This multidisciplinary research program aims to advance the feasibility of tooth recycling for reconstructive dentistry.

Short Term

Long Term

Principal Investigator
Dr Varawan Sae-Lim, Department of Restorative Dentistry

Local Collaborators
A/Prof Lim Tit Meng, Dept of Biological Sciences, NUS
Dr Dietmar W Hutmacher, Division of Bioengineering, & Department of Orthopaedic Surgery, NUS
A/Prof Kelvin Foong, Dept of Preventive Dentistry, NUS
Dr Cao Tong, Faculty of Dentistry, NUS

Overseas Collaborator
Dr. Yin Xiao, Tissue BioRegeneration and Integration, School of Life Science, Queensland University of Technology, Garden Point Campus, Brisbane, Australia

Embryonic Stem Cells
To study the efficacy of differentiation of embryonic stem cells to target somatic lineage.

Project 2:
Mesenchymal Stem Cells for Osteogenic and Chondrogenic Tissue Regeneration.

Human Embryonic Stem Cells

Programme Title (1)

The establishment of human embryonic stem cell lines of diverse ethnic origin, to be utilized for broad-ranging therapeutic and non-therapeutic applications

Programme Objectives

1. To establish new human embryonic stem cell (hESC) lines from donated cryopreserved embryos of diverse ethnic origin.
2. To investigate whether hESC lines of different ethnic origin would differ in their transcriptional and proteomic profiles, as well as in their epigenetic characteristics.
3. To develop the isolated hESC lines of diverse ethic origin as ideal cell sources for diverse therapeutic and non-therapeutic applications.
4. To improve the current methodology of deriving hESC lines from cryopreserved embryos.

Specific Problem areas and definition

1. Current restrictive legislation in the United States have severely hampered international efforts in hESC research, in particular the establishment of new lines with different HLA genotypes, which would be crucial for future therapeutic applications. Hence, our group will attempt to develop new hESC lines from donated cryopreserved embryos leftover from fertility treatment. This would be obtained from a number of different fertility clinics around the world, in Europe and Asia.
2. HESC lines of different ethnic origin could differ in their transcriptional and proteomic profiles, as well as in their epigenetic characteristics. The donated embryos will originate from couples of diverse ethnicity. The data obtained could have important implications for cell-based therapy as well as the newly-developing field of pharmacogenetics – that is the individualized tailoring of medication based on the genetic background of the patient.
3. In recent years, there has been growing awareness that the genetic background of an individual based on ethnicity, is a major factor in pre-determining susceptibility to various diseases, as well as pharmacological response to medication. The next major challenge would be to individually tailor the prescription of different types and dosages of medication according to the genetic background of the patient. In this regard, hESC lines of diverse ethnic origin could provide a useful tool for characterizing and comparing genetic and developmental mechanisms within different human ethnic populations. This in turn could have important implications (NIH, 2001) in; (1) gene/protein delivery therapy; (2) cell based transplantation therapy; (3) the development of toxicity screening tests for biomaterials, drugs, food, household and personal care products; (4) environmental analysis of water, soil, air, and natural/artificial products and analysis of bio-chemical toxins and anti-toxin system; (5) disease study and drug discovery, particularly in the screening of different types and dosages of newly-developed drugs, prior to commencement of actual clinical trials.
4. Current techniques for deriving hESC lines from cryopreserved embryos are still sub-optimal with relatively low-efficiency. It is believed that the most crucial time point is the first few days in which the newly-isolated inner cell mass (ICM) clump from blastocyst stage embryos is placed within in vitro culture. A systematic approach will therefore be used to evaluate different culture conditions and the supplementation of novel growth factors and extracellular matrix, to enhance the efficiency of establishing hESC lines from cryopreserved embryos.

Deliverables

1. The program, if successful, will make very significant scientific and clinical contribution and international impact in developing hESC lines of diverse ethnic origin for various therapeutic and non-therapeutic applications.
2. The project hopes to produce a tremendous amount of intellectual property through publications and patents.
3. The intellectual property of the project can be invested to scale-up production for potential application/commercialization of hESC lines of diverse ethnic origin.
4. The experience obtained from this project can be used in the development of other ethnic-specific hESC lines for biomedical research.

HESC of specific ethnic origin may be best suited for both clinical and non-clinical applications in human sub-populations of that particular ethnicity.

1. The study of genetic and developmental mechanisms;
2. Gene/protein delivery therapy to cure tissue and organ lesions;
3. Cell-injection therapy for tissue and organ repair;
4. Cell transplantation-based tissue and organ reconstruction and regeneration;
5. The development of toxicity screening tests for biomaterials and drugs; food, household and personal care products;
6. Environmental analysis of water, soil, air, and natural/artificial products and analysis of bio-chemical toxins and anti-toxin system;
7. Disease study and drug discovery.
   

