Intro

For the past few years studying stem cells and stem cell therapy have been of great importance for
scientists. Their pluripotent and multipotent characteristics have been used for tissue regeneration and
repair and they have been vastly used in tissue engineering technics. In order to classify a cell as a “stem
cell” it must have two defining features: 1- no limit in self-renewal and; 2- a unique ability to
differentiate. One way of categorizing stem cells is to classify them in two general categories including
pluripotent and multipotent stem cells. A pluripotent stem cell can differentiate into all type of cells and
they are present in the body for a short period of time before turning into more specialized multipotent
stem cells. These multipotent cells will then become cells of germlines or a particular tissue. (15)

Dental pulp tissue is of neural crest origin and acquiring, isolating and preserving it long-term has been
made easier due to noninvasive and novel procedures (collecting it from extracted wisdom teeth and
exfoliated deciduous teeth). (8) Mesenchymal stem cells are a group of adult stem cells that can be
found in bone marrow in low quantities and they will differentiate into mesodermal tissues. They have a
potential to turn into all three germ layers. (2, 16) A human dental pulp stem cell is a multipotent stem
cell that can be easily isolated from pulp tissue and differentiate into several groups of cells such as
adipocytes, osteocytes, glial cells and neurons because of their heterogeneous nature. Hence, they are a
great noninvasive, ethically approved source for mesenchymal-like stem cells. (9,10) Due to their neural
crest origin, hDPSCs have a neuro-ectodermal specification. For instance, they can produce multiple
neurotrophic factors like nerve growth factor (NGF) and glial cell line-derived neurotrophic factor
(GDNF) which gives them some neuroprotective features. As mentioned before, hDPSCs have a
multipotent ability and are able to differentiate into Neuronal-like stem cells. These cells can express
some of the typical neuronal markers (e.g., PAX6, MAP2, Vimentin, neurofilaments) and Nestin that can
be used to evaluate their neurogenic ability. (4,6)

The nervous system’s functional units are neurons and their job is to make electrical impulse
transmissions easier. Thereupon, any damages or injuries involving these cells results into multiple
neurological disorders. The type of the disorder depends on the type of the involved neuron. (8) They
can be the result of a damaged Central Nervous System or Peripheral Nervous System (e.g., Parkinson’s
disease, spinal cord injury, Alzheimer’s disease and etc.). (11,12) Treatment of these neurodegenerative
disorders is complicated because of the limited ability of self-repairing of the injured neurons and
replenish of the lost ones. Therefore, scientists have tried cell-transplant therapy to enhance
endogenous nerve regeneration. They direct the cell division towards increasing the population of cells
to have a vast number of homogenous cells that will be delivered to the patient for regeneration of the
injured tissue. (15) One of the best options of repairing these lesions with help of tissue engineering
technics are hDPSCs. Their ability to turn into neurons on one hand and their potential for tolerating
toxic environments of the lesions on the other hand makes them a great option for this purpose. (1) But
one important challenge is delivering these cells effectively to the desired area and creating a
biocompatible environment for the cell differentiation. So, the search for a suitable biomaterial for
transferring these cells and creating an environment for guiding their differentiation to the intended
cells is crucial in cell therapy. (11, 14)

Scaffolds along with cells and growth factors are the essential agents for making a structure to be used
in tissue engineering. (12) Scaffolds are means of keeping the cells in place and facilitate growth and
differentiation. They are also an environment for cell migration and adhesion. Therefore, they must have
specific features. They should have high biocompatibility and biodegradability and they need to be non-
toxic and non-inflammatory. Some of the most used scaffolds are chitosan scaffolds, fibrin, collagen,
fibronectin, polylactic acid scaffolds (3DP-PLAS) and silk. Some are natural and others are synthetic.
Consideration of the pore size, texture and mechanical strength of these scaffolds are also necessary.
Two problems of these structures are their low capacity of holding cells and improper extracellular
matrix (ECM). (1,4,5,7) One way to improve these downfalls is to add exogenous biomaterials and coat
the scaffold with biomolecules and proteins in order to upgrade the scaffold into a better acting
environment that will provide enough material and surface for the stem cells to grow and differentiate.
Another way to solve this problem is to create an ECM-like structure as hydrogels or decellularized ECM
that also provides the required material and surface. (2,5,6)

As explained above, the function of neurons is incredibly important due to their mission in transferring
electrical signals across the nervous system. So, any dysfunction of these cells will result in dysfunction
in the whole nervous system. Therefore, the need for a safe, ethically approved way of regenerating
neurons and nerves can be clearly seen. The hDPSCs are a great option for tissue engineering in the
treatment of injured neurons and neurodegenerative disorders due to the non-invasive way of getting
them. Hence, optimum use and culture of these cells are necessary. In this study we aim to collect and
report research that focused on creating environments that will effectively transfer these cells to the
intended site and guide their differentiation to the desired neuron cells.

