Introduction with trypsin and DMEM / F12 (Gibco-Life Technologies,

Introduction

Spinal cord injury (SCI) is a kind of
neurological trauma, which leads to sensory and motor functions deficiency. No
proper treatment has been found for SCI to date, but cell- based therapy has
been introduced as a novel effective treatment for SCI. In this regard various
cell types were transplanted in the SCI model such as embryonic stem cell, bone
marrow-derived mesenchymal stem cell and neural stem cell.

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Human
dental pulp stem cells (hDPSCs) originate from neural crest and can be
differentiated into the osteoblasts, adipocytes, chondrocytes and myoblasts. hDPSC
can easily be acquired from removed or discarded teeth without any ethical
problems for regenerative medicine. The positive therapeutic effects of DPSCs
administration is widely accepted in the various diseases such as myocardial
infarction, Alzheimer’s disease and ischemia. Nowadays, DPSC transplantation
was introduced as a trustworthy procedure for the healing the central nervous
system (CNS) disorders, by releasing neurotropic factors and increasing
neurogenesis.  DPSCs have demonstrated
the multifaceted neuro-regenerative abilities including anti-inflammatory
activity, regeneration of the injured axon, anti-apoptotic activity and cell
replacement activity. Recently, neural differentiations of hDPSCs have been
exhibited in the several studies. In the trauma of spinal cord, DPSCs are
capable to regenerate the nerves and can differentiate into the neuron like
cells and glial cells.

Cerebrospinal
fluid (CSF) mainly secreted by the choroid plexus, in addition brain parenchyma
also has limited secretion potential. CSF contains various Ions, vitamins,
certain peptides and proteins which fills the brain ventricles and surrounds the
CNS. There are some studies that indicated the effects of CSF on the cell
survival, cell proliferation, neuronal and glial differentiation. However, the
impact of CSF on survival and differentiation of stem cells is not well
described.

Recent
findings have emphasized the effects of stem cell-based transplantation
technique as a promising therapeutic method for ameliorating the SCI. In
regenerative medicine, hDPSCs are considered as a reliable therapeutic source
for stem cell therapy. The purpose of this study is to evaluate the effects of
DPSCs administration in rat SCI model, in addition to the CSF role to enhance
the beneficial effects of the treatment, by increasing survival and
differentiation rate.

 

Materials
and Methods

Isolation
and culture of hDPSCs: hDPSCs
were extracted from normal human (18-25 years old) third molar teeth, which were collected from adults at the dental
clinic of Mazandaran University of Medical Sciences. Teeth were cut up with
surgery cutter to reveal the pulp chamber, and then pulp tissues were gently
separated by sterile forceps. Pulp tissues were digested mechanically and
enzymatically with scalpel and trypsin 0.25% (Gibco, USA), respectively. After
5 minutes incubation of tissues with trypsin and DMEM / F12 (Gibco-Life
Technologies, Canada) in incubator, the falcon tube was centrifuged and the
supernatant was removed, then tissues were cultured in 25 ml cell culture
flasks containing DMEM/F12 supplemented with 15% FBS, streptomycin /penicillin,
and L-glutamine. Cell growth was exanimated with an inverted microscope. hDPSCs
at passages 3-4 were used for transplantations in this study.

Collection
of CSF: Newborn rat race Sparagu Dawly was
used for CSF
collection. Cerebrospinal fluid was collected
from the cisterna magna of each animal. After a short centrifugation (1,400 rpm),
the supernatant were transferred into the sterile microtube, and then all samples
were frozen in liquid nitrogen and stored at ?80 °C.   

MTT assay: Optimal CSF concentration was obtained
by calculating the viability rate
of cells. In this regard, we used a colorimetric
assay based on the reduction of tetrazolium dye 3-(4,
5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The optimum
dose and time of using CSF were assessed by adding the hDPSCs to culture medium
containing the CSF concentrations of in 0%, 2.5%, 5%, 10%, and 20% for 8 days. In
the next step, the MTT (Sigma-Aldrich, USA) was dissolved in PBS solution at a
concentration of 5 mg/ml. After the incubation of the cells in the 96-well
plate with MTT (3 hours at 37°C), dimethyl sulfoxide (Sigma) was added to each
well to dissolve formazan crystals.  After
solubilization was complete, the absorbance (570 nm) of formazon
products was measured by Eliza plate reader (Synergy H11, BioTek, Winooski, VT,
USA).