UNIVERSIDADE ESTADUAL PAULISTA
“JÚLIO DE MESQUITA FILHO”
Instituto de Ciência e Tecnologia
Campus de São José dos Campos
ORIGINAL ARTICLE DOI: https://doi.org/10.4322/bds.2025.e4296
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Braz Dent Sci 2025 Jan/Mar;28 (1): e4296
This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
A new approach in bone tissue regeneration: in vivo study of the
impact of calcium aluminate cement scaffolds incorporated with
mesenchymal cells
Uma nova abordagem na regeneração do tecido ósseo: estudo in vivo do impacto de
scaffolds
de cimento de aluminato de
cálcio incorporado com células mesenquimais
Carla da Silveira e Oliveira BRONZE1, Letícia Adrielly Dias GRISANTE1 , Juliani Caroline Ribeiro de ARAÚJO1 ,
Rafaella Souza GUARDIA1 , Iranel de Las Nievez González VICUNA2 , Ivone Regina de OLIVEIRA2 ,
Luana Marotta Reis de VASCONCELLOS1 ,
1 - Universidade Estadual Paulista, Instituto de Ciência e Tecnologia, Patologia Bucal. São José dos Campos, SP, Brazil.
2 - Universidade do Vale do Paraíba, Instituto de Pesquisa e Desenvolvimento. São José dos Campos, SP, Brazil.
How to cite: Bronze CSO, Grisante LAD, Araújo JCR, Guardia RS, Vicuna ILNG, Oliveira IR, Vasconcellos LMR. A new approach in bone
tissue regeneration: in vivo study of the impact of calcium aluminate cement scaffolds incorporated with mesenchymal cells. Braz Dent
Sci. 2025;28(1):e4296. https://doi.org/10.4322/bds.2025.e4296
ABSTRACT
Objective: The objective of this study was to evaluate the bone regeneration potential of CAC-based scaffolds,
with or without mesenchymal stem cells (MSC), in bone defects created in rat femurs. Material and Methods:
Forty-eight CAC scaffolds and their blends of tricalcium phosphate (TCP), zinc oxide (ZNO), and zirconia (ZIRC)
were produced, with half of these incorporated with MSC. Twenty-three Wistar rats were used, with 3 for MSC
isolation and 20 for creating bone defects in both femurs. Five animals were assigned to each group, and during
the defect surgery and material insertion, the animals received MSC-incorporated scaffolds on the left side
and non-incorporated scaffolds on the right side, with the same type of material used in each animal to avoid
different systemic effects (n=5); they were euthanized 21 days after the surgical procedure. Results: In the
scanning electron microscopy analysis of the scaffolds, structures with open and interconnected pores, as well
as cell adhesion, were observed in all groups. In the histological analysis, all groups showed newly formed bone
trabeculae interspersed with bone marrow cells and connective tissue. Conclusion: In the histomorphometry,
for the scaffolds not incorporated with MSC, the ZIRC group showed greater bone formation, and in the MSC-
incorporated scaffolds, the TCP group demonstrated better results, both exhibiting a statistically signicant
difference from the other groups (p<0.05).
KEYWORDS
Biocompatible materials; Bone cements; Bone regeneration; Bone tissue; Mesenchymal stem cells.
RESUMO
Objetivo: O objetivo neste trabalho foi avaliar o potencial de regeneração óssea de
scaffolds
à base de CAC,
incorporados ou não com células mesenquimais (MSC) em defeitos ósseos realizados em fêmures de ratos.
Material e Métodos: Foram produzidos 48
scaffolds
de CAC e suas blendas fosfato tricálcico (FOSF), óxido
de zinco (ZNO) e zircônia (ZIRC), sendo que metade destes foram incorporados com MSC. Vinte e três ratos
Wistar
foram utilizados, sendo 03 para isolamento das MSC e 20 para confecção de defeitos ósseos em ambos
os fêmures. Foram separados 5 animais para cada grupo, sendo que durante a cirurgia de defeito e inserção do
marterial os animais receberam
scaffolds
incorporados com MSC do lado esquerdo e não incorporados do lado
direito, em cada animais foi utilizado material de mesmo tipo para que não houvessem diferentes efeitos sitêmicos
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Bronze CSO et al.
A new approach in bone tissue regeneration: in vivo study of the impact of calcium aluminate cement scaffolds incorporated with mesenchymal cells
Bronze CSO et al. A new approach in bone tissue regeneration: in vivo study of
the impact of calcium aluminate cement scaffolds incorporated
with mesenchymal cells
INTRODUCTION
Regenerative medicine aims to restore
organs, tissues, or cells to recover compromised
mechanical and biological functions due to
trauma, tumors, infections, degenerative
diseases, and aging [1,2]. Tissue bioengineering
accelerates tissue regeneration and repair by
developing new biomaterials to restore, enhance,
or prevent tissue function deterioration [3,4].
Biomaterials, interacting with biological systems,
can treat, enhance, or replace tissues, organs, or
body functions [5].
With increased life expectancy, degenerative
diseases and pathological conditions causing
tissue loss, such as neoplasms and tumors, are
growing, along with a higher probability of
trauma and bone fractures. A major challenge of
bioengineering is developing biomaterials to assist
in bone tissue recovery [6]. In recent decades,
new synthetic biomaterials have been developed
to promote and accelerate bone regeneration
without damaging healthy tissues or increasing
contamination risks [7-11]. Biomaterials used
as bone substitutes must be biocompatible
and osteoconductive, allowing the migration
of osteoprogenitor cells to the injured site and
providing support for bone neoformation [5,12].
They can be presented in various forms such as
powders, solid blocks, membranes, hydrogels,
sponges, and scaffolds, with different origins
and chemical compositions [13,14]. Three-
dimensional porous scaffolds mimic the
extracellular matrix environment, guiding cell
migration, differentiation, and proliferation to
form new tissue [15-18]. The size and quantity of
pores of the scaffolds have an important inuence
on the progression of osteogenesis, since the
greater quantity and size of pores result in greater
bone growth [19-21]. The interconnection
between these pores promote a key role in the
migration and proliferation of blood vessels - a
primary condition for tissue growth. In addition
to supplying nutrients, vascularization will
coordinate the activity of bone cells and their
migration to the implantation site [22].
Calcium aluminate cement is an excellent
material for filling bone defects, acting as a
barrier against bacteria [23]. Scaffolds based on
calcium aluminate cement (CAC) release calcium
ions, favoring osteogenic differentiation and
mineralization during bone regeneration [9,24].
Additionally, they form a layer similar to apatite
or carbonated hydroxyapatite on their surface,
improving osteointegration [25,26]. Other
materials such as tricalcium phosphate (TCP),
zinc oxide, and zirconia, when associated with
CAC, show positive results in osteoblastic cell
viability