C o n s t r u c t s a n d Sc a ff o l d s

Employed to Regenerate
Dental Tissue
Peter E. Murray, PhD

KEYWORDS
 Constructs  Scaffolds  Dental tissue  Tissue grafting

KEY POINTS
 There are 8 key elements that are needed to create tissue constructs for dental
regeneration.
 The creation of tissue constructs requires scaffolds to be seeded with stem cells in
a controlled environment with growth factors.
 The type of stem cells and selection of scaffolds are based on the need to regenerate
mucosa, gingiva, pulp or other dental tissue.

Dental tissue injury and regeneration affects the daily lives of almost everyone. People
who have missing teeth and dental tissues have difficulty eating1 and speaking,2 suffer
a lower health-related quality of life,3 are less likely to be employed,4 more likely to be
depressed,5 and can suffer emotional problems because of their unhealthy appear-
ance.6 Dental implants and dentures can be aesthetically and functionally effective
and also satisfy patients.7 But dental implants are not able to completely replace all
the functions of natural teeth.8 Gingiva, skin, and bone can be replaced by transplant-
ing donor tissues, but these therapies are limited by donor shortage,9 or sometimes
the limited regenerative ability of donated tissues.10 Tissue engineering is emerging
as a promising therapy to regenerate missing teeth and dental tissues.10 A key
element of tissue engineering is to generate a functional tissue construct, to replace
lost or damaged tissues. Most dentists believe that regenerative dental treatments
involving the delivery of stem cells and tissue constructs will become common within
the next 10 to 20 years.11 Most dentists are willing to receive training to be able to
provide regenerative endodontic procedures for their patients.12 Half of dentists
already use membranes, scaffolds, or bioactive materials.12 It was surprising that
55% of dentists were unsure whether regenerative procedures would be successful.
The surveys11,12 indicate broad support among dentists for using scaffolds and

Department of Endodontics, College of Dental Medicine, Nova Southeastern University, 3200

South University Drive, Fort Lauderdale, FL 33328-2018, USA
E-mail address: petemurr@nova.edu

Dent Clin N Am 56 (2012) 577–588

doi:10.1016/j.cden.2012.05.008 dental.theclinics.com
0011-8532/12/$ – see front matter Ó 2012 Elsevier Inc. All rights reserved.
578 Murray

constructs to regenerate tissues, but also the need for strong evidence that demon-
strates the reliability of using these new therapies.

REGENERATION OF DENTAL TISSUE

The aim of regenerative dental therapies is to restore patients to full oral health. This
means restoring normal function to tissue that has been damaged or missing due to
trauma, disease, cancer, or a congenital condition. Greenwood and colleagues13
described regenerative medicine as the “emerging interdisciplinary field of research
and clinical applications focused on the repair, replacement, and regeneration of cells,
tissues, or organs to restore impaired function resulting from any cause, including
congenital defects, disease, trauma, and aging.” Regeneration approaches use
a combination of scaffolds, stem cells, growth factors, tissue engineering, organ tissue
culture, transplantation, and tissue grafting. There are 8 key elements to create and
use tissue constructs for tissue regeneration.
Step #1. The size, shape, functions, and aesthetics of the missing or defective
tissue, such as a bone defect, needs to be assessed using cone beam micro-
computer tomography14 and radiographs.
Step #2. Stem cells, such as dental pulp stem cells from an exfoliated baby tooth,15
need to be obtained from the host patient or a donor to serve as the building
blocks for tissue regeneration.15
Step #3. Stem cells account for a very small percentage of the cells within tissues.
The stem cells must be identified using surface markers and be isolated from all
the donated cells using fluorescent cell sorting.16
Step #4. Millions of stem cells are needed to create functional tissues; this requires
that they be expanded using cell culture.17
Step #5. The activity of the stem cells must be controlled by growth factors during
cell culture to ensure that the stem cells differentiate into a useful cell type (eg,
bone or periodontal ligament).18
Step #6. Cells grown in culture lack a 3-dimensional scaffold necessary to function
and have the correct size and shape to generate a tissue; therefore the cells
need to be seeded onto a scaffold to form a tissue construct that gives the cells
the characteristics of a tissue, such as dental pulp stem cells seeded onto poly-
mer and collagen scaffolds to generate replacement pulp tissue.19,20
Step #7. The tissue construct is maintained in cell culture until a functional tissue is
generated.21
Step #8. The tissue construct is grafted or implanted into the donor site,21 where
the regenerated tissue is required.
These steps to create and use tissue constructs are shown in Fig. 1.

Fig. 1. Steps to create and use constructs to for tissue regeneration.