Methods and materials

This systematic review was developed based on PRISMA guidelines.

Search strategy and selection

this systematic review was developed to detect and assess the studies done on neural differentiation of
dental stem cells. 13 studies were chosen for this purpose by searching all studies in databases such as
pubmed, scopus and web of science using the formula in table 1. There was no search limitation. The
search was done by hand. All the studies related to the subject were searched and detected. The
searched phrase included four primary keywords "neural", "differentiation", "dental" and "stem cells"
and all related keywords based on MeSH terms. Endnote software was used to manage the searched
studies and references based on reviewing the title and the abstract of every study and duplicate and
unrelated studies were detected and got eliminated. Duplicate entries were searched by considering the
title of published articles, the year of publication and authors. All studies related to the matter were
checked and only the results related to human subjects were chosen. The evaluation and selection of
the studies related to the matter was done by two of the researchers independently (SMK and DA) and
the elimination of unrelated studies was defined based on the exclusion and inclusion criteria and one
researcher checked the accuracy (RRD)

Exclusion and inclusion criteria

Every study that investigated the neural differentiation of dental stem cells was included in this
systematic review. Duplicate citations, studies that the full text was not available, animal studies and
unrelated outcomes were excluded.

Data collection and extraction

 The data found by searching was entered into the endnote software and the initial collection of the
required information was done by two researchers (SMK and DA) by reviewing the title and the abstrats
of the articles. the studies that were performed on animals were excluded. Then the first author (SMK)
reviewed the full texts of the chosen articles and collected the data that was needed.

The information that the researcher extracted was based on (1) authors and publication year (2) the
source of cells (3) type of study (4) the biomaterial (5) the impact on differentiation (6) differentiation
review technique (7) neural expression marker check (8) resulted neuron type

Quality of studies

The first author (SMK) evaluated the studies and reported the required data based on STROBE
(strengthening the Reporting Studies in Epidemiology) statement.

Results
On our initial search we identified 731 literatures in PubMed (151), Web of Science (132) and Scopus
(448). 290 duplicates were found automatically and 237 duplicates were found manually. 168 studies
were assessed according to their title and abstract, 43 references were chosen for further study based
on their full texts. After assessing their full texts, 13 articles were identified as qualified for the purpose
of this systematic review and data extraction. All the extracted data is available at Table1. Papers that
included stem cells extracted from a patient with disorder were excluded. One article was not accessible
and was excluded.

The most used biomaterial in the literatures is “chitosan porous scaffold” which was used in 4 articles. 3
articles didn’t mention using of any scaffolds and they mostly focused on the hydrogels and cultures
used in the differentiation process.

1 paper used PLCL scaffold, 1 used TPS-PN, 1 used CSM, 1 used decellularized ECM scaffold, 1 used 3D
PLAS, 1 used poly-L-lysine-coated coverslips, 2 used hydrogels, 2 used 3D floating sphere technique and
1 used neurosphere technique in the differentiation procedure. All of the used techniques and materials
were effective and produced neurons or neuron-like cells and had at least 1 positive neuronal
expression marker check.

All the extracted data and obtained results will be useful for scientists all over the world to use at their
researches about neuron regeneration therapy and cell engineering.

Reference Authors’ Year of Source Type Biomaterial Impact Differentiati Neuronal Resulted
Name Publication of of on on Review Expression Neuron Type
Cells Study Scaffold Growth Factors/ Different Technique Marker
Cultures iation Check
1 Pineda, J. 2022 hDPSC in vitro Nanopatterned Plastic-adherent Indirect ICC the neural Not
R., et al. poly(lactide-co- monolayer FC stem cell mentioned
caprolactone) cultures, Floating marker
(PLCL) dentosphere Nestin, glial
scaffolds cultures markers: glial
fibrillary
acidic protein
(GFAP), and
S100ß, and
neuronal
markers:
NeuN and
doublecortin
(DCX)

2 Gao, Y., et 2022 hDPSC in vitro TPS-PN plates rVT, LN, PLO, Indirect flow βIII-tubulin, Not
al. (poly-N- PBS, PS, DPBS, cytometry, nestin mentioned
isopropylacryla immunostaini
mide-co-butyl Trypsin-EDTA, ng
acrylatecoated α-MEM, FBS,
TPS plates)
Neurobasal-A,
bFGF, EGF, B27,
PN

