Three-dimensional printed PLA scaffold and human gingival stem cell-derived extracellular vesicles: a new tool for bone defect repair
Diomede et al. Stem Cell Research & Therapy
Three-dimensional printed PLA scaffold and human gingival stem cell-derived extracellular vesicles: a new tool for bone defect repair
Francesca Diomede 3
Agnese Gugliandolo 2
Paolo Cardelli 3
Ilaria Merciaro 3
Valeria Ettorre 1
Tonino Traini 3
Rossella Bedini 7
Domenico Scionti 2
Alessia Bramanti 2 6
Antonio Nanci 5
Sergio Caputi 3
Antonella Fontana 1
Emanuela Mazzon 2
Oriana Trubiani 0 4
0 Department of Medical, Oral and Biotechnological Sciences, University “G. d'Annunzio” , Via dei Vestini, 66100 Chieti , Italy
1 Department of Pharmacy, University “G. d'Annunzio” , Chieti , Italy
2 IRCCS Centro Neurolesi “Bonino Pulejo” , Messina , Italy
3 Department of Medical, Oral and Biotechnological Sciences, University “G. d'Annunzio” , Chieti , Italy
4 Department of Medical, Oral and Biotechnological Sciences, University “G. d'Annunzio” , Via dei Vestini, 66100 Chieti , Italy
5 Laboratory for the study of Calcified Tissues and Biomaterials, Department of Stomatology, Faculty of Dentistry, Université de Montréal , Montréal, Québec , Canada
6 Institute of Applied Science and Intelligent Systems “ISASI Eduardo Caianiello”, CNR , Messina , Italy
7 National Centre of Innovative Technologies in Public Health, Italian National Institute of Health , Rome , Italy
Background: The role of bone tissue engineering in the field of regenerative medicine has been a main research topic over the past few years. There has been much interest in the use of three-dimensional (3D) engineered scaffolds (PLA) complexed with human gingival mesenchymal stem cells (hGMSCs) as a new therapeutic strategy to improve bone tissue regeneration. These devices can mimic a more favorable endogenous microenvironment for cells in vivo by providing 3D substrates which are able to support cell survival, proliferation and differentiation. The present study evaluated the in vitro and in vivo capability of bone defect regeneration of 3D PLA, hGMSCs, extracellular vesicles (EVs), or polyethyleneimine (PEI)-engineered EVs (PEI-EVs) in the following experimental groups: 3D-PLA, 3D-PLA + hGMSCs, 3D-PLA + EVs, 3D-PLA + EVs + hGMSCs, 3D-PLA + PEI-EVs, 3D-PLA + PEI-EVs + hGMSCs. Methods: The structural parameters of the scaffold were evaluated using both scanning electron microscopy and nondestructive microcomputed tomography. Nanotopographic surface features were investigated by means of atomic force microscopy. Scaffolds showed a statistically significant mass loss along the 112-day evaluation. Results: Our in vitro results revealed that both 3D-PLA + EVs + hGMSCs and 3D-PLA + PEI-EVs + hGMSCs showed no cytotoxicity. However, 3D-PLA + PEI-EVs + hGMSCs exhibited greater osteogenic inductivity as revealed by morphological evaluation and transcriptomic analysis performed by next-generation sequencing (NGS). In addition, in vivo results showed that 3D-PLA + PEI-EVs + hGMSCs and 3D-PLA + PEI-EVs scaffolds implanted in rats subjected to cortical calvaria bone tissue damage were able to improve bone healing by showing better osteogenic properties. These results were supported also by computed tomography evaluation that revealed the repair of bone calvaria damage. Conclusion: The re-establishing of the integrity of the bone lesions could be a promising strategy in the treatment of accidental or surgery trauma, especially for cranial bones.
Human gingival mesenchymal stem cells; 3D scaffold; Extracellular vesicles; Bone regeneration
Bone defects are serious consequences of conditions
such as trauma, infection, surgical resections, and other
systemic problems that negatively affect the bone healing
]. In particular, calvarial bone lesions due to
accidental or surgery trauma represent a major and
difficult health concern in reconstructive surgery [
Indeed, it is well known that the spontaneous calvaria
regeneration occurs only in children less than 2 years
]. Thus, surgeons have been trying for many years
to restore functionality and the aesthetic appearance
using autografts, allografts, and even xenografts without
satisfactory outcomes [
]. Consequently, there has been
considerable effort towards developing new strategies to
improve bone growth, bone healing, and repair cranial
defects. In this context, tissue engineering has become a
promising approach for bone regeneration. In particular,
the use of scaffolds represents an integral part of bone
tissue engineering [
]. Scaffolds offer mechanical
support and three-dimensional (3D) support that favor cell
adhesion, migration, and differentiation in vivo [
However, to be used in the regeneration of tissues, these
devices must have some fundamental features including
biocompatibility, biodegradability, mechanical strength,
and matrix properties. Fiber and pore sizes may
influence some cellular responses, including migration,
proliferation, and differentiation [
]. For these reasons,
there is increasing interest in designing new biomaterials
that could be used in the form of scaffolds as bone
substitutes conceived to induce minimal or no immune
response and for encouraging implant/tissue interaction
]. The most used synthetic and biodegradable
scaffolds are poly(ε-caprolactone), poly(glycolic acid) and
poly(lactide) (PLA) scaffolds as well as their copolymers
]. Among these, PLA is widely used in the
regenerative medicine field due to its biodegradability,
biocompatibility, thermal plasticity, and suitable mechanical
Human dental mesenchymal stem cells (MSCs)
derived mainly from gingiva and periodontal ligament hold
great promise for bone regeneration owing to the less
invasive method for tissue explant collection and their
capacity to be a simple autologous MSC resource tool
]. These cells possess a high capacity for
expansion and the ability to differentiate into osteogenic cells
that can grow on biocompatible substrates [
therapeutic efficacy has been evaluated in regenerative
medicine relative to both dental and nondental
There are now considerable data supporting the
concept of paracrine signaling of extracellular vesicles (EVs)
as an important factor for stem cell therapy [
are small membrane vesicles containing functional
proteins, lipids, and nucleic acids, such as mRNA and
microRNA (miRNA), which are released by a variety of
cell types [
]. The soluble bioactive molecules present
in EVs directly activate target cells, inhibit apoptosis and
fibrosis, and stimulate tissue-intrinsic progenitor cell
]. Recently, Qin et al. demonstrated that
human MSC-derived EVs enter the osteoblasts and deliver
osteogenic miRNA by endocytosis, thus modulating
osteogenic gene expression. Therefore, human MSCs are
recognized as being able to promote bone regeneration in
Sprague-Dawley rats with calvarial defects [
] or fracture
healing in a mouse model [
], and are potently
]. Nevertheless, in these studies EVs were either
directly injected to the fracture [
], or applied using
hydrogel as a delivery system [
]. Only Xie et al. [
used a proper scaffold (decalcified bone matrix) to
properly deliver EVs.
Our aim was to evaluate the regenerative effects of
3D PLA scaffolds enriched with human gingival MSCs
(hGMSCs) and complexed with EVs as well as with
engineered EVs. In particular, we sought to engineer
EVs to improve the adhesiveness of EVs onto the 3D
PLA scaffold and to favor the intracellular release of
EVs content. We have directed our attention towards
the achievement of a coating of polyethyleneimine
(PEI), a biocompatible polymer that is known in
PEIcomplexed nucleic acids to induce osmotic swelling (i.
e., a proton-sponge effect) and promote the endosomal
content release without the need for an additional
endosomolytic agent [
]. Moreover, PEI has been
used for activating PLA scaffolds and has been
demonstrated to have an affinity for them [
our in vivo and in vitro analyses show that EVs and
PEI-EVs can be advantageously used with PLA
scaffolds to promote bone regeneration.
