Wen Lin Chai

Tissue-engineered Oral Mucosa

Advances in tissue engineering have permitted the three-dimensional (3D) reconstruction of human oral mucosa for various in vivo and in vitro applications. Tissue-engineered oral mucosa have been further optimized in recent years for clinical applications as a suitable graft material for intra-oral and extra-oral repair and treatment of soft-tissue defects. Novel 3D in vitro models of oral diseases such as cancer, Candida, and bacterial invasion have been developed as alternatives to animal models for investigation of disease phenomena, their progression, and treatment, including evaluation of drug delivery systems. The introduction of 3D oral mucosal reconstructs has had a significant impact on the approaches to biocompatibility evaluation of dental materials and oral healthcare products as well as the study of implant-soft tissue interfaces. This review article discusses the recent advances in tissue engineering and applications of tissue-engineered human oral mucosa.

Development of a Novel Model for the Investigation of Implant–Soft Tissue Interface

BACKGROUND In dental implant treatment, the long-term prognosis is dependent on the biologic seal formed by the soft tissue around the implant. The in vitro investigation of the implant-soft tissue interface is usually carried out using a monolayer cell-culture model that lacks a polarized-cell phenotype. This study developed a tissue-engineered three-dimensional oral mucosal model (3D OMM) to investigate the implant-soft tissue interface. METHODS A 3D OMM was constructed using primary human oral keratinocytes and fibroblasts cultured on a skin-derived scaffold at an air-liquid interface. A titanium implant was inserted into the engineered oral mucosa and further cultured to establish epithelial attachment. The 3D OMM was characterized using basic histology and immunostaining for cytokeratin (CK) 10 and CK13. Histomorphometric analyses of the implant-soft tissue interface were carried out using a light-microscopy (LM) examination of ground sections and semi-thin sections as well as scanning electron microscopy (SEM). RESULTS Immunohistochemistry analyses suggests that the engineered oral mucosa closely resembles the normal oral mucosa. The LM and SEM examinations reveal that the 3D OMM forms an epithelial attachment on the titanium surface. CONCLUSION The 3D OMM provided mimicking peri-implant features as seen in an in vivo model and has the potential to be used as a relevant alternative model to assess implant-soft tissue interactions.

The biological seal of the implant–soft tissue interface evaluated in a tissue-engineered oral mucosal model

For dental implants, it is vital that an initial soft tissue seal is achieved as this helps to stabilize and preserve the peri-implant tissues during the restorative stages following placement. The study of the implant–soft tissue interface is usually undertaken in animal models. We have developed an in vitro three-dimensional tissue-engineered oral mucosal model (3D OMM), which lends itself to the study of the implant–soft tissue interface as it has been shown that cells from the three-dimensional OMM attach onto titanium (Ti) surfaces forming a biological seal (BS). This study compares the quality of the BS achieved using the three-dimensional OMM for four types of Ti surfaces: polished, machined, sandblasted and anodized (TiUnite). The BS was evaluated quantitatively by permeability and cell attachment tests. Tritiated water (HTO) was used as the tracing agent for the permeability test. At the end of the permeability test, the Ti discs were removed from the three-dimensional OMM and an Alamar Blue assay was used for the measurement of residual cells attached to the Ti discs. The penetration of the HTO through the BS for the four types of Ti surfaces was not significantly different, and there was no significant difference in the viability of residual cells that attached to the Ti surfaces. The BS of the tissue-engineered oral mucosa around the four types of Ti surface topographies was not significantly different.

Modified silk fibroin scaffolds with collagen/decellularized pulp for bone tissue engineering in cleft palate: Morphological structures and biofunctionalities

