Cementoblastic lineage formation in the cross-talk between stem cells of human exfoliated deciduous teeth and epithelial rests of Malassez cells.

OBJECTIVES: The purpose of this study was to evaluate the synergistic effect of epithelial rests of Malassez cells (ERM) and transforming growth factor-ß1 (TGF-ß1) on proliferation, cementogenic and osteogenic differentiation of stem cells derived from human exfoliated deciduous teeth (SHED). MATERIALS AND METHODS: SHED were co-cultured with ERM with/without TGF-ß1. Then, SHED proliferation, morphological appearance, alkaline phosphatase (ALP) activity, mineralization behaviour and gene/protein expression of cemento/osteoblastic phenotype were evaluated. RESULTS: TGF-ß1 enhanced SHED proliferation when either cultured alone or co-cultured with ERM. ERM induced the cementoblastic differentiation of SHED which was significantly accelerated when treated with TGF-ß1. This activity was demonstrated by high ALP activity, strong mineral deposition and upregulation of cementum/bone-related gene and protein expressions (i.e. ALP, collagen type I, bone sialoprotein, osteocalcin and cementum attachment protein). CONCLUSIONS: ERM were able to induce SHED differentiation along the cemento/osteoblastic lineage that was triggered in the presence of TGF-ß1. CLINICAL RELEVANCE: The cemento/osteoblastic differentiation capability of SHED possesses a therapeutic potential in endodontic and periodontal tissue engineering.

Isolation and enhancement of a homogenous in vitro human Hertwig's epithelial root sheath cell population.

Hertwig's epithelial root sheath (HERS) cells play a pivotal role during root formation of the tooth and are able to form cementum-like tissue. The aim of the present study was to establish a HERS cell line for molecular and biochemical studies using a selective digestion method. Selective digestion was performed by the application of trypsin-EDTA for 2 min, which led to the detachment of fibroblast-like-cells, with the rounded cells attached to the culture plate. The HERS cells displayed a typical cuboidal/squamous-shaped appearance. Characterization of the HERS cells using immunofluorescence staining and flow cytometry analysis showed that these cells expressed pan-cytokeratin, E-cadherin, and p63 as epithelial markers. Moreover, RT-PCR confirmed that these cells expressed epithelial-related genes, such as cytokeratin 14, E-cadherin, and ΔNp63. Additionally, HERS cells showed low expression of CD44 and CD105 with absence of CD34 and amelogenin expressions. In conclusion, HERS cells have been successfully isolated using a selective digestion method, thus enabling future studies on the roles of these cells in the formation of cementum-like tissue in vitro.

In vitro models in biocompatibility assessment for biomedical-grade chitosan derivatives in wound management.

One of the ultimate goals of wound healing research is to find effective healing techniques that utilize the regeneration of similar tissues. This involves the modification of various wound dressing biomaterials for proper wound management. The biopolymer chitosan (beta-1,4-D-glucosamine) has natural biocompatibility and biodegradability that render it suitable for wound management. By definition, a biocompatible biomaterial does not have toxic or injurious effects on biological systems. Chemical and physical modifications of chitosan influence its biocompatibility and biodegradability to an uncertain degree. Hence, the modified biomedical-grade of chitosan derivatives should be pre-examined in vitro in order to produce high-quality, biocompatible dressings. In vitro toxicity examinations are more favorable than those performed in vivo, as the results are more reproducible and predictive. In this paper, basic in vitro tools were used to evaluate cellular and molecular responses with regard to the biocompatibility of biomedical-grade chitosan. Three paramount experimental parameters of biocompatibility in vitro namely cytocompatibility, genotoxicity and skin pro-inflammatory cytokine expression, were generally reviewed for biomedical-grade chitosan as wound dressing.

A 20-year experience of immediate mandibular reconstruction using free fibula osteocutaneous flaps following ameloblastoma resection: Radical resection, outcomes, and recurrence.