Scaffold development and three-dimensional scaffold constructs
The scaffold was obtained from a commercial poly(lactide)
(PLA; Keytech srl, Ancona, Italy). This material was
provided as a 1.75-mm diameter filament. Industrial-grade
PLA is generally a stereocopolymer with some percentage
of D-units in the poly (L-lactide) chain and has higher
molar mass distribution. This material is also formulated
with additives, such as a stabilizer and a nucleating agent,
the latter being important for processing and stability [
Technical specifications of the PLA filament are reported
in Table 1.
Different scaffold designs were investigated in this
study. The main differences were 3D structure, pore
size, and porosity. Height and diameter of the samples
were kept constant at 12 and 6 mm, respectively. The
different design characteristics for each sample are
reported in Fig. 1f. Each sample was developed with a
commercial CAD software (Rhinoceros 5, McNeel
Europe, Barcelona, Spain); the projects were then
applied to a printing slicing software (Cura 15.04, Ultimaker
B.V., Geldermalsen, The Netherlands). The sliced project
was finally transferred to a commercial fuse filament
fabrication 3D printer (DeltaWASP 2040; CSP srl, Massa
Structural scaffold evaluation
Scaffold scanning electron microscope characterization
A scanning electron microscope (EVO 50 XVP; Zeiss,
Jena, Germany) was used to image the surfaces of the
scaffold; the specimens were sputtered with 4–8 nm of
gold and then mounted on carbon tape dots [
In vitro osteogenesis performance
hGMSCs were seeded at 8 × 103 cells/cm2 in chemically
defined MSC growth medium (MSCGM-CD) (control
medium) (Lonza, Basel, Switzerland) and in osteogenic
differentiation medium (Lonza) in the presence of all
scaffold designs (Fig. 1a–e). To evaluate the performance of
different scaffold designs in terms of osteogenic
differentiation, the expression of RUNX2 after 1 week of
culture was performed by Western blotting analysis (Fig. 1g).
In vitro degradation
Scaffolds were immersed into 20 mL of
phosphatebuffered saline (PBS; 0.01 M, pH 7.4) and ascorbic acid
(0.01 M) to evaluate in vitro degradation as previously
reported. The function of the ascorbic acid was to
stabilize the scaffold degradation byproducts. Vials were
kept at 37 °C on a shaker table at 75 rpm. At each
evaluation time point (days 1, 7, 14, 28, 56, 84, and 112), pH
was measured, PBS was replaced, and scaffolds were
evaluated for residual mass after vacuum drying. To
evaluate mechanical and structural characteristics of the
degrading scaffolds, wet scaffolds were evaluated in
compression (n = 5); scaffold mean pore size, trabecular
thickness, and porosity were evaluated using
microcomputed tomography (mCT) at day 0 (n = 3).
Scaffolds from each group underwent compressive
mechanical testing at each degradation time point using
a universal testing machine (LR 30 K; Lloyd Instruments
Ltd., Bognor Regis, UK). At each time point, the
scaffolds were removed from PBS and immediately tested in
air except on day 0 (n = 5), when testing was performed
before immersion in PBS. For all tests the machine was
equipped with a 500 N load cell. Samples were loaded at
a crosshead speed of 0.5 mm/min and experimental data
were acquired every 10 ms. Yield strength and
compressive modulus were evaluated. Moreover, strength at 15%
strain was measured since no bending of the specimens
was observed until this strain value.
mCT was used to obtain noninvasive images of the
scaffolds. mCT analysis was performed on a Skyscan 1072
(Bruker MicroCT, Kontich, Belgium) operated at 70 kV/
200 mA, 0.45° rotation step, with a total rotation angle
Cell culture and derived extracellular vesicles
Written approval for gingival biopsy collection was
obtained from the Medical Ethics Committee at the
Medical School, “G. d’Annunzio” University, Italy, and each
participant gave informed consent. Gingival tissue
biopsies were obtained from healthy adult volunteers with no
gingival inflammation. The gingival specimens were
deepithelialized with a scalpel for the exclusion of most of
the keratinocytes resident in the gingival tissue [
brief, the connective tissue was grinded and then washed
several times with PBS (Lonza) and subsequently
cultured using TheraPEAK™MSCGM-CD™ BulletKit
serumfree, chemically defined (MSCGM-CD) medium for the
growth of human MSCs (Lonza) [
]. The medium was
changed twice a week, and cells spontaneously migrating
from the explant fragments after reaching about 80% of
confluence were detached using Triple Select (Lonza).
hGMSCs at passage 2 were stained with toluidine blue
and observed with a Leica DMIL10 (Leica Microsystem,
Milan, Italy) inverted light microscope; images were
captured using a Leica EC3 digital camera apparatus [
Scaffold cell performance evaluation
The scaffold morphology, pore size, and cellular
attachment were evaluated using scanning electron
microscopy (SEM). hGMSCs were seeded onto the 3D PLA
scaffold and incubated at 37° for 24 h. They were
subsequently fixed for 30 min with 2.5% (v/v) glutaraldehyde
in 0.1 M sodium cacodylate and dehydrated in a graded
series of ethanol. Afterward, the samples were mounted
onto metallic stubs with carbon tape and then
sputtercoated with gold using an Emitech K550 sputter coater
]. The design scaffold “C” (Fig. 1c) had been chosen
to be studied.
Cytotoxicity of degradation byproducts
The scaffolds were immersed in PBS (0.01 M, pH 7.4),
with 0.01 M of ascorbic acid, in a ratio of 6:100 scaffold
for in vitro degradation. An aliquot of PBS from
degrading PLA scaffolds of the various groups was removed at
days 1, 7, 14, 28, 56, and 112 to generate an extract for
cytotoxic evaluation of the degradation byproducts. The
cellular response to the eluates was evaluated using
Cells were plated and grown to 80% confluency before
initiating the assays. Once at confluence, the eluates
were combined with hGMSC cell culture media (in one
of three ratios: 1:99, 10:90, and 50:50) and added to
hGMSCs that were cultured on tissue culture
polystyrene well plates. The eluates and cell culture media
solutions were not further adjusted for pH or osmolarity.
Cultured cells were exposed to extract and media
solution at 37 °C and 5% CO2 for 24 h; the cytotoxicity was
then quantitatively evaluated with the MTT cell
metabolic activity assay. hGMSC culture media without
degradation byproducts was used as a control.
The viability of hGMSCs seeded with eluates
combined with MSCGM-CD was evaluated in three ratios: 1:
99, 10:90, and 50:50. The cellular response was
measured by the quantitative colorimetric MTT
test) (Promega, Milan, Italy) [
]. The cells cultured with
MSCGM-CD (Lonza) were used as a control. Cells (2 ×
103cells/well) were seeded into a 96-well culture plate
with MSCGM-CD medium (Lonza) and, after 24 h of
incubation at 37 °C, 15 μl/well MTT was added to the
culture medium and the cells were incubated for 3 h at 37 °
]. The supernatants were read at a 650-nm
wavelength using an ND-1000 NanoDrop Spectrophotometer
(NanoDrop Technologies, Rockland, DE, USA). The
MTT assay was performed in three independent
experiments, with six replicate wells for each experimental
hGMSC extracellular vesicles (EVs) isolation
The conditioned medium (CM; 10 mL) after 48 h of
incubation were collected from hGMSCs at passage 2. The
CM was centrifuged at 3000 × g for 15 min to eliminate
suspension cells and debris. For the EVs extraction we
used an ExoQuick TC commercial agglutinant (System
Biosciences, Euroclone SpA, Milan, Italy). Briefly, 2 mL
ExoQuick TC was added to 10 mL of conditioned
medium recovered from hGMSCs. The mix was
incubated overnight at 4 °C without rotation; one
centrifugation step was performed at 1500 × g for 30 min to
sediment the EVs and the pellets were resuspended in
200 μL PBS. The detection of EVs whole homogenate
protein was used as a confirmation of the presence of
release of EVs in hGMSCs.
Engineered EVs preparation
The EVs pellet (100 μL) was resuspended with 2 mL
PBS, and 2 mL of branched polyethyleneimine solution
(PEI; MW 25,000; Sigma-Aldrich, Milan, Italy) (0.05 mg/
mL in 0.3 M NaCl) was added to the EVs suspension in
PBS and the mixture was incubated for 20 min at room
temperature. The suspension was then centrifuged at
4000 rpm for 15 min and the supernatant was removed
to get rid of the excess PEI. The precipitate was
resuspended in 2 mL PBS. The engineered EVs (PEI-EVs)
suspension was characterized using dynamic light scattering
(DLS) experiments and ζ-potential measurements.