Programme Title (2)

Directed somatic differentiation of human embryonic stem cells into various mesodermal, endodermal and ectodermal lineages for diverse clinical and non-clinical applications

Programme Objectives

1. To develop novel culture conditions that would bias early human embryonic stem cells (hESC) differentiation into either one of the three major germ layer lineages: endoderm, mesoderm and ectoderm
2. With hESC-derived mesodermal progenitors, in vitro culture conditions will be further optimised for controlled differentiation into the osteochondral lineage, as well as into endothelial cells and cardiomyocytes
3. With hESC-derived endodermal progenitors, in vitro culture conditions will be further optimised for controlled differentiation into the hepatic lineage
4. With hESC-derived ectodermal progenitors, in vitro culture conditions will be further optimised for controlled differentiation into the keratinocyte lineage
5. Phenotypic assessment of the various differentiated mesodermal, endodermal and ectodermal progeny lineages.

Specific Problem areas and definition

1. A major challenge in developing clinical and non-clinical applications for hESC is to achieve their controlled differentiation into well-defined lineages. This would be particularly important for clinical therapy, since it is likely that the transplantation of differentiated hESC progenies would result in higher engraftment efficiency and enhanced clinical efficacy, compared to the transplantation of undifferentiated hESC. Moreover, this would reduce the risk of teratoma formation, and avoid spontaneous differentiation of hESC into undesired lineages at the transplantation site. The very first step in achieving this objective will be develop novel culture conditions that would bias early hESC differentiation into either of the three major germ layer lineages: endoderm, mesoderm and ectoderm, during embryoid body formation in suspension culture. Further differentiation into a specific lineage can then be carried out with progenitor cells of each of the three germ layer lineages.
   
2. Among the lineages that can be derived from hESC-derived mesodermal progenitors are: osteoblasts, chondroblasts, endothelial precursors and cardiomyocytes, all of which can be utilized for cell-transplantation therapy and tissue engineering. It must be remembered that stem-cell based therapy for osteochondral defects and repair of the infarcted myocardium, are major areas of regenerative medicine that attract much attention and research funding. HESC-derived endothelial precursors are envisioned to have diverse and wide-ranging applications in tissue-engineering. A systematic approach will be used to evaluate different culture conditions and novel cytokine and extracellular matrix supplements to achieve the controlled differentiation of mesodermal progenitors into these various lineages.
   
3. Of particular interest among the lineages that can be derived from hESC-derived endodermal progenitors is the hepatic lineage. The focus here will not be on clinical application, but instead on pharmacokinetic and cytotoxicity screening. This is because the liver is the primary organ for detoxification in the human body. Additionally, liver metabolization of pharmaceuticals determines their subsequent efficacy and side-effects. A systematic approach will be used to evaluate different culture conditions and novel cytokine and extracellular matrix supplements to achieve the controlled differentiation of endodermal progenitors into the hepatic lineage.
   
4. Of particular interest among the lineages that can be derived from hESC-derived ectodermal progenitors is the keratinocyte lineage. Because the skin is the first physical barrier of the human body to the external environment, hESC-derived keratinocytes would be particularly useful for cytotoxicity screening. A systematic approach will be used to evaluate different culture conditions and novel cytokine and extracellular matrix supplements to achieve the controlled differentiation of ectodermal progenitors into the keratinocyte lineage.
   
5. Several different techniques will be used for phenotypic assessment of the various differentiated mesodermal, endodermal and ectodermal progeny lineages. This would include: RT-PCR, immunohistochemistry, flow-cytometry, as well as functional in vivo testing on live animal models.

Deliverables

HESC-derived somatic cells are the best source of human tissue and organ-forming cell that can be utilized for broad-ranging therapeutic and non-therapeutic purposes. The major applications are:

1. The study of genetic and developmental mechanisms;
2. Gene/protein delivery therapy to cure tissue and organ lesions;
3. Cell-injection therapy for tissue and organ repair;
4. Cell transplantation-based tissue and organ reconstruction and regeneration;
5. The development of toxicity screening tests for biomaterials and drugs; food, household and personal care products;
6. Environmental analysis of water, soil, air, and natural/artificial products and analysis of bio-chemical toxins and anti-toxin system;
7. Disease study and drug discovery.

To date, purified somatic cells of specific lineages have not yet been derived from hESC (Web of Science and PubMed database).

1. The program, if successful, will make very significant scientific and clinical contribution and international impact in stem cell research.
2. The project hopes to produce a tremendous amount of intellectual property through publications and patents.
3. The intellectual property of the project can be invested to scale-up production for potential applications/commercialization of hESC-derived somatic cell lineages.
4. The experience obtained from this project can be used in the development of other hESC lines for biomedical research.

Biocompatibility of Novel Biomaterials
To evaluate the biocompatibility of:

1. Novel (developmental) biomaterial scaffolds for guided tissue regeneration and tissue engineering,
2. Novel (developmental) dental biomaterials

Project 2
Biocompatibility of novel scaffold biomaterials

Project 3
Biocompatibility of developmental dental pulp repair materials and root sealer cement

1. To research and capitalize the major mechanisms involved in the photo-modulation of biomineralization at molecular, tissues, individual, and population level.
2. To apply photo-chemical interaction in imaging and characterizing biophysical and mechanical properties of enamel.