 Regenerate Dental Tissue 579

DENTAL REGENERATION

Dental regeneration can be defined as the process in people by which specialized

dental tissues are replaced by the recruitment, proliferation, migration, and differenti-
ation of dental stem cells.22 It is important that the newly regenerated tissues recapit-
ulate the architecture and function of the missing or damaged dental tissue. However,
ideal reconstructive goals, such as a complete return to original clinical form and func-
tion, are frequently not completely achieved.23 Regeneration can occur more readily in
some tissue types compared with others (eg, the oral mucosa can readily regenerate
without scarring, while most other dental tissues heal by granulation tissue, which can
eventually form fibrous scar tissue).24 Dental tissue regeneration depends on the
activity of progenitor cells or stem cells to be seeded within a scaffold to generate
a new tissue construct25 (eg, artificial skin that can serve as a scaffold for the regrowth
of dermal tissue using the hosts own cells).26 Another example is freeze-dried bone,
which provides a scaffold for the host’s own osteoblasts to regenerate bone defects.27
By combining different types of stem cells with different types of scaffolds and control-
ling the cell culture and tissue engineering conditions, the outcome of the construct
can be altered to create various types of dental tissues, from teeth to salivary glands,
shown in Fig. 2.

HISTORY OF DENTAL REGENERATION

From the beginnings of dentistry in Egypt almost 5000 years ago,28 dentists have been
seeking improved procedures to generate replacement teeth, and to regenerate
dental tissues for their patients. In 1952, BW Hermann29 was reported to have
made one of the first attempts at dental tissue regeneration. Herman used calcium
hydroxide (calxyl) to promote dentin bridge formation for vital pulp therapy following
partial pulp amputation.29 In 1961, Nygaard-Ostby30 established a blood clot to use
as a scaffold to revascularize tissue within the root canals of teeth (Fig. 3). During
recent decades, many more dental procedures and scaffolds have been developed
to promote dental tissue regeneration, especially in the field of periodontics to guide
bone and tissue regeneration. The concepts of guided tissue and bone regeneration
(GTR/GBR) were first published by Melcher in 1976,31 who outlined the necessity of
using barrier membranes to exclude unwanted cell lines from healing sites to allow
tissue growth. The first clinical case reports of GTR/GBR were presented by Nyman
and colleagues32 through the intraoral application of Millipore bacterial filters com-
posed of a cellulose acetate as the first barrier membranes used to achieve

Fig. 2. Generation of dental tissue constructs.

580 Murray

Fig. 3. Blood clot revascularization within the root canal of a tooth.

regeneration of periodontal tissues as a direct alternative to surgical resection proce-

dures to reduce periodontal pocket depths.
Next came the introduction of platelet-rich plasma (PRP) to use as a natural fibrin
scaffold as part of oral surgery by Whitman and colleagues33 in 1997. PRP is thought
to activate platelets and release growth factors to enhance wound healing.33 PRP
became more popular after Marx and colleagues34 demonstrated that combining
PRP with autogenous bone in mandibular continuity defects resulted in significantly
faster radiographic maturation and histomorphometrically denser bone regenera-
tion.34 PRP has also been used in the field of regenerative endodontics, with the recent
case report of Torabinejad and Turman,35 which demonstrated the merits of PRP as
a natural scaffold for pulp revascularization.35

SCAFFOLDS

People and animals have a natural scaffold that surrounds cells and provides struc-
tural support for the formation and maintenance of tissues and organs. The scaffold
is mainly composed of extracellular matrix proteins (ECMPs). The key ECMPs are
collagen, vitronectin, fibronectin, and laminin, which provide cells with anchorage,
sequestration of growth factors, and signal cells to migrate, differentiate, and prolif-
erate through integrin receptor-mediated signaling pathways.36 ECMPs have impor-
tant roles in dental regeneration. Laminin promotes odontoblast differentiation,37
and a recent study by Howard and colleagues38 demonstrated that it is an important
factor in dental pulp stem cell migration. Fibronectin has been shown to increase
ameloblast growth and differentiation, while vitronectin provides a structural frame-
work.39 Collagen is the predominant structural component of all tissues and has
been observed to immobilize growth factors to regulate cell proliferation and
differentiation.40
Natural ECMP scaffolds have varying chemical and physical characteristics which
contribute to the specific functions of the tissue in which they reside. Scaffolds for
tissue engineering have been created with a range of physical properties; these
include porosity, pore size, weight, and hydration capacity, as shown in Table 1. A
 Regenerate Dental Tissue 581