To evaluate cytotoxicity of the PEI-EVs on hGMSCs,
vesicles were incubated with Alexa Fluor 488 Wheat
Germ Agglutinin (Life Technologies, Milan, Italy) for
10 min at 37 °C and subsequently analyzed with confocal
laser scanning microscopy (CLSM; LSM510 META,
Zeiss) after 12 h of incubation.
Atomic force microscopy measurements
To evaluate EVs and PEI-EVs surface morphology,
atomic force microscopy (AFM) measurements were
performed using a Multimode 8 Bruker AFM
microscope with Nanoscope V controller (Bruker AXS, Marne
La Vallee, France). It is worth highlighting that AFM
analyses were performed to visualize the prevailing
smallest exosomal objects since it is very difficult to visualize
very irregular micrometric surfaces such as those that
one could expect for aggregated microvesicles.
Nevertheless, several authors have already used this technique
to visualize EVs, thus exploiting the mild experimental
conditions under which it is possible to visualize them
and avoiding the high vacuum of transmission electron
microscopy measurements [
]. Silicon cantilever and a
RTESPA-300 tip (spring constant = 40 N/m and resonant
frequency 300 Hz) were used in a tapping in air mode.
The specimen was prepared by dropping a solution of
EVs and PEI-EVs on a SiO2 wafer followed by air drying
at 37 °C for 1 h. The solutions of EVs and PEI-EVs
dropcasted onto SiO2 water had a different concentration
because we wanted to avoid the formation of big aggregates,
particularly for the less charged, and consequently more
prone to aggregate, PEI-EVs.
Dynamic laser light scattering
EVs dispersions were characterized using DLS
experiments and ζ-potential measurements using a
Brookheaven Zeta Plus.
In vitro osteogenesis performance
hGMSCs were seeded at 8 × 103 cells/cm2 in
MSCGMCD culture medium (control medium) (Lonza) and in
the presence of 3D-PLA, 3D-PLA + EVs, and 3D-PLA +
PEI-EVs. Scaffolds were pretreated for 48 h under
agitation (MacsMix, Milthenyi, Bologna, Italy) with 5 mL
PBS of EVs and PEI-EVs. Evaluation of calcium
deposition and extracellular matrix (ECM) mineralization was
obtained by Alizarin Red S staining assay performed
after 6 weeks. Cells were washed with PBS, fixed in 10%
(v/v) formaldehyde (Sigma-Aldrich) for 30 min, and
washed twice with abundant dH2O prior to the addition
of 0.5% Alizarin Red S in H2O, pH 4.0, for 1 h at room
temperature. After cell incubation under gentle shaking,
cells were washed with dH2O four times for 5 min. For
staining quantification, 800 μL 10% (v/v) acetic acid was
added to each well. Cells incubated for 30 min were
scraped from the plate, transferred into a 1.5-mL vial,
and vortexed for 30 s. The obtained suspension, overlaid
with 500 μL mineral oil (Sigma-Aldrich), was heated to
85 °C for 10 min and then transferred to ice for 5 min,
carefully avoiding opening of the tubes until fully cooled,
and centrifuged at 20,000 × g for 15 min. Then 500 μL
of the supernatant was placed into a new 1.5-mL vial
and 200 μL of 10% (v/v) ammonium hydroxide was
added (pH 4.1–4.5); 150 μL of the supernatant obtained
from cultures were read in triplicate at 405 nm by a
spectrophotometer (Synergy HT, BioTek, Bad
Friedrichshall, Germany) [
Purified EVs were treated as previously described to protein
]. Proteins were extracted from EVs and
PEIEVs, from hGMSCs, 3D-PLA+ hGMSCs, 3D-PLA + EVs +
hGMSCs, and 3D-PLA + PEI-EVs + hGMSCs after 6 weeks
of culture. Proteins were separated on sodium dodecyl
sulfate-polyacrylamide minigels and transferred onto PVDF
membranes (Immobilon-P Transfer membrane, Millipore,
Billerica, MA, USA), blocked with PBS containing 5%
nonfat dried milk (PM) for 45 min at room temperature,
and subsequently probed at 4 °C overnight with specific
CD9 (1:500; Santa Cruz Biotechnology Inc., Santa Cruz,
CA, USA), CD63 (1:500; Abcam, Cambridge, UK), CD81
(1:500; Santa Cruz), and TSG101 (1:500; Santa Cruz) for
EVs, RUNX2 (1:500; Santa Cruz), BMP2/4 (1:500; Santa
Cruz), and β-Actin (1:750; Santa Cruz) for hGMSCs,
3DPLA + hGMSCs, 3D-PLA + EVs + hGMSCs, and 3D-PLA
+ PEI-EVs + hGMSCs in 1× PBS, 5% (w/v) nonfat dried
milk, 0.1% Tween-20 (PMT). Horseradish peroxidase
(HRP)-conjugated goat anti-mouse IgG was incubated as a
secondary antibody (1:2000; Santa Cruz) for 1 h at room
]. The relative expression of protein bands
was visualized using an enhanced chemiluminescence
system (Luminata Western HRP Substrates, Millipore) and
protein bands were acquired and quantified with the
ChemiDoc MP System (Bio-Rad, Hercules, CA, USA) and a
computer program UVIband-1D gel analysis software
(Uvitec, Cambridge, UK), respectively.
Total RNA was isolated from hGMSCs, 3D-PLA + EVs
+ hGMSCs, and 3D-PLA + PEI-EVs + hGMSCs cultured
for 1 week using the Total RNA Purification Kit (Norgen
Biotek Corp., Ontario, CA, USA) according to the
manufacturer’s protocol. Total RNA was quantified by means
of the BioSpectrometer (Eppendorf, Milan, Italy) using
μCuvette G1.0 (Eppendorf ).
To analyze the osteogenic differentiation ability, RUNX2
and BMP2/4 markers were evaluated by means of
RTPCR as previously reported by Diomede et al. [
RNA sequencing and library preparation
To prepare RNA sequencing libraries the TruSeq RNA
Access library kit (Illumina, Inc., San Diego, CA, USA)
was used according to the manufacturer’s instructions.
Briefly, 50 ng of RNA from each sample was fragmented
at 94 °C for 8 min. In a first strand phase, the cDNA
was synthesized by using random hexamers and the
SuperScript II Reverse Transcriptase (Invitrogen, Milan,
Italy) at 25 °C for 10 min, 42 °C for 15 min, and 70 °C
for 15 min.
In a second strand of cDNA synthesis, the RNA
templates were removed and a second replacement strand
was generated by dUTP internalization to produce
double-strand cDNA. Afterwards, to purify the
bluntended cDNA, AMPure XP beads (Beckman Coulter,
Brea, CA, USA) were used. The 3′ ends of the cDNA
were adenylated to permit the adaptor ligation in the
subsequent step. After that, the libraries were purified
with AMPure XP beads. A first PCR amplification step
was performed to enrich those fragments of DNA that
have adaptors on both ends, and also to enhance the
quantity of DNA in the library (15 cycles of 98 °C for
10 s, 60 °C for 30 s, and 72 °C for 30 s). The library has
been validated using the Agilent Technologies 2100
Bioanalyzer. After that, 200 ng of each DNA library was
combined and the first hybridization step was performed
using exome capture probes according to a standardized
protocol (18 cycles, starting at 94 °C, and then
decreasing by 2 °C for every cycle). To eliminate nonspecific
binding, magnetic beads coated with streptavidin were
used to capture probes hybridized to the target regions.