Cleft palate is a congenital malformation that generates a maxillofacial bone defect around the mouth area. The creation of performance scaffolds for bone tissue engineering in cleft palate is an issue that was proposed in this research. Because of its good biocompatibility, high stability, and non-toxicity, silk fibroin was selected as the scaffold of choice in this research. Silk fibroin scaffolds were prepared by freeze-drying before immerging in a solution of collagen, decellularized pulp, and collagen/decellularized pulp. Then, the immersed scaffolds were freeze-dried. Structural organization in solution was observed by Atomic Force Microscope (AFM). The molecular organization of the solutions and crystal structure of the scaffolds were characterized by Fourier transform infrared (FT-IR) and X-ray diffraction (XRD), respectively. The weight increase of the modified scaffolds and the pore size were determined. The morphology was observed by a scanning electron microscope (SEM). Mechanical properties were tested. Biofunctionalities were considered by seeding osteoblasts in silk fibroin scaffolds before analysis of the cell proliferation, viability, total protein assay, and histological analysis. The results demonstrated that dendrite structure of the fibrils occurred in those solutions. Molecular organization of the components in solution arranged themselves into an irregular structure. The fibrils were deposited in the pores of the modified silk fibroin scaffolds. The modified scaffolds showed a beta-sheet structure. The morphological structure affected the mechanical properties of the silk fibroin scaffolds with and without modification. Following assessment of the biofunctionalities, the modified silk fibroin scaffolds could induce cell proliferation, viability, and total protein particularly in modified silk fibroin with collagen/decellularized pulp. Furthermore, the histological analysis indicated that the cells could adhere in modified silk fibroin scaffolds. Finally, it can be deduced that modified silk fibroin scaffolds with collagen/decellularized pulp had the performance for bone tissue engineering and a promise for cleft palate treatment.

A review of histomorphometric analysis techniques for assessing implant-soft tissue interface

Abstract The success of dental implant treatment depends on the healing of both hard and soft tissues. While osseointegration provides initial success, the biological seal of the peri-implant soft tissue is crucial for maintaining the long term success of implants. Most studies of the biological seal of peri-implant tissues are based on animal or monolayer cell culture models. To understand the mechanisms of soft tissue attachment and the factors affecting the integrity of the soft tissue around the implants, it is essential to obtain good quality histological sections for microscopic examination. The nature of the specimens, however, which consist of both metal implant and soft peri-implant tissues, poses difficulties in preparing the specimens for histomorphometric analysis of the implant-soft tissue interface. We review various methods that have been used for the implant-tissue interface investigation with particular focus on the soft tissue. The different methods are classified and the advantages and limitations of the different techniques are highlighted.

Clinicopathologic study of odontogenic keratocysts in Singapore and Malaysia

This was a retrospective study of odontogenic keratocysts in people from the Singapore-Malaysian region. The purpose of this study was to present the clinicopathologic features of odontogenic keratocysts in the Oriental population and to compare these data with those from other reported studies. Biopsy records from 1981 to 1992 of 61 cases of odontogenic keratocysts from patients in Malaysia and Singapore showed that 42.6% of patients were female and 57.4% of patients were male. Among patients with cysts, 75.4% were Chinese, 6.6% were Malays, 9.8% were Indians and 8.2% were other ethnic groups. The mean age of these patients was 26.98 +/- 15.38 years with a peak incidence occurring in the second to fourth decades. The location of the lesions was more often in the mandible (65.5%) than the maxilla (31.0%). There was a marked predilection for lesions to occur in the posterior mandible. Histologically, 90.2% of the cysts were lined with a para-keratinized stratified squamous epithelium while only 3.3% of the cysts were lined with orthokeratinized stratified squamous epithelium. Mixed para-keratinized and orthokeratinized epithelial linings were observed in 4 cases (6.5%). The cyst linings were mainly uninflamed (95.1%). Inflammation of the cyst wall was found in 42 cases (68.8%). Twelve (19.7%) cases contained keratin in the lumen. A satellite cyst was observed in only 6 cases (9.8%). In conclusion, most clinical and histological features seen in this study were similar to those found for Caucasians. The only clinical feature that was different was the peak age incidence, that ranged from the second to fourth decades, with an absence of a second peak. Odontogenic keratocysts presenting at the site of the dentigerous cyst were observed in 7 cases (11.5%).

Ultrastructural analysis of implant-soft tissue interface on a three dimensional tissue-engineered oral mucosal model.

A three dimensional tissue-engineered human oral mucosal model (3D OMM) used in the investigation of implant-soft tissue interface was recently reported. The aim of this study was to examine the ultrastructural features of soft tissue attachment to various titanium (Ti) implant surfaces based on the 3D OMM. Two techniques, that is, focus ion beam (FIB) and electropolishing techniques were used to prepare specimens for transmission electron microscopic (TEM) analysis of the interface. The 3D OM consisting of both epithelial and connective tissue layers was constructed by co-culturing human oral keratinocytes and fibroblasts onto an acellular dermis scaffold. Four types of Ti surface topographies were tested: polished, machined (turned), sandblasted, and TiUnite. The specimens were then processed for TEM examination using FIB (Ti remained) and electropolishing (Ti removed) techniques. The FIB sections showed some artifact and lack of details of ultrastructural features. In contrast, the ultrathin sections prepared from the electropolishing technique showed a residual Ti oxide layer, which preserved the details for intact ultrastructural interface analysis. There was evidence of hemidesmosome-like structures at the interface on the four types of Ti surfaces, which suggests that the tissue-engineered oral mucosa formed epithelial attachments on the Ti surfaces.