Table 1
Physical properties of 3-dimensional tissue engineering scaffolds

Porosity
Scaffold Hydration (Pores Per Average Weight/Wet
Type Average Dimensions Capacity Linear Inch) Pore Size Weight
Polymer 5  4 mm 0.039 cm3 30 mL 120 /120 100–200 mm 5.2 mg/32 mg
Collagen 4.5  4.2 mm 0.039 cm3 25 mL 120 /120 100–200 mm 3.5 mg/45 mg
Calcium 5  3 mm 0.058 cm3 30 mL 60 /110 200–400 mm 45 mg/99 mg
Phosphate

high porosity and sufficient pore size are necessary to facilitate cell seeding and diffu-
sion of cells and nutrients throughout scaffold.41 The other important properties are
biocompatibility and biodegradability. Some scaffolds are permanent, while for other
scaffolds, they need to be absorbed by the surrounding tissues to avoid interfering
with the regenerated tissue. The rate of degradation should coincide with the rate of
tissue formation.42 The latest generations of scaffolds have been engineered to
have ideal properties and functional customization: injectability, synthetic manufac-
ture, biocompatibility, nonimmunogenicity, transparency, nano-scale fibers, low
concentration, and high resorption rates.43 Scaffolds for tissue engineering can be
created from synthetic materials such as polymers, similar to absorbable surgical
sutures,44 or by collagen, a natural ECMP scaffold material,45 or by calcium phos-
phate.46 The architecture of these scaffolds is shown in Fig. 4.
Scaffolds for dental tissue regeneration are comprised of a very diverse group of
natural tissues such as skin and bone sourced from human donors, to synthetic mate-
rials designed to be used as skin and bone scaffolds. Some scaffolds are injectible,
which makes them easy to deliver; however these hydrogel and nanofiber scaffolds
are often not able to maintain good cell survival. Spongy scaffolds include absorbable
collagen and polymers, which can maintain good stem cell survival, but which lack the
structural strength needed for load bearing and muscle movement. The advantages
and limitations of the common types of scaffolds for dental regeneration are summa-
rized in Table 2.

DENTAL TISSUE CONSTRUCTS

Dental pulp and periodontal tissue constructs have been created by seeding dental
stem cells onto scaffolds made from bovine collagen, open polylactic acid (polymer),
and calcium phosphate, followed by cell culture and tissue engineering.20 Tissue engi-
neered dental pulp constructs can be created by seeding stem cells from human exfo-
liated deciduous (SHED) teeth onto scaffolds, which are then implanted into human
teeth (Fig. 5).19 Tissue-engineered periodontal constructs can be created by seeding
periodontal stem cells onto several different types of scaffolds. The most successful
scaffolds for maintaining periodontal ligament stem cell (PDLSC) survival appear to
be the spongy scaffolds, particularly scaffolds created from polymer or collagen, as
shown in Figs. 6 and 7. Calcium phosphate scaffolds appear to be less effective at
maintaining periodontal stem cell survival (see Figs. 6 and 7). The injectible scaffolds
are comprised of a hydrogel, Pepgen P15, or DBX are the easiest to apply in the
mouth, but unfortunately appear to be the least effective for maintaining the survival
of the periodontal stem cells (see Figs. 6 and 7). The survival of stem cells within scaf-
folds is essential for the success of regenerating functional dental tissues.
582 Murray

Fig. 4. Photomicrograph of scaffold structures. (A) Photomicrograph of the collagen scaf-

fold structure. The 3-dimensional collagen composite scaffold is a natural scaffold manufac-
tured from a mixture of collagens that are derived from bovine hide. Overall, this material
exhibits collagen fibrillar architecture, which is representative of the structure of collagen
within the interstitial matrix. (B) Photomicrograph of the polymer scaffold structure. The
3D OPLA (Open-Cell Polylactic Acid) scaffold is a synthetic polymer scaffold that is synthe-
sized from D,D-L,L polylactic acid. This material has a facetted architecture, which is
effective for culturing high density cell suspensions. (C) Photomicrograph of the calcium
phosphate scaffold structure. The 3-dimensional calcium phosphate scaffold is a proprietary
mineralized calcium phosphate bioceramic that is ideal for in vitro analysis of bone metab-
olism and cartilage regeneration.

PERIODONTAL LIGAMENT CONSTRUCTS

The regeneration of severe periodontal bone defects used to require bone grafts. To-
day, several types of injectable, cement, ceramic, and spongy scaffold materials are
available as an alternative to bone grafts; these scaffolds provide a framework for
the patient’s own osteoblasts and stem cells to repair bone defects.47,48 The scaffolds
can also be seeded with autologous PDLSCs to create a tissue construct that can en-
hance periodontal regeneration in animals.49 Periodontal and bone regeneration can
be accelerated by using bioactive materials containing enamel matrix proteins such as
Emdogain50 (Straumann USA, Andover, MA, USA) and growth factors such as platelet-
derived growth factor,51 insulin-derived growth factor,52 and platelet-rich plasma.53
PDLSCs were isolated and characterized by Seo and colleagues54; these stem cells
were found to be capable of forming all components of the periodontal apparatus
including cementoblasts, osteoblasts, and fibroblasts.54 PDLSCs readily attach to
the root surface of teeth, even in the absence of growth factors.55 The seeding of
PDLSCs onto the root surfaces of teeth can be used to bioengineer functional peri-
odontal ligament in rodents.56 This demonstrates that the success of bioactive mate-
rials for the regeneration of small bone defects may be supplemented in the future by
seeding PDLSCs onto scaffolds and the roots of teeth to regenerate large bone
defects.
 Regenerate Dental Tissue 583