Another capture round with streptavidin-coated beads
was performed, followed by two heated washes to
discharge the nonspecific binding from the beads. Then,
the enriched libraries were eluted from the beads and
were ready for a second cycle of hybridization. This
hybridization step was necessary to obtain a wide
specificity of regions of capture. After that, the libraries were
purified through the AMPure XP bead, and amplified
according to the protocol (10 cycles; incubation at 98 °C
for 10 s, incubation at 60 °C for 30 s, and incubation at
72 °C for 30 s), followed by a purification step. Libraries
were quantified by the qPCR using KAPA Library
Quantification Kit—Illumina/ABI Prism_ (Kapa Biosystems,
Inc., Wilmington, MA, USA) and certified with the
Agilent High Sensitivity Kit on a bioanalyzer. The size of
the DNA fragments has been set in a range 200–650 bp
and peaked around 250 bp. Libraries were normalized to
12pM and subjected to cluster, and single read
sequencing was executed for 150 cycles on a MiSeq instrument
(Illumina) following the protocol guidelines. The
produced libraries were loaded for clustering on a MiSeq
Flow Cell v3 and then sequenced with a MiSeq
Instrument (Illumina) [
]. The cluster density validation had
been executed by the software of the instrument
throughout the run.
Next-generation sequence data processing
Data obtained by next-generation sequencing (NGS)
analysis were processed. Specifically, the sequence reads
were subjected to the demultiplexing process to have a
separation of the sequence reads in different files for
each index tag/sample by using the CASAVA algorithm
(CASAVA, version 1.8.2; Illumina, Inc., San Diego, CA,
USA). Then, for the alignment of sequences, the
RNASeq Alignment version 1.0.0 (Illumina) and the reference
sequence “Homo sapiens UCSC hg19” for the read
mapping the TopHat 2 (Bowtie 1) were used. The fragments
per kilobase of exon per million fragments mapped
(FPKM) values were calculated for each sample using
the normalized read counts for each annotated gene:
([1000 × read count] / [number of gene covered bases ×
number of mapped fragments in million]). Unmapped
reads were deleted, preserving only read pairs with both
reads aligned to the reference sequence “Homo sapiens
UCSC hg19.” The comparison between two different
specimens was performed by a scatter plot of the log2 of
Statistical analysis was accomplished using analysis of
variance (ANOVA) and Tukey’s post-hoc analysis (p < 0.
05). The statistical data on the read counts were carried
out with the Cufflinks Assembly&DE package version 2.
0.0 to establish the proportion of differentially expressed
genes for a q value < 0.05. The gene ontology (GO)
analysis of the genes differentially expressed between
experimental groups were performed by the free tools “Gene
Ontology Consortium” (available online at http://www.
Male Wistar rats weighing 300–350 g were used for this
experiment. Animals were acquired from Harlan, Milan,
Italy, and housed in individually ventilated cages and
maintained under 12-h light/dark cycles at 21 ± 1 °C and
50–55% humidity with food and water ad libitum.
To implant the scaffold, rats were first anesthetized with
a combination of tiletamine and xylazine (10 mL/kg,
intraperitoneally). Afterwards, the implant site was
prepared with iodopovinone (Betadine) after trichotomy.
Following a median sagittal incision of about 2.5 cm
from the occipital region, a total thickness cut was
applied; the calvaria was then exposed in the frontal area
and in the parietal areas. The circular section bone
receiving site, with a diameter of 5 mm and a height of 0.
25 mm, was injured by means of a rotary instrument at
a controlled speed (trephine milling machine, Alpha
BioTec, HTD Consulting S.r.l., Siena, Italy) under constant
irrigation of a physiological solution.
For their texture and flexibility, 3D-PLA, 3D-PLA +
hGMSCs, 3D-PLA + EVs, 3D-PLA + PEI-EVs, 3D-PLA +
EVs + hGMSCs, and 3D-PLA + PEI-EVs + hGMSCs were
easily inserted in contact with bone tissue to cover the
damaged area. The skin flap was then sutured with
Caprosyn 6-0 synthetic monofilament adsorbable sutures
(Covidien AG, Neuhausen am Rheinfall, Switzerland)
using interrupted points. Standard feeding and hydration
were maintained as a constant throughout the
The design scaffold “C” was chosen to be implanted in
the host tissue.
Rats were randomly distributed into the following
groups (n = 24 total animals):
1. 3D-PLA (n = 4): rats subjected to scraping of the
cortical calvarial bone tissue and implant of 3D-PLA;
2. 3D-PLA + hGMSCs (n = 4): rats subjected to
scraping of the cortical calvarial bone tissue and
implant of 3D-PLA + hGMSCs;
3. 3D-PLA + EVs (n = 4): rats subjected to scraping of
the cortical calvarial bone tissue and implant of
3D-PLA + EVs;
4. 3D-PLA + EVs + hGMSCs (n = 4): rats subjected to
scraping of the cortical calvarial bone tissue and
implant of 3D-PLA + EVs + hGMSCs;
5. 3D-PLA + PEI-EVs (n = 4): rats subjected to scraping
of the cortical calvarial bone tissue and implant of
3D-PLA + PEI-EVs.
6. 3D-PLA + PEI-EVs + hGMSCs (n = 4): rats subjected
to scraping of the cortical calvarial bone tissue and
implant of 3D-PLA + PEI-EVs + hGMSCs.
After 6 weeks the animals were euthanized by
intravenous administration of Tanax (5 mL/kg body weight)
and their calvariae were processed for morphological
For each experiment we used 2 × 106 hGMSCs stained
with PKH-26 and EVs and PEI-EVs stained with
PKH67, according to the Sigma procedures.
The specimens were fixed for 72 h in 10% formalin
solution, dehydrated in ascending graded alcohols, and
embedded in LR White resin (Sigma-Aldrich). After
polymerization, undecalcified oriented cut sections of
50 μm were prepared and ground down to about
30 μm using the TT System (TMA2, Grottammare,
The sections were analyzed before staining with CLSM
(LSM510 META, Zeiss) and, after a double-staining
procedure with methylene blue and fuchsin acid solutions,
they were observed under a light microscope [
The investigation was carried out by means of a
bright-field light microscope (Leica Microsystem)
connected to a high-resolution digital camera DFC425B
Leica (Leica Microsystem). Histomorphometry, shown as
the percentage of the newly formed bone, was carried
out using a digitizing pad (Matrix Vision GmbH,
Oppenweiler, Germany) and a histometry software package
with image capturing capabilities (Image-Pro Plus 4.5,
Media Cybernetics Inc., Immagini & Computer Snc,
Three-dimensional reconstruction was obtained by
means of ZEN2 software (Zeiss). Data and statistical
analysis were performed with the Statistical Package for
Social Science (SPSS, v.21.0, IBM Analytics, Armonk,
To confirm the presence of hGMSCs in the sample
groups 3D-PLA + hGMSCs, 3D-PLA + EVs + hGMSCs,
and 3D-PLA + PEI-EVs + hGMSCs, the sections were
rehydrated for 30 min and subsequently stained with
human anti-ANA (1:200; Merck Millipore, Milan, Italy)
and observed with CLSM (LSM800, Zeiss).
Computed tomography (CT)
CT was used to evaluate the bone repair. CT analysis of
calvariae was performed on a Siemens Somatom
Definition AS operated at 70 kV/350 mA. The thickness of
acquisition was 0.6 mm with a range of reconstruction of
3D PLA scaffold characterization
The 3D design of the five different generated scaffolds are
shown in Fig. 1a–e. Surface morphology, pore and
trabecular dimensions, and total porosity of the 3D printed
scaffolds were characterized using SEM (Fig. 1a1–e1) and
mCT (Fig. 2). During printing, the scaffolds exhibited a
reduction in real pore size and trabecular separation
compared to their design, resulting in a concurrent reduction
in total porosity. There was also a reduction compared to
planned values for design D and E (Fig. 2). Furthermore,
Fig. 2 Microcomputed tomography three-dimensional rendering and evaluation of porosity, pore size, and wall thickness at different degradation
time point. Scaffolds with a design A, b design B, c design C, d design D, and e design E at T0, T14, and T28. mCT values at T0 for porosity, trabecular
thickness (TT), and trabecular separation (TS)
scaffolds with larger filament/pore dimensions showed a
mild layering within each trabecular area (Fig. 1f ). Specific
bands of RUNX2 were present in all samples of different
design, while a higher expression of osteogenic-related
marker is clearly visible in hGMSCs cultured with design
scaffold C (Fig. 1g).