BACKGROUND: The mandible is an important structure that is located in the lower third of the face. Large mandibular defects after tumor resection cause loss of its function. This study assessed the outcomes and tumor recurrence after immediate mandibular reconstruction using a free fibula osteocutaneous flap following radical resection of ameloblastoma. METHODS: This is a retrospective non-randomized study of outcomes and tumor recurrence of all patients diagnosed with mandibular ameloblastoma from August 1997 until August 2017 (20 years) requiring free fibula osteocutaneous flap reconstruction at a single institution. The patients were identified through an electronic operative database; subsequently, their medical records and photo documentation were retrieved. RESULTS: Twenty-seven patients were included in this study. Eighteen patients were male, while nine were female. The majority of the patients (48.1%) were in their third decade of life when they were diagnosed with ameloblastoma. All of them underwent radical resection of the tumor with a surgical margin of 2 cm (hemimandibulectomy in cases with a large tumor) and immediate mandibular reconstruction with a free fibula osteocutaneous flap. Two patients required revision of a vascular anastomosis due to venous thrombosis postoperatively, while one patient developed a flap recipient site infection. The flap success rate was 100%. There was no tumor recurrence during a mean follow-up period of 5.6 years. CONCLUSIONS: Mandibular ameloblastoma should be treated with segmental mandibulectomy (with a surgical margin of 2 cm) to reduce the risk of recurrence. Subsequent mandibular and adjacent soft tissue defects should be reconstructed immediately with a free fibula osteocutaneous flap.

Combination of vascularized outer-table calvarial bone graft based on the superficial temporal vessels and allomatrix for the repair of an orbito-frontal blow-out fracture in a child.

CASE PRESENTATION: A vascularized outer-table calvarial bone graft was used for repairing a Posnick type 2 traumatic orbito-frontal bone defect supported by the use of a calcium-based putty (Allomatrix) in a 7-year-old girl. Gaps between the donor and recipient sites were filled with Allomatrix containing demineralized bone matrix particles. Four years later there was a good cosmetic result using an artificial left eye. DISCUSSION: Orbito-frontal defects have been repaired using iso-, allo- or xenografts as well as synthetic materials. Anatomical studies have pointed to the importance of the superficial temporal vessels for the vascular supply of a graft to the calvaria. CONCLUSION: The vascularized outer-table calvarial bone graft based on the superficial temporal vessels is a reliable option for repairing bony defects in the craniofacial skeleton especially in irradiated or scarred areas where there is a paucity of well-vascularized tissues. It also has the potential for bony growth in children.

Physical properties and biocompatibility of oligochitosan membrane film as wound dressing.

BACKGROUND: The physical and biological characteristics of oligochitosan (O-C) film, including its barrier and mechanical properties, in vitro cytotoxicity and in vivo biocompatibility, were studied to assess its potential use as a wound dressing. METHODS: Membrane films were prepared from water-soluble O-C solution blended with various concentrations of glycerol to modify the physical properties of the films. In vitro and in vivo biocompatibility evaluations were performed using primary human skin fibroblast cultures and subcutaneous implantation in a rat model, respectively. RESULTS: Addition of glycerol significantly influenced the barrier and mechanical properties of the films. Water absorption capacity was in the range of 80%-160%, whereas water vapor transmission rate varied from 1,180 to 1,618 g/m2 per day. Both properties increased with increasing glycerol concentration. Tensile strength decreased while elongation at break increased with the addition of glycerol. O-C films were found to be noncytotoxic to human fibroblast cultures and histological examination proved that films are biocompatible. CONCLUSION: These results indicate that the membrane film from O-C has potential application as a wound-dressing material.

Synergistic effects of chitosan scaffold and TGFß1 on the proliferation and osteogenic differentiation of dental pulp stem cells derived from human exfoliated deciduous teeth.

OBJECTIVE: Multipotent stem cells derived from human exfoliated deciduous teeth (SHED) represent a promising cell source for tissue regeneration. In the present study we decided to test the inductive effect of chitosan and transforming growth factor-ß1 (TGFß1) as a scaffold/factor combination on SHED proliferation and osteogenic differentiation. DESIGN: Cell proliferation was quantitatively assessed by PrestoBlue, live/dead assay was performed and cell attachment to chitosan scaffold was examined by scanning electron microscopy (SEM). For osteogenic differentiation analysis, alkaline phosphatase activity was quantified, cells were stained with Alizarin Red, and the lineage specific genes/proteins ALP, COL I, BSP, and OCN were analysed by real-time PCR and Western blot. RESULTS: SHED remained viable and attached well to the chitosan structure. Moreover, TGFß1 significantly enhanced the proliferative activity of SHED on the chitosan scaffold. Our data further revealed that chitosan and TGFß1 enhanced the osteogenic differentiation of SHED, as evidenced by high ALP activity, strong mineral deposition, and the up-regulation of ALP, COL I, BSP, and OCN gene/protein expression. CONCLUSION: Together, data from our study indicate that the combination of chitosan scaffolds and TGFß1 enhanced proliferation and osteogenic differentiation of SHED. These findings suggest that the combined application of chitosan scaffold and TGFß1 in conjunction with SHED might be beneficial for in vivo bone regeneration.