3 Drewry, 2022 Exclud --------- ------------------- ---------------------- ----------- ----------------- ----------------- -----------------
M. D., et ed
al. (due
to not
being
accessi
ble)
4 Zheng, K., 2021 hDPSC in vitro Chitosan Porous Low glucose Indirect western blot, GFAP, Not
et al. Scaffolds Dulbecco Immunofluor S100β, β- mentioned
modified Eagle escence tubulin III
medium Staining,
(DMEM), fetal Reverse
Accepted transcription-
Manuscript polymerase
bovine serum chain reaction
(FBS), penicillin, (RT-PCR)
streptomycin
5 Luo, L. H., 2021 hDPSC in cellulose/soy GelMA-bFGF Indirect Immunohisto GFAP, β- Not
et al. vitro/ protein isolate hydrogels chemical tubulin III mentioned
in vivo composite analysis,
membranes Western blot
(CSM)
6 Laudani, 2020 hDPSC in vitro decellularized fetal bovine serum Indirect Cytofluorimet MAP2, b-III Not
S., et al. ECM scaffold (FBS), penicillin, ric, tubulin, mentioned
streptomycin, and Immunofluor neurofilament
fungizone, escent, SEM, -heavy (NF-
H), VIM, NF-
trypsin/EDTA, a- RT-PCR
L, PAX6
MEM, Ascorbic
acid
7 Hsiao, D., 2020 hDPSC in vitro 3DP-PLAS ---------------------- Indirect Immunofluor glial fibrillary neural
et al. escence acidic protein precursor
staining, (GFAP), cells,
nestin, neurogenic
neurofilament structures,
-M (NF-M), astrocyte-like
bIII-tubulin, cells
microtubule
associated
protein 2
(MAP2)
8 Kang, Y. 2019 hDPSC in vitro ------------------- Protocol 1: β- Indirect flow ChAT, HB9, cholinergic
mercaptoethanol cytometry, ISL1, BETA- neuronal-like
 N., et al. (BME) and nerve cell cycle 3, MAP-2 cells
growth analyses, RT-
factor (NGF) qPCR,
Protocol 2: Immunocytoc
tricyclodecane-9- hemistry
yl-xanthogenate (ICC),
(D609) Analysis of
Protocol 3: basic acetylcholine
fibroblast growth (Ach)
factor secretion in
(bFGF), sonic culture
hedgehog (SHH), media
and retinoic acid
(RA)

9 Pisciotta, 2018 hDPSC in vitro ------------------- Three- Indirect immunofluore nestin, neural
A., et al. Dimensional scence CD271, SOX- progenitor
Floating Sphere analysis, 10, b- cells, neural
Culture System/ Western blot IIITubulin, crest stem
EGF, b-FGF, analysis, MAP-2 cells
DMEM/F12 pseudocolor
culture medium, analysis
L-glutamine
penicillin,
streptomycin, B27
10 Bojnordi, 2018 hDPSC in vitro poly-L-lysine- neurosphere Indirect Flow nestin, NF68, neuroprogenit
M. N., et coated technique/basic cytometry, MAP-2, β- or cells,
coverslips fibroblast growth Immunofluor tubulin mature
al.
factor, epidermal escent neurons
growth factor, staining
B27, N2

11 Harnidab 2017 hDPSC in vitro Chitosan- ---------------------- Indirect real-time Oct-4, Nestin, neuronlike
adi, H. G., Intercalated PCR (RT- NF-M, NF-H, cells
Montmorillonite PCR), MAP2, βIII-
et al.
/Poly (vinyl immunostaini tubulin
alcohol) ng
Nanofibers
12 Zhang, J., 2016 hDPSC in vitro chitosan porous Dulbecco Indirect polymerase CNPase, Oligodendroc
et al scaffolds modified Eagle chain reaction MAP-2, ytes,
medium (RT-PCR), GFAP astrocytes,
(DMEM), Western neurons
fetal bovine serum blotting,
(FBS), penicillin, immunofluore
streptomycin scence assays
13 Martens, 2014 hDPSC in vitro ------------------- collagen Indirect Immunocytoc laminin, p75, Schwann cell-
W., et al. hydrogels (cell hemistry, GFAP, like
suspension, ELISA CD104, phenotype
MEM, nestin
type I rat tail,
sodium
hydroxide.)/
alpha
modification
(Alpha-MEM)
fetal bovine
serum,
glutamine,
penicillin,
streptomycin,
beta-
mercaptoethanol,
trans-retinoic acid
(RA), forskolin,
basic
fibroblast growth
factor (b-FGF),
platelet-derived
growth factor AA
 (PDGFaa),
heregulin-beta-1
(NRG)
14 Feng, X. 2014 hDPSC in vitro 3D highly neural induction Indirect RT-PCR, Nestin, MAP-2+
M., et al. porous chitosan medium (NIM)- western CNPase, neurons,
scaffold DMEM/F- blotting, MAP-2, GFAP+
12 medium, B27, immunofluore GFAP astrocytes and
N2, BDNF, scence CNP+
NGF, bFGF oligodendroc
ytes