses. After 1 and 2 weeks, 3 rats from the sham group were also killed. On hematoxylin and eosin-stained sections, the cell density and the number of giant cells were determined. Blood vessels, inflammation, and the presence of myofibroblasts were detected by immunohistochemistry. An influx of inflammatory cells started after 1 week but had completely subsided after 8 weeks. Myofibroblasts were observed within the scaffolds only in the first 2 weeks. Angiogenesis already started after 1 week and showed a peak after 4 weeks, slowly declining afterward. The scaffolds were gradually integrated within the host tissue and only elicited a mild and transient inflammatory response. The scaffolds were biocompatible and seemed to be promising for future applications in cleft palate surgery.
Journal of Craniofacial Surgery 05/2008; 19(3):599-608. · 0.82 Impact Factor
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ABSTRACT: To compare the tissue reactions to implanted collagen scaffolds in the palate and the skin of rats.
Crosslinked collagen scaffolds were implanted submucoperiosteally in the palate, and subcutaneously on the skull and on the back of 25 rats and evaluated after up to 16 weeks. On H&E-stained sections, the cell density and the number of giant cells within the scaffolds were determined. Blood vessels, inflammatory cells, and myofibroblasts were detected by immunohistochemistry.
A faster ingrowth of myofibroblasts and blood vessels in the palate was found during the first week compared with the skin. A more severe inflammatory response was initially found in the back skin. Furthermore, about twice as much giant cells were present in these scaffolds.
The oral environment seems to promote the ingrowth of myofibroblasts and blood vessels into the scaffolds. Mechanical stimuli seem to enhance the initial inflammatory response. Overall, the scaffolds were gradually integrated within the host tissue, eliciting only a transient inflammatory response. The scaffolds were biocompatible and are promising for future applications in oral surgery.
Archives of Oral Biology 04/2008; 53(4):376-87. · 1.60 Impact Factor
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ABSTRACT: Tissue shortage complicates the surgery of cleft palate (CP) anomalies. The healing of defects on the palate impairs growth of the dento-maxillary complex due to scar tissue formation. Implantation of grafts into the wound area might reduce this adverse effect of surgery. The aim of this study was to evaluate a cultured autologous mucosal substitute, which can be used as a graft material. Two different types of cultured mucosal substitutes composed of skin-derived substrates (unprocessed dermis and AlloDerm) and autologous oral keratinocytes were implanted in palatal wounds in six beagle dogs (1-1.5 years of age). The cultured substitutes were compared with a sham and a control group. The animals were sacrificed in pairs 1, 3, and 12 weeks after surgery. Epithelial regeneration, inflammatory response (leucocyte protein L1), ingrowth of (myo-) fibroblasts, collagen type III, and formation of a basal membrane (JM 403) were evaluated. The results demonstrated that all cultured substitutes possessed a multilayered epithelium, closely resembling normal palatal epithelium. After implantation, however, the epithelium was lost and an inflammatory response was observed in the first week. After 3 and 12 weeks, the implanted substitutes had completely disappeared and epithelial migration occurred from the wound margins. It is possible to culture an autologous epithelium on a skin-derived substrate and implant it as an oral mucosal substitute in palatal wounds. However, these substitutes do not improve the healing of palatal wounds. It is suggested that the revascularization of the wound area is too slow to allow survival and integration of the substitutes.
The European Journal of Orthodontics 03/2008; 30(1):1-9. · 0.89 Impact Factor
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ABSTRACT: Tissue shortage complicates surgery of cleft lip and palate. The healing of defects on the palate impairs growth of the dentoalveolar complex because of scar tissue formation. Implantation of a matrix into the wound might overcome this adverse effect. Integra with and without a silicone top layer was implanted into standardized full-thickness wounds (Ø 6 mm) in the palatal mucoperiosteum in beagle dogs. In some wounds, the silicone layer was removed after 14 days. Control wounds did not have an implant. At 2 and 4 weeks post-surgery, the wounds were assessed for epithelialization, inflammation (hematoxylin and eosin, leucocyte protein L1), number of myofibroblasts (alpha smooth muscle actin), and general histological characteristics. Wounds filled with Integra without the silicone layer showed fewer myofibroblasts and inflammatory cells than the sham wounds. Collagen fibers were more randomly orientated in these wounds than in the sham group. Wound closure was found to be retarded, and many inflammatory cells were present when Integra with silicone was implanted. The silicone layer was lost within 4 weeks in these wounds. We conclude that, in the moist oral environment, the silicone of Integra is not required. Re-epithelialization and tissue integration proceed more favorably without it. Further research in the dentoalveolar development with Integra will be conducted in a simulated cleft palate repair in the dog model.
Tissue Engineering 12/2007; 13(11):2689-98. · 4.02 Impact Factor