• Investigating laser-induced prevention of enamel demineralization-establishing innovative “organic blocking theory” and developing new device/technique for preventing tooth decay.
• Exploring biophotonic technologies to promote fluoride-incorporation in enamel hydroxyapatite crystals for enamel remineralization.
• Studying the major physicochemical principles guiding nano-scale biomineralization .
• Imaging enamel microstructures with second harmonic generation and two-/ three-photon excitation.
• Validating early detection of enamel demineralization using Diagnodent
• Quantifying the beneficial and detrimental potentials of laser bleaching (without chemical agents).

Local collaboration
A/P Liu XY, Dept of Physics, NUS
A/P Shen Ze Xiang, Dept of Physics, NUS
A/P Chuah Gaik Khuan, Dept of Chemistry, NUS
Dr. Pan Jisheng, Institute of Material Research and Engineering
Dr. Pramoda, Institute of Material Research and Engineering, Institute of Microelectronic
A/P Henry Yu, Dept of Physiology,NUS
A/P Osipowicz, Thomas, Research Centre for Nuclear Microscopy:
Dr. Hong MingHui, Data Storage Institute

Intra-faculty collaborators
A/P Keng Siong Beng, Dept of Restorative Dentistry, NUS
Dr. Rashid Tahir, Dept of Preventive Dentistry, NUS

In the area of Biomaterials development, advanced ionomeric cements and a functional graded dental post has been developed in collaboration with the Institute of Materials Research and Engineering, Faculty of Engineering and Advanced Materials Research Centre (NTU). A total of four patents have been filed or awarded for the fore mentioned work. While the short-term goal is to develop more novel technologies with dental applications (spherical glasses and nanofillers for glass ionomer cements; functionally graded composite brackets and wires), the longer-term goal is to see these technologies commercialized and applied in dental products in collaboration with Multinational Dental Corporations.

Characterization of biomaterials is divided into in-vitro (laboratory based) and in-vivo (clinical trials) testing. In-vitro characterization involves the evaluation of mechanical, physical, chemical and biological properties of materials. Emphasis is placed on studying environmental effects and manipulative procedures on materials. While physico-mechanical and biological characterization is done in-house, chemical and electrochemical testing is done in collaboration with the Faculty of Science, NUS. The list of tests and facilities are shown in the attached table.

With regards to the in-vitro characterization work, the development of micro-testing strategies for characterization of materials warrants particular attention. A collaborative project has been established with Instron Corporation to develop depth-sensing micro indentation techniques and instruments. This allows for testing of materials using specimens that are clinically size-scale appropriate. Although the programme has started only in Jan 2002, Instron Corporation has already licensed one of the devices developed.

In-vivo characterization conducted through clinical trials examines the clinical behaviour, performance and longevity of restorations. The correlation and symbiosis between the two facets of testing greatly enhances understanding of clinical materials. Much of the above work has been supported by Industry including NMC like 3M-ESPE, GC Corporation and Dentsply.

The short-term goal of the programme is to attain international recognition in the field of biomaterials testing. This has been achieved in part. The longer-term goal is to be a global leader in micro testing of dental materials and to provide alternatives to current standards testing (ISO, BSI etc.) which are clinically more realistic.

Department of Preventive Dentistry
A/Prof Stephen Hsu; Dr Rashid Tahir; Dr Betty Mok

Dean’s Office (FOD)
Dr Cao Tong

Faculty of Science
A/Prof Siow Kok Siong; A/Prof Lee Hian Kee; Dr Daniel Blackwood

Faculty of Engineering
Dr Lim Chwee Teck; Dr Quan Chenggen

RESEARCH AREA

1. To create patient-specific 3D virtual human head models by synthesizing conventional craniofacial and oral imaging modalities.
2. To utilize computer vision technologies to develop 3D imaging applications for craniofacial visualization and simulation.
3. To develop these technologies and applications for adoption in routine clinical practice of craniofacial and oral rehabilitation.

The programme of the CICL focuses on conducting fundamental and applied research and technology development in 3D oral/craniofacial imaging, visualization, and simulation. It combines conventional imaging modalities used in clinical practice for the development of patient-specific 3D models. The programme has a two-pronged approach:

Development of clinical parameters, acquisition of patient data, and validation
1. Acquisition of patient data through routine clinical records
2. Development of 3D image archive for static and functional data
3. Establishment of morphometric and functional/dynamic clinical parameters
4. Take measurements from clinical parameters
5. Validation of computer-based visualization applications

Research and Development of 3D imaging and visualization applications
1. Merging of different image formats
2. Visualization of 3D human head
3. Registration of functional information with 3D head model
4. Treatment simulations
5. Refinement of 3D head models through additional patient data

Faculty of Dentistry
1. Assoc Prof Kelvin Foong
2. Assoc Prof Keng Siong Beng

Faculty of Engineering
1. Assoc Prof Ong Sim Heng
2. Assoc Prof Ashraf Kassim