Keloid pathogenesis via Drosophila similar to mothers against decapentaplegic (SMAD) signaling in a primary epithelial-mesenchymal in vitro model treated with biomedical-grade chitosan porous skin regenerating template.

The effects of locally produced chitosan (CPSRT-NC-bicarbonate) in the intervention of keloid pathogenesis were investigated in vitro. A human keratinocyte-fibroblast co-culture model was established to investigate the protein levels of human collagen type-I, III and V in a western blotting analysis, the secreted transforming growth factor-ß1 (TGF-ß1) in an enzyme-linked immunosorbent assay (ELISA) and the mRNA levels of TGF-ß1's intracellular signaling molecules (SMAD2, 3, 4 and 7) in a real-time PCR analysis. Keratinocyte-fibroblast co-cultures were maintained in DKSFM:DMEM:F12 (2:2:1) medium. Collagen type-I was found to be the dominant form in primary normal human dermal fibroblast (pNHDF) co-cultures, whereas collagen type-III was more abundant in primary keloid-derived human dermal fibroblast (pKHDF) co-cultures. Collagen type-V was present as a minor component in the skin. TGF-ß1, SMAD2 and SMAD4 were expressed more in the pKHDF than the pNHDF co-cultures. Co-cultures with normal keratinocytes suppressed collagen type-III, SMAD2, SMAD4 and TGF-ß1 expressions and CPSRT-NC-bicarbonate enhanced this effect. In conclusion, the CPSRT-NC-bicarbonate in association with normal-derived keratinocytes demonstrated an ability to reduce TGF-ß1, SMAD2 and SMAD4 expressions in keloid-derived fibroblast cultures, which may be useful in keloid intervention.

Chitosan dermal substitute and chitosan skin substitute contribute to accelerated full-thickness wound healing in irradiated rats.

Wounds with full-thickness skin loss are commonly managed by skin grafting. In the absence of a graft, reepithelialization is imperfect and leads to increased scar formation. Biomaterials can alter wound healing so that it produces more regenerative tissue and fewer scars. This current study use the new chitosan based biomaterial in full-thickness wound with impaired healing on rat model. Wounds were evaluated after being treated with a chitosan dermal substitute, a chitosan skin substitute, or duoderm CGF. Wounds treated with the chitosan skin substitute showed the most re-epithelialization (33.2 ± 2.8%), longest epithelial tongue (1.62 ± 0.13 mm), and shortest migratory tongue distance (7.11 ± 0.25 mm). The scar size of wounds treated with the chitosan dermal substitute (0.13 ± 0.02 cm) and chitosan skin substitute (0.16 ± 0.05 cm) were significantly decreased (P < 0.05) compared with duoderm (0.45 ± 0.11 cm). Human leukocyte antigen (HLA) expression on days 7, 14, and 21 revealed the presence of human hair follicle stem cells and fibroblasts that were incorporated into and surviving in the irradiated wound. We have proven that a chitosan dermal substitute and chitosan skin substitute are suitable for wound healing in full-thickness wounds that are impaired due to radiation.

Dentoalveolar relationships of Malay children with unilateral cleft lip and palate.

OBJECTIVE: To determine the treatment outcome based on dentoalveolar relationships among Malay children born with nonsyndromic complete unilateral cleft lip and palate (UCLP). DESIGN: Retrospective cohort study. SETTING AND SAMPLE POPULATION: The Department of Orthodontics at the School of Dental Science and the Reconstructive Sciences Unit, School of Medical Science, Universiti Sains Malaysia. Dental study models of 82 UCLP Malay children aged 8 to 10 years were evaluated. All subjects had their cleft lip and palate repaired, but no alveolar bone graft or any orthodontic treatment was performed. OUTCOME MEASURE: The outcome of dental arch relationships was assessed using the Goslon Yardstick Index: a dental measure with outcomes ranked on a scale ranging from 1 to 5. Agreement of rating was assessed with weighted kappa statistics; both intraexaminer and interexaminer agreements were high, indicating good reproducibility. RESULTS: A total of 2.4% of the sample was in grade 1, 24.4% in grade 2, 35.4% in grade 3, 31.7% in grade 4, and 6.1% in grade 5. The mean Goslon index score was 3.15. CONCLUSION: Dentoalveolar relationship outcomes of UCLP Malay children are intermediate according to the Goslon Yardstick. Interpretation of results should consider the ethnic differences in the craniofacial complex.