Stem Cells & Regenerative Medicine

Stem Cells & Regenerative Medicine

• Stem cells can self-renew over time and differentiate into specialized cells like muscle, blood, and brain cells.
• Stem cells are classified by their plasticity (developmental versatility).
• Totipotent stem cells can differentiate into all cell types and an entire organism.
• They are fertilized eggs and cells after few divisions.
• Pluripotent stem cells can create all tissue types but not an entire organism
• These cells, arising from the inner cell mass, need the outer layer (placenta) to function fully.
• Multipotent stem cells are less versatile, differentiating into a limited range of cells within a tissue type
• Offspring of pluripotent cells become progenitors (e.g., blood, skin, nerve cells).
• Adult stem cells are multipotent, replacing damaged or lost cells in differentiated tissues.
• They are undifferentiated cells in differentiated tissues
• Adult stem cells have been identified in various tissues (blood, neural, endothelial, muscle, mesenchymal, gastrointestinal, and epidermal).
• Tissue-specific stem cells are harder to isolate and less easily maintained in culture than embryonic stem cells.
• Induced pluripotent stem cells (iPSCs) are engineered in a lab by reprogramming tissue-specific cells (like skin) into pluripotent cells mirroring embryonic stem cells.
• Viruses are used in the process, raising cancer risks.
• iPSCs have been sourced from dermal fibroblasts, skin keratinocytes, amniotic fluid-derived cells, CD34 blood cells, and mesenchymal stem cells.
• Mesenchymal stem cells (MSCs) are a type of adult stem cell extensively studied, often found in bone marrow, but can also be isolated from fat or cord blood.
• MSCs can generate osteogenic, chondrogenic, and adipogenic cells.
• In vitro odontogenesis involves inducing dental epithelial-like cells from mouse ES cells, or iPSCs differentiating into tooth-related structures, including dental mesenchymal cells used in regeneration.
• Advantages of dental mesenchymal stem cells include high reprogramming efficiency, multipotency, ease of acquisition, and low morbidity at the donor site.
• Applications of dental stem cells are focused on repairing damaged teeth structures (including dental pulp regeneration, periodontal complex regeneration, osseous regeneration, and other tissue regeneration in medicine).
• Regenerative medicine aims to recreate fully functioning tissues and organs to replace those lost due to disease, injury, or aging, including hair follicles and mammary glands.

Regenerative Dentistry

• In vitro odontogenesis involves three-dimensional bioengineered teeth and tooth germ generation.
• Bioengineered teeth (in vitro) have correct tooth structure, masticatory function, and nervous response to mechanical stress.
• Tooth development relies on reciprocal interactions between ectoderm-derived epithelium and neural crest-derived mesenchyme.
• In the lab, tooth formation requires epithelial cells for initial signals to mesenchyme cells and the presence of both to develop a tooth.
• Stem cells are a source for lab-induced teeth. These stem cells of focus are epithelial stem cells (ameloblasts) and mesenchymal stem cells (odontoblasts, cementoblasts, osteoblasts and fibroblasts).

Human Postnatal Mesenchymal Stem Cells

• Postnatal stem cells include Dental pulp stem cells (DPSCs), cells from exfoliated deciduous teeth (SHEDs), periodontal ligament stem cells (PDLSCs), cells from apical papilla (SCAPS), and dental follicle progenitor cells (DFPCs).
• These cells have slightly different potencies and some cells are derived from neural crest or mesoderm.

Dental Pulp Stem Cells (DPSCs)

• DPSCs are a mesenchymal type of cell found inside dental pulp, differentiating into osteoblasts, smooth muscle cells, adipocyte-like cells, neurons, dentin, and dentin-pulp-like complexes.
• In vitro experiments have shown chondrogenic potentials.
• Overall, DPSCs show more suitability than BMSCs for mineralized tissue/bone regeneration.
• DPSCs can differentiate into neural and vascular endothelial cells.

Stem Cells from Human Exfoliated Deciduous Teeth (SHEDs)

• SHEDs are progenitor cells from the deciduous tooth pulp remnants.
• SHEDs exhibit a higher proliferation rate and differentiation capacity than BMSCs, and even some DPSCs in several studies.
• SHEDs can differentiate into osteoblasts, odontoblasts, adipocytes, and neural cells.

Periodontal Ligament Stem Cells (PDLSCs)

• PDLSCs originate from the neural crest.
• They exhibit characteristics similar to MSCs and can develop into osteoblasts, cementoblasts, adipocytes, chondrocytes, Periodontal ligament, and cementum-like tissue in vivo.