DETECTION OF HOST-SPECIFIC IMMUNOGENIC PROTEINS IN THE SALIVA OF PATIENTS WITH ORAL SQUAMOUS CELL CARCINOMA

The main purpose of this article is to develop a new and reliable saliva-based clinical diagnostic method for the early detection of oral squamous cell carcinoma (OSCC). This study used an immunoproteomic approach which allowed the detection of immunogenic host proteins in patients’ samples using pooled human antibodies. In an attempt to investigate potential biomarkers of OSCC, two-dimensional electrophoresis (2-DE) followed by immunoblotting of saliva from patients and controls were compared. The protein spots of interest were analyzed using 2-DE image analyzer and subsequently subjected to MALDI-TOF/TOF and then matched against NCBI database. The result showed that four protein clusters, namely Human Pancreatic Alpha-amylase (HPA), Human Salivary Amylase (sAA), keratin-10 (K-10), and Ga Module Complexed with Human Serum Albumin (GA-HSA), had exhibited immunoreactivity in western blot. The results are suggestive of the potential use of the differentially expressed saliva protein as tumor biomarkers for the detection of OSCC. However, further studies are recommended to validate this finding.

Modified porous scaffolds of silk fibroin with mimicked microenvironment based on decellularized pulp/fibronectin for designed performance biomaterials in maxillofacial bone defect.

Maxillofacial bone defect is a critical problem for many patients. In severe cases, the patients need an operation using a biomaterial replacement. Therefore, to design performance biomaterials is a challenge for materials scientists and maxillofacial surgeons. In this research, porous silk fibroin scaffolds with mimicked microenvironment based on decellularized pulp and fibronectin were created as for bone regeneration. Silk fibroin scaffolds were fabricated by freeze-drying before modification with three different components: decellularized pulp, fibronectin, and decellularized pulp/fibronectin. The morphologies of the modified scaffolds were observed by scanning electron microscopy. Existence of the modifying components in the scaffolds was proved by the increase in weights and from the pore size measurements of the scaffolds. The modified scaffolds were seeded with MG-63 osteoblasts and cultured. Testing of the biofunctionalities included cell viability, cell proliferation, calcium content, alkaline phosphatase activity (ALP), mineralization and histological analysis. The results demonstrated that the modifying components organized themselves into aggregations of a globular structure. They were arranged themselves into clusters of aggregations with a fibril structure in the porous walls of the scaffolds. The results showed that modified scaffolds with a mimicked microenvironment of decellularized pulp/fibronectin were suitable for cell viability since the cells could attach and spread into most of the pores of the scaffold. Furthermore, the scaffolds could induce calcium synthesis, mineralization, and ALP activity. The results indicated that modified silk fibroin scaffolds with a mimicked microenvironment of decellularized pulp/fibronectin hold promise for use in tissue engineering in maxillofacial bone defects.

A biocompatibility study of injectable poly(caprolactone-trifumarate) for use as a bone substitute material

The need for bone graft alternatives has led to the development of numerous bone graft substitutes. Here, the authors have synthesized a biodegradable poly(caprolactone-trifumarate) (PCLTF) polymer solution that could be injected into any bony defect. This polymer solution was synthesized using polycaprolactone-triol and fumaryl chloride (FCl). PCLTF is a multiple-branching, unsaturated and cross-linkable in situ material. The surface microstructure of PCLTF was investigated using a field emission scanning electron microscope. The incorporation of double bonds originating from FCl into the poly(caprolactone) backbone was confirmed in the Fourier transform infrared spectra. The in vitro cytotoxic effects of PCLTF, its leachable extracts and degradation products were evaluated in direct and indirect contact tests against human oral fibroblasts. Cell viability was evaluated using the microculture tetrazolium assay and cytotoxicity evaluations of PCLTF were tested in accordance with ISO 10993-5 standards. The results showed that there was evidence of reasonable cell growth, good cell viability and intact cell morphology after exposure to PCLTF, its extracts and degradation products. There was no evidence of critical cytotoxic effects.