Table 2
Common types of scaffolds for dental regeneration

Scaffold
Type Properties Advantages Limitations
Hydrogel A colloid jelly-like Injectable, Low stem cell survival,
scaffold biocompatible, and lacks functional
Nanofiber Self-assembling absorbed by body strength, variable
peptides often outcomes
mixed with hydrogel
Collagen Sponge Easy to handle, Lacks functional
Polymer clinically effective strength to support
Calcium Phosphate Brittle absorbed by body tissues or muscle
Silk Fibers movement
Fibrin Centrifuged from Easily made scaffold
peripheral blood from host peripheral
blood
Bone Sourced from donor Bone grafts are Expensive and requires
and freeze-dried clinically very a donor, risk of
into a powder effective contamination
Synthetic bone Bone defect filling Clinically effective and Not as effective as
material safe natural bone
Skin Sourced from a Clinically effective Need cadavers or
cadaver or host donor skin, risk of
contamination
Synthetic skin Wound dressing Clinically useful and Temporary fix, patient
material safe will still need skin
grafts

DENTAL PULP CONSTRUCTS

The seeding of polymer scaffolds with dental pulp stem cells (DPSCs) to create dental
pulp constructs, which were implanted subcutaneously into a New Zealand rabbit,
was able to regenerate dentin/pulp-like tissues including and osteodentin.57 The
seeding of DPSCs on polymer scaffolds inside tooth slices, which have been

Fig. 5. Tissue engineered dental pulp constructs in the root canal of teeth. (A) Tooth with
a cleaned and shaped root canal. (B) Tooth with a dental pulp construct.
584 Murray

Fig. 6. Periodontal stem cell survival in scaffolds to create tissue engineered periodontal
constructs: (A) Polymer scaffold. (B) Collagen scaffold. (C) Calcium phosphate scaffold. (D)
Hydrogel scaffold. (E) Pepgen P15 scaffold. (F) DBX scaffold.

implanted subcutaneously in mice, has been successful at angiogenesis58 and for

regenerating odontoblast-like cells and dental pulp tissues.59 The roots of teeth
were regenerated by seeding stem cells onto scaffolds that were then implanted
into tooth sockets in minipigs.60 After 3 months, the construct, called a bioroot, had
a post inserted and porcelain crown cemented.60 This demonstrated the potential
to bioengineer replacement teeth.
The regeneration of pulpal tissue can potentially revitalize already-existing end-
odontically treated teeth; the prospect of regenerating entire teeth or bio-roots may
one day provide an alternative to dentures, fixed prosthetics, and dental implants to
replace missing teeth.

Fig. 7. Bar chart of periodontal and dental pulp stem cell survival in tissue engineered
constructs.
 Regenerate Dental Tissue 585

Fig. 8. The role of tissue engineered constructs in future dental treatment.

Autologous cell homing is an underrecognized approach in tissue regeneration61

that offers an alternative to the allotransplantation of stem cells from a donor.
Promoting a patient’s own stem cells to home to a scaffold and regenerate tissue
could help avoid the time-consuming and expensive steps of ex vivo stem cell isola-
tion, harvesting, and manipulation and expansion. Simplifying tissue regeneration by
promoting patient stem cell homing may accelerate regulatory, commercial, and clin-
ical processes. Stem cell homing has been demonstrated to generate new pulp tissue
in human molars that have been implanted into mice.61 In another rodent study, the
grafting of scaffolds was able to create teeth and periodontal ligament by cell
homing.62

SUMMARY

Dental regenerative treatments and guided tissue regeneration already save millions of
teeth each year. Synthetic bone regeneration materials are routinely used to regen-
erate the bone defects of dental patients. Researchers are aiming to develop en-
hanced regenerative therapies that have the capability to regenerate teeth and
tissues beyond current limits. This will require the development of new protocols using
stem cells, scaffolds, and growth factors.63 Several bioengineering approaches in-
volving stem cells, scaffolds, and growth factors can be combined to regenerate
the different tissues needed to generate a tooth and its supporting tissues. The role
of tissue engineered constructs in future dental treatment is shown in Fig. 8.

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