Stem Cells from Apical Papilla (SCAPs)

• SCAPs are cells isolated from the apical root of developing teeth.
• They exhibit characteristics of MSCs and can differentiate into osteoblasts, adipocytes, chondrocytes, and neurons under the right conditions.

Dental Follicle Progenitor Cells (DFPCs)

• DFPCs are stem cells from the dental follicle (surrounding the tooth germ).
• The follicle contains ectomesenchymal cells that develop into periodontium.
• DFPCs can differentiate into osteoblasts, adipocytes, chondrocytes, and neural cells.

Dental Epithelial Stem Cells

• Ameloblasts are not readily found in postnatal dental tissues, being lost during tooth eruption.
• Recent studies show that the epithelial cell rest of Malassez (ERM) can differentiate into ameloblast-like cells, expressing cytokeratin and amelogenin proteins in vitro.
• Enamel-like tissues developed eight weeks after transplantation, indicating the presence of developed ameloblasts.

Odontogenic Potential and Competence

• Odontogenic potential refers to a tissue's ability to induce gene expression in an adjacent tissue and initiate tooth development.
• Initially, odontogenic potential resides in the dental epithelium but later shifts to the mesenchyme (as shown in mice).
• Odontogenic competence is a tissue's ability to reciprocate odontogenic signals and support tooth/dentin development; only neural crest-derived mesenchyme possess this competence.
• Combining early epithelial tissue with non-neural crest-derived mesenchyme results in no tooth being formed.
• Recombination of first branchial arch epithelium and second arch mesenchyme (or certain neural crest tissues) does result in tooth formation.

Partial Tooth Repair

• Gradual enamel/dentin loss and pulp exposure prompts living odontoblasts to produce reactionary dentin.
• Large lesions lead to odontoblast death, releasing TGFβ growth factors.
• These factors recruit pulp stem cells, which migrate and produce reparative dentin.
• Enamel proteins (primarily amelogenin) are found in odontoblasts implying synthesis and deposition of enamel.
• Adult stem cells and their cultivation/multiplication in a biodegradable scaffold can guide odontoblast and ameloblast differentiation.
• In vitro and in vivo studies have shown promising partial tooth repair results.

Is Human Tooth Regeneration a Prospective Clinical Reality or a Fantasy?

• Some animals continually replace teeth throughout life (polyphyodonty).
• In some animals (mice, voles), damaged teeth can be repaired or replaced with stem cells.

Bioengineered Tooth Implant Development

• Developing a bioengineered tooth into the correct structure in an oral cavity.
• Successfully erupting teeth 37-49 days after transplantation in mice.
• Reaching the occlusal plane with opposing teeth in mice
• Bioengineered mature teeth require bone integration after transplantation to maintain periodontal ligament (40 days in studies).
• Bioengineered tooth enamel and dentin hardness are within normal ranges.

Replacement Methods for a Whole Tooth

• Tooth-related cells, preferably stem cells.
• Odontogenic epithelial cells (of human origin, like ERM, impacted teeth, etc).
• Using impacted or developing teeth or combining dissociated epithelial/mesenchymal tissues or intact dental epithelium/mesenchyme to generate tooth-like structures.

Culture Techniques

• Supporting 3D tooth-like growth leading to tooth-like bud or even replicas.
• Utilizing artificial, tooth-shaped biodegradable scaffolds.

Surgical Techniques

• Ability to implant bioengineered tissue/tooth germ into the prepared empty alveolar socket
• Resulting in proper root, periodontal ligament, and osteointegration development.
• Enabling bioengineered teeth growth, morphogenesis, and eruption

Novel Three-Dimensional Cell Manipulation Methods (for Whole Tooth Regeneration)

• Methods are illustrated by diagrams and include steps such as 3D cell manipulation, bioengineered germ tooth transplantation, tooth eruption, and bioengineered tooth unit engraftment.

Engraftment of Bioengineered Tooth

• Shown via diagrams, highlighting steps in cell manipulation, bioengineered tooth unit (including components like dentin and enamel), and methods of visualization and observation (e.g., microscopy, micro-CT scanning).

Transplantation of Bioengineered Tooth Germ

Methods and diagrams illustrating cell preparation, collagen gel formation, extraction, wound healing, and transplantation. Also includes images for observation at various time points post-implantation