3D PLA in vitro degradation
Degradation in ascorbic acid solution was measured at
day 112 by the following changes in mass and pH. The
lowest values in pH were recorded at day 112 for all
groups (Fig. 3a). Mass loss increased throughout the study,
with the biggest increase after 28 days (Fig. 3b). The
various scaffold designs generally maintained their
compressive mechanical properties during degradation in vitro.
For strength at 15% of strain at T0, design E had
significantly lower values compared with the other four groups,
while design D values were statistically higher than those
obtained for designs B, C, and E. On the other hand,
design A preserved a significantly higher strength at the end
of degradation compared with designs B and C at the
same time point. Designs A and C showed the best
performances in terms of strength after degradation (Fig. 3c). In
terms of yield strength at T0, design D showed
significantly higher values compared with designs B, C, and E;
specimens from design E had significantly lower values
compared with the other four scaffolds. On the other
hand, design B showed a decrease in terms of yield
strength (Fig. 3d). As far as the compressive modulus at
T0 is concerned, design D showed the highest values, with
a statistically significant difference compared with design
C. After 112 days, this group also showed a significant
difference compared with design B that showed a decrease in
compressive modulus (Fig. 3e).
MTT evaluation of cytotoxicity of degradation byproducts
The metabolic activity of cells exposed to the extract of
degrading PLA scaffolds was statistically different from
the control group for all PBS extract concentrations,
scaffold design, and degradation times. However, designs
A and C showed no difference in terms of cell metabolic
activity between T1 and T112, irrespective of extract/cell
ratio. Designs B, D, and E showed a difference between
T1 and T112 for all the ratios. Positive peaks in cell
activity within a degradation time point could not be
confirmed for the three extract/cell ratios used, as reported
in more detail by MTT graphs (see Additional file 1).
3D PLA and hGMSC interaction
Since synthesis design C demonstrated the best features in
terms of mechanical and chemical properties, this was
used to analyze the interaction of hGMSCs with 3D PLA.
Morphological analysis by SEM demonstrated numerous
extensions of cytoplasmic processes which enabled
cellular anchorage (see Additional file 2, section A). Cells
spread and extended on the uneven surface and across the
filaments to create extended contact areas between them,
organizing a multilayer covering the 3D substrate. The
biomaterial surface covered with hGMSCs is evident when
compared with 3D PLA without cells (see Additional file 2,
inset in section A). At high magnification, an increased
number of cellular bridges were demonstrated (see
Additional file 2, section B). The scaffold material did
not induce visible changes in cellular morphology.
EV and PEI-EV characterization
The DLS analysis shows the presence of a heterogeneous
population of EVs, spanning from 100 to 1200 nm. In
particular, two main dimensional populations could be
identified, the average diameter of the first population
being 93 ± 24 nm and that of the second population
being 1200 ± 400 nm (Fig. 4b, e1). Both populations
increased in dimension after the addition of PEI, with the
final size being 250 ± 50 nm and 3600 ± 500 nm,
respectively (Fig. 4b, e2). The EV suspension had a ζ-potential
of −10.7 ± 0.9 mV, whereas after the addition of the
cationic polyelectrolyte PEI, there was an increase to −1.2
± 0.9 mV coating (Fig. 4b). Although these size increases
are particularly high for a simple coating of PEI, they
also show the occurrence of a PEI coating. Indeed, the
decrease in ζ-potential, consequent to the PEI adsorption
(see below), may favor the formation of small aggregates.
EVs and PEI-EVs were also analyzed by AFM. Figure
4a1 highlights the presence of a large number of
globular EVs of different dimensions with a central depression,
thus confirming previous reports on the shape of EVs
] as well as DLS data. Some debris or aggregated
vesicles were also observed. Very interestingly, the surface of
EVs appeared relatively smooth. On the other hand, the
specimen of PEI-EVs (Fig. 4a2) shows objects of
homogeneous dimensions, with no central depression, and
characterized by a less smooth surface with respect to
pure EVs; likely these latter findings confirm the
adsorption of PEI onto the EVs surface.
hGMSCs were incubated with EVs and PEI-EVs stained
with WGA Alexa Fluor 488. After 2 days, CLSM images
were captured and the viability was estimated to be in the
range 80–90%. Very interestingly, the number of vesicles
in the cells is higher when the PEI-EVs (Fig. 4d) rather
than nonengineered EVs are used (Fig. 4c), indicating a
higher capacity of EVs to enter the cells when coated. At
high magnification, the vesicles in the cytoplasmic
compartment are clearly visible (Fig. 4c, d). Western blot
analysis performed on EVs showed a positivity for CD9,
CD63, CD81, and TSG101 molecules (Fig. 4f ).
Low-magnification photographs were used to verify the
Alizarin Red S staining (Fig. 5a); meanwhile, light
microscopy imaging was used to highlight cell osteogenic
differentiation at high magnification under four different
culture conditions (Fig. 5b). The best results in terms of
production of calcium deposits were shown by hGMSCs
cultured in the presence of 3D-PLA + EVs and 3D-PLA
Data were quantified using spectrometric analysis after
6 weeks of osteogenic induction (Fig. 5c). RT-PCR was
performed to analyze changes in RUNX2 and BMP2/4
gene expression in all groups after 6 weeks of culture.
We observed increases in RUNX2 and BMP2/4 mRNA
expression in the 3D-PLA + EVs and 3D-PLA + PEI-EVs
group, respectively, which were significantly higher than
the control group (Fig. 5d). Western blotting indicated
consistent findings for RUNX2 and BMP2/4 protein
expression (Fig. 5e).
The transcriptome of hGMSCs, 3D-PLA + EVs + hGMSCs,
and 3D-PLA + PEI-EVs + hGMSCs was investigated using
high-throughput sequencing with an Illumina MiseqDx.
Statistical analysis revealed that 31 genes were differentially
expressed between the examined groups. Specifically, the
analysis of these genes expressed among hGMSCs,
3DPLA + EVs + hGMSCs, and 3D-PLA + PEI-EVs + hGMSCs
was performed by inserting the selected genes on the online
database “Gene Ontology Consortium” identify a putative
GO class. According to GO analysis, 31 genes involved
in “regulation of ossification” and “ossification” were
upregulated in the 3D-PLA + PEI-EVs + hGMSCs group
compared to the hGMSCs group (false discovery rate
(FDR) = 8.14 × 10−35). Moreover, among these genes,
16 genes were upregulated in the 3D-PLA + EVs +
hGMSCs group compared with the hGMSCs group
(FDR = 3.03 × 10−15) (see Additional file 3A and
Additional file 4: Table S1).
GO analysis also showed that 19 genes belonging to the
family GO “regulation of osteoblast differentiation” and
“osteoblast differentiation” were upregulated in the
3DPLA + PEI-EVs + hGMSCs group compared with the
hGMSCs group (FDR = 2.56 × 10−15). Among these genes,
10 genes were upregulated in the 3D-PLA+ EVs +
hGMSCs group compared with the hGMSCs group (FDR
= 4.42 × 10−14) (see Additional file 3B and Additional file 4:
Table S2). Finally, the comparison between the two big GO
families (ossification and osteoblast) showed 19 genes that
were common to the both families.
Moreover, 9 genes reported to be upregulated during
osteogenesis were more expressed in 3D-PLA + PEI-EVs +
hGMSCs compared with hGMSCs [
] (see Additional file 4:
Table S3 and Additional file 5). Furthermore, some genes
reported to be involved in osteoblast differentiation
through transforming growth factor (TGF)-β signaling
were upregulated in 3D-PLA + PEI-EVs + hGMSCs
(TGFBR1, SMAD1, BMP2, MAPK1, MAPK14, and
] (see Additional file 4: Table S3 and
Additional file 5).
In addition, gene expression profiles of adhesion
molecules and ECM were evaluated in the 3D-PLA + PEI-EVs
+ hGMSCs group compared with control cells by NGS
analysis. There were a total of 20 differentially expressed
genes, including 9 upregulated genes and 11
downregulated genes (see Additional file 4: Table S4 and
Additional file 6). As shown in Additional file 6,
genes that encode integrin, basement membrane
laminins, membrane proteins that mediate
cell-tocell and cell-to-matrix interactions, and inhibitor of
the matrix metalloproteinases were upregulated in
3D-PLA + PEI-EVs + hGMSCs. In parallel, genes that
encode for cell-cell adhesion and for the ECM
constituents of basement membranes were
downregulated in 3D-PLA + PEI-EVs + hGMSCs.
3D PLA in vivo evaluation
Overall, the scaffolds harvested after 6 weeks of
implantation in calvaria of rats contained 3D PLA scaffold
infiltrated or not with hGMSCs. Histological samples after
staining with acid fuchsin and methylene blue solution
showed a different response to various substrates. In
3DPLA samples, ECM without signs of mineralization was
observed (Fig. 6a), while in 3D-PLA + hGMSCs samples
the integration process had started, with deposition of
new ECM (Fig. 6b).
More host tissue ingrowth in the implant site was
observed in 3D-PLA + EVs and 3D-PLA + EVs + hGMSCs
when compared with the 3D-PLA and 3D-PLA +
hGMSCs (Fig. 6).
Abundant ECM and nodules of new bone formation
stained with acid fuchsin were present in both samples,
while blood vessels formation was visible in 3D-PLA +
EVs samples (Fig. 6d) when compared with 3D-PLA +
EVs (Fig. 6c).
In samples grafted with 3D-PLA + PEI-EVs and
3DPLA + PEI-EVs + hGMSCs there was a significant
difference in host tissue response. New bone deposition and
ECM areas, blood vessels formation, and osteoblast-like
cells are valuable in bone defects grafted with 3D-PLA +
PEI-EVs + hGMSCs (Fig. 6f ). In the 3D-PLA + PEI-EVs
samples, numerous bone nodules were visible as well as
various blood vessels of different dimensions indicating
a new vascular network formation (Fig. 6e).
CT evaluation showed that bone damage was still
present in the 3D-PLA, 3D-PLA + hGMSCs, and
3DPLA + EVs samples. However, in the 3D-PLA + EVs +
hGMSCs, 3D-PLA + PEI-EVs, and 3D-PLA + PEI-EVs +
hGMSCs samples, the complete repair of the calvarial
defect was visible (Fig. 7).
Histomorphometry analysis showed that newly formed
bone represented 12.27%, and the total surface area
constituted by ECM and biomaterial residual graft material
87.72%, as reported in Table 2.
To demonstrate the presence and the ability for bone
regeneration of hGMSCs and EVs in the host tissue, cells
were stained with PKH-26 and EVs or PEI-EVs were
(See figure on previous page.)
Fig. 6 Histological evaluation. Samples harvested at 6 weeks after the calvarial defects were transplanted with a 3D-PLA scaffold or b 3D-PLA +
human gingival mesenchymal stem cells (hGMSCs). Left panels (1): The images at low magnification (4×) showed 3D-PLA and 3D-PLA + hGMSCs
integrated smoothly with the host tissue. Middle panels (2): High-magnification images (10×) showing the contact area between 3D-PLA and
3D-PLA + hGMSCs with bone calvaria grafted at 6 weeks postsurgery. Right panels (3): Images obtained at 40× objective showing the connective
tissue between 3D-PLA and 3D-PLA + hGMSCs and bone host tissue. Samples harvested at 6 weeks after the calvarial defects was transplanted
with c 3D-PLA + extracellular vesicles (EVs) scaffold or d 3D-PLA + EVs + hGMSCs. Left panels (1): The images at low magnification (4×) showed
3D-PLA + EVs and 3D-PLA + EVs + hGMSCs integrated smoothly with the host. Middle panels (2): High-magnification images (10×) showing the
new bone formation stained with acid fuchsin in both samples grafted at 6 weeks postsurgery. Right panels (3): Images obtained at 40× objective
showed a zone with new mineralized matrix inside 3D PLA scaffold for both samples. In particular, in the 3D-PLA + EVs (c3) sample in the contact
zone some blood vessels are valuable. Samples harvested at 6 weeks after the calvarial defects was transplanted with e 3D-PLA + polyethyleneimine
(PEI)-EVs scaffold or f 3D-PLA + PEI-EVs + hGMSCs. Left panels (1): The images at low magnification (4×) showed 3D-PLA + PEI-EVs and 3D-PLA +
PEIEVs + hGMSCs integrated smoothly with the host. Middle panels (2): High-magnification images (10×) showing the new bone formation stained with
acid fuchsin in both samples grafted at 6 weeks postsurgery. Right panels (3): Images obtained at 40× objective showed new bone formation inside
the scaffold structure. In particular, in 3D-PLA + PEI-EVs there were numerous blood vessels present around the new bone deposition area. Scale bars
= 10 μm. *, scaffold; V, vessels; B, new bone. Acid fuchsin-toluidine blue staining
stained with PKH-67. Confocal analysis further
demonstrated the presence of hGMSCs grafted with the
3DPLA (Fig. 8d1, e1, f1), of EVs (Fig. 8b1, e1) and of
PEIEVs (Fig. 8c1, f1).
3D reconstruction of each microscopy image showed
the spatial organization between the scaffold, cells, and
EVs or PEI-EVs in the grafted site (Fig. 8a2–f2).
Human GMSCs nuclei labeled with human anti-ANA
green fluorescent conjugate are clearly visible in the rat
calvaria, confirming the presence of living transplanted
cells (Fig. 9).
Three-dimensional printing now allows fabrication of
complex scaffold designs with different
interconnections, porosity, and pore shape that were previously
difficult to build [
]. In this study, five innovative
original designs of 3D-PLA scaffolds have been
printed and their filament/pore sizes have been
characterized. The broad range of tested porosities
represents the novelty of this work. These scaffolds were
then doped with hGMSCs-derived extracellular
vesicles and tested for their ability to regenerate bone
defects induced in rat calvaria.
In addition, absorbable polymers for bone tissue
engineering must ensure mechanical stability while degrading,
thus keeping defect site stability during bone regeneration
]. We examined here which 3D-PLA printed
porous scaffold designs provided the best mechanical stability
over time during their degradation.
Early investigations on 3D-PLA scaffolds suggested an
ideal pore size in the 100-μm range, based on cell size.
Subsequent studies suggested that larger pores (100–
350 μm) improve cell migration and blood vessel
]. SEM examination revealed that the
3D PLA printed scaffolds showed larger filaments and
reduced pore size with respect to the features of the
PLA polymer during extrusion and subsequent cooling.
As a consequence, the scaffolds produced in this study
should show adequate cellular migration and provide
nutrients after in vivo implantation .
Since the influence of nanoscale topography is crucial
for bone substitute biomaterials, its role on our 3D-PLA
scaffolds must be considered [
]. The scaffold
degradation was revealed by mass loss and decrease of extract
pH, and tested at increasing times over a 122-days
period. At the end of the study, the mass was
significantly reduced in all groups. The pH reduction
confirmed the lactide loss [
Noteworthy, the degradation did not have a major
impact on the mechanical properties over the 112-days
Scaffold-host tissue compatibility is a fundamental
characteristic. Therefore, when using a degradable material its
degradation byproducts must not induce a cytotoxic
response. This work evaluated the potential cytotoxicity
elicited during the absorption of PLA. Lactic acid is one of
the PLA hydrolytical degradation byproducts [
]. This is
a key point, as one indication of degradation of polyesters
is a decrease in pH as seen in this study. The reduced cell
metabolic activity after incubation with the eluates was
statistically lower than control cells; however, it must be
considered that this result was not influenced by the
increasing degradation time for scaffold design C. The slow
absorption of the tested PLA allowed for mechanical
stability over the estimated bone formation and maturation
time. Moreover, this pattern reduced the cytotoxicity
potential due to slow release of acid. The slow absorption
rate and host metabolization of lactic acid are
fundamental for a low cytotoxic response in vivo. For PLA in vivo,
the acidic byproduct is lactic acid and it would be
metabolized through the Cori cycle; this process cannot be
replicated in vitro [
]. Eventually, our study demonstrated
that the increase of PLA byproducts over time did not
induce an increased cytotoxic response for design C and
SEM evaluation of 3D-PLA + hGMSCs constructs
demonstrated good anchorage, morphological characteristics,
and bridging of the cultured cells. Moreover, the
biochemical analysis showed an increased expression of RUNX2 in
MSCs have been used for autologous therapy in
combination with platelet-rich plasma and/or scaffolds in
distraction osteogenesis [
]. Among the various tissues from
which MSCs can be isolated, growing attention has been
paid to dental tissues including periodontal ligament, dental
pulp, and gingiva owing to the minimally invasive
procedure involved in their collection, and remarkable
differentiation ability towards neurogenic and other cells .
Recently, animal data confirm that periodontal ligament
stem cells seeded onto 3D scaffolds in the mouse calvaria
undergo precocious osteointegration and vascularization
]. This effect may be due to the ability of the stem
cells to undergo osteogenic differentiation, but also to their
immunomodulatory and anti-inflammatory properties.
In particular, several findings suggest that, in general,
MSCs exert their action mainly through paracrine
signaling by EVs and their soluble secretory products, also
called a ‘secretome’, containing a pool of soluble
]. Released membrane vesicles from eukaryotic
cells, as exosomes, microparticles, microvesicles, and
apoptotic bodies, can be retained as a dynamic
extracellular vesicular compartment, strategic for their paracrine or
autocrine biological effects in tissue metabolism [
are characterized by the presence of specific
membraneassociated proteins, such as CD9, CD63, CD81, and tumor
suppressor gene 101 (TSG101) [
In vitro, mineralizing osteoblast exosomes are capable
of entering into bone marrow stromal cells to induce
them to the osteoblast phenotype, with trough
upregulation of b-catenin in recipient cells representing a
potential therapeutic approach [
]. Recently, it has also been
demonstrated that the beta tricalcium phosphate
(βTCP) scaffold functionalized with human-induced
pluripotent stem cell-derived MSCs promotes bone repair
and regeneration in a rat model of calvarial bone defects
]. The underlying mechanism through which
exosomes enhance the osteoinductive activity of β-TCP and
promote bone regeneration could be due to the
activation of endogenous bone marrow MSCs in the bone
defect site. This was suggested by the observation that
exosomes could be released from the exosome/β-TCP
complex and then be internalized by bone marrow
To improve the internalization and performance of the
EVs we have complexed them with PEI. As far as
PEIengineered EVs are concerned, many factors such as
molecular weight, degree of branching, zeta potential,
particle size, cationic charge density, molecular structure,
sequence, and conformational flexibility have all been
shown to affect PEI efficiency and cytotoxicity when
used as a transfecting agent [
]. We found that the best
compromise between effectiveness and toxicity was a
concentration of 0.05 mg/mL PEI that conferred to EVs
a slight increase in zeta-potential from −10.7 ± 0.9 mV
to −1.2 ± 0.9 mV with a tolerable viability of the
recipient cells (5–10%). The improved internalization of
PEIEVs with respect to EVs may be ascribed to the capacity
of PEI, being a cationic species, to favor internalization
via proteoglycan binding [
]. The subsequent
internalization mechanisms proposed for EVs are attachment or
fusion with the target cell membrane, delivering
exosomal proteins to the recipient cell [
internalization by the recipient cells by mechanisms such as
endocytosis . In the present study, labeled PEI-EVs
were demonstrated to be internalized mainly by
endocytosis, being highly represented after incubation in the
recipient cells. The “proton sponge” effect that characterize
PEIs is then essential for the endosomal content escape
ability and could be related to the better efficiency of
PEIEVs as compared with nonfunctionalized EVs [
Our report based on in vitro and in vivo functional
studies focuses on the behavior of the living construct
constituted from 3D-PLA + hGMSCs and engineered or
nonengineered EVs. Alizarin Red S staining showed that
after 6 weeks of culture calcium depositions are more
evident in the presence of 3D-PLA and EVs or PEI-EVs.
In fact, mounting evidence suggests that EVs alone are
capable of promoting proliferation and migration of cells
as well as osteogenesis and angiogenesis [
transcriptome profile of genes involved in osteoblast
differentiation and ossification in a 3D-PLA living construct
enriched with EVs and PEI-EVs was examined by the
NGS platform. Our results showed that 31 genes were
differentially expressed between the examined groups.
Specifically, the GO analysis demonstrated that
biomaterial enriched with PEI-EVs + hGMSCs induced
upregulation of all 31 identified genes involved in the regulation
of ossification as well as in the ossification processes
(FHL2, BMP2, TWSG1, CCDC47, FAM20C, ERCC2,
LEP, TOB2, IMPAD1, CHRDL1, MINPP1, HIRA,
MYBBP1A, JAG1, MEF2C, SUCO, SFRP1, SOX9, SIX2,
RHOA, PDLIM7, IFT80, SMAD1, HDAC7, ASF1A, ID3,
SNAI1, PEX7, RPL38, BMP2K, and BCAP29) when
compared with hGMSCs.
Likewise, our results showed that, among the examined
31 genes, 19 genes involved in the “regulation of osteoblast
differentiation” and “osteoblast differentiation” (FHL2,
BMP2, TWSG1, CCDC47, FAM20C, HIRA, MYBBP1A,
JAG1, MEF2C, SUCO, SFRP1, PDLIM7, SMAD1, IFT80,
HDAC7, ASF1A, ID3, SNAI1, and BCAP29) were
upregulated by the biomaterial in PEI-EVs + hGMSCs compared
Moreover, we found that 9 genes that were reported to
be upregulated during osteogenesis and some genes
reported to be involved in osteoblast differentiation through
TGF-β signaling were expressed at higher levels in
3DPLA + PEI-EVs + hGMSCs compared with hGMSCs [
]. In addition, we evaluated the gene expression profiles
of ECM and adhesion molecules in the 3D-PLA +
PEIEVs + hGMSCs group compared with control cells. A total
of 20 differentially expressed genes were found in the
3DPLA + PEI-EVs + hGMSCs group. Upregulated genes were
those that encode for integrin (ITGA6), basement
membrane laminins (LAMB3, LAMA1, and LAMC1),
membrane proteins that mediate cell-to-cell and cell-to-matrix
interactions (CTNNA1, VCAN, CD44, and THBS2), and
inhibitor of the matrix metalloproteinases (TIMP3).
Downregulated genes were those that encode for cell-cell
adhesion (ITGA3, ITGB5, ITGAV, ACTB, CTNNB1, and
CTGF) and for the ECM constituents of basement
membrane (LAMA3, TNC, GAPDH, and COL4A2). All these
results suggest the prospective role of 3D-PLA biomaterial
enriched with PEI-EVs + hGMSCs in inducing the
regulation of adhesion molecules, ECM, and osteogenic genes.
In light of the above findings, we investigated the
osteogenic effects of 3D-PLA scaffold infiltrated or not with
hGMSCs-derived extracellular vesicles. In vivo studies
testing biomaterials in the complex environment of the
body are considered strategic to understanding the
biological machinery involved in bone regeneration.
Previously, we have demonstrated that periodontal ligament
stem cells implanted in the mouse do not show
immunogenic effects, and after 3 weeks a massive number
of cells with different sizes and features and maturation
degrees were detected in the mouse calvaria implanted
with hPDLSCs/DB [
]. Here, we demonstrated that
hGMSCs seeded onto 3D-PLA can induce a
regenerative bone process but the presence of EVs and,
principally, PEI-EVs also activates local bone induction
significantly contributing to the regeneration process.
In fact, the 3D-PLA scaffold implanted in the mouse
calvaria does not show immunogenic effects and, after
6 weeks, numerous cells secrete ECM that initiates
osteogenic mineralization. In addition, the presence of
the EVs and, more specifically, PEI-EVs linked to the
3D-PLA scaffold improve the mineralization process as
well as the development of an extensive vascular
network that is indicative of osteointegration, as recently
shown by Xie et al. [
]. The key role of PEI-EVs in
bone repair was also shown by CT analysis, with a
complete repair seen in both the 3D-PLA + PEI-EVs
and 3D-PLA + PEI-EVs + hGMSCs groups. CT analysis
showed the presence of damage in the 3D-PLA + EVs
group, but in the 3D-PLA + EVs + hGMSCs group the
bone regeneration was similar to the groups with
PEIEVs. These findings suggest that the EVs fraction can
contribute to osteogenic regeneration and, in particular,
that the PEI-engineered EVs are responsible for a more
rapid evolution and a major degree of maturation of
new bone tissue. In fact, after 6 weeks of in vivo
implantation, formation of new bone nodules and blood
vessels were evident in calvariae samples grafted with
3D-PLA + PEI-EVs. Our data are therefore in
agreement with the promising results on the pro-osteogenic
impact of EVs visualized in animal models; EVs
stimulate bone regeneration whereas PEI-EVs induce the
bone apposition and emphasize the proangiogenic
capacity, thus showing that scaffolds coated with PEI-EVs
could represent a new tool in critical-size bone defects
]. Similarly, PEI-decorated graphene oxide is a potent
inducer of stem cell osteogenesis leading to nearly double
the alkaline phosphatase expression and mineralization
]. Along these lines, it could be concluded that PEI-EVs
play a critical role in cell fate determination; they are not
], but rather they enrich the chemical and physical
properties of our novel 3D-PLA printed porous scaffolds.
With the limitations of this study, we have shown that
3D-PLA printed porous scaffolds and the beneficial
effect of EVs and PEI-EVs on osteogenic commitment
could represent a novel platform in the study of
personalized stem cell-free therapy in bone tissue regeneration.
Additional file 1: Cytotoxicity of degradation byproducts. (A–E) Graphs
reporting the metabolic activity of cells exposed to the extract of degrading
PLA scaffolds at different endpoint for each design. (F) MTT assay performed
on hGMSCs directly exposed to the 3D PLA scaffold at different endpoint.
(JPEG 1168 kb)
Additional file 2: 3D PLA and hGMSCs interactions. SEM micrographs at
low (A) and high magnifications (B) showing cell adhesion on the scaffold
surface. Scaffold surface without hGMSCs are reported in the inset in
section A. A, magnification 300×; B, magnification 750×. Scale bar = 10 μm.
(JPEG 1188 kb)
Additional file 3: Gene expression. (A) The expression value of genes
for “regulation of ossification” and “ossification” differentially expressed
between 3D-PLA+ EVs + hGMSCs and 3D-PLA+ PEI-EVs + hGMSCs and
compared with hGMSCs. (B) The expression value of genes for “regulation
of osteoblast differentiation” and “osteoblast differentiation” differentially
expressed between 3D-PLA+ EVs + hGMSCs and 3D-PLA+ PEI-EVs + hGMSCs
and compared with hGMSCs. (JPEG 769 kb)
Additional file 4: Table S1. The differential gene expression between
3D-PLA+ EVs + hGMSCs and 3D-PLA+ PEI-EVs + hGMSCs compared with
hGMSCs is given as expression value and fold change expressed in
logarithm with base 2 (FC Log2). Gene ontology (GO) processes indicate the
gene classification in the “regulation of ossification” and “ossification”.
Instead, the statistical significance is indicated by the false discovery rate
(FDR), q values ≤ 0.05 were considered statistically significant. Table S2. The
differential gene expression between 3D-PLA+ EVs + hGMSCs and 3D-PLA+
PEI-EVs + hGMSCs compared with hGMSCs is given as expression value
and fold change expressed in logarithm with base 2 (FC Log2). Gene
ontology (GO) processes indicate the gene classification in the “regulation of
osteoblast differentiation” and “osteoblast differentiation”. Instead, the
statistical significance is indicated by the false discovery rate (FDR), q values ≤ 0.05
were considered statistically significant. Table S3. The differential gene
expression between 3D-PLA+ EVs + hGMSCs and 3D-PLA+ PEI-EVs + hGMSCs
compared with hGMSCs is given as expression value and fold change
expressed in logarithm with base 2 (FC Log2). The statistical significance is
indicated by the false discovery rate (FDR), q values ≤ 0.05 were considered
statistically significant. Table S4. The differential gene expression in 3D-PLA
+ PEI-EVs + hGMSCs compared with hGMSCs is given in fold change
expressed in logarithm with base 2 (FC Log2). The statistical significance is
indicated by the false discovery rate (FDR), q values ≤ 0.05 were considered
statistically significant. (DOCX 59 kb)
Additional file 5: Gene expression. Expression value of genes activated
during osteogenesis and osteoblast differentiation in 3D-PLA+ EVs +
hGMSCs and 3D-PLA+ PEI-EVs + hGMSCs and compared with hGMSCs
(q ≤ 0.05, Benjamini–Hochberg false discovery rate). (JPEG 319 kb)
Additional file 6: Gene analysis. Differential regulation of genes coding
for adhesion molecules and ECM proteins in the 3D-PLA + PEI-EVs +
hGMSCs group compared with hGMSCs cells. Upregulated transcripts
shown in red, downregulated transcripts shown in green (q ≤ 0.05,
Benjamini–Hochberg false discovery rate). (JPEG 271 kb)
3D: Three-dimensional; AFM: Atomic force microscopy; CLSM: Confocal laser
scanning microscopy; CM: Conditioned medium; DLS: Dynamic laser light
scattering; ECM: Extracellular matrix; EVs: Extracellular vesicles; GO: Gene
ontology; hGMSCs: Human gingival mesenchymal stem cells;
mCT: Microcomputed tomography; MSCGM-CD: Mesenchymal stem cell
growth medium—chemically defined; MSC: Mesenchymal stem cell; NGS:
Nextgeneration sequencing; PBS: Phosphate-buffered solution;
PEI: Polyethyleneimine; PEI-EVs: Polyethyleneimine-engineered extracellular
vesicles; PLA: Poly(lactide); SEM: Scanning electron microscopy; TCP: Tricalcium
This work has been supported by OT ex 60% University of Chieti fund, and
partly by PRIN 20102ZLNJ5 “Stem cells and 3D scaffolds: a novel construct in
bone regeneration” financed by the Ministry of Education, University, and
Research (M.I.U.R.), Rome, Italy.
Availability of data and materials
All data generated and/or analyzed during this study are included in this
published article and its supplementary Additional files.
OT: study design, data analysis, writing, and revising the paper. EM: data
analysis, writing, and revising the paper. AF: data analysis, vesicles
engineering and evaluation, writing, and revising the paper. FD: scaffold
development, scaffold structural, chemical and physical analysis, performing
in vitro experiments, in vitro and in vivo data evaluation, and writing the
paper. AG: gene analysis, in vitro and in vivo data evaluation, and revising
the paper. PC: scaffold development, scaffold structural, chemical and
physical analysis, and writing the paper. RB: scaffold structural, chemical and
physical analysis. IM: performing in vitro experiments and histological
processing. DS: gene analysis and EV engineering and evaluation. TT:
histological processing. SC: revising text. AB: revising text. AN: revising text.
All authors read and approved the final manuscript.
Ethics approval and consent to participate
All experimental procedures were approved by the Ethics Committee of
the University “G. d’Annunzio”, Chieti-Pescara and Ministry of Health
(Italy). Written informed consent was obtained from all donors. All animal
care and use was performed according to the European Organization
Guidelines for Animal Welfare. The study has been authorized by the
Ministry of Health “General Direction of animal health and veterinary
drug” (Authorization 768/2016-PR 28/07/2016- D.lgs 26/2014). The experiments
were planned in such a way as to minimize the total number of rats needed for
Consent for publication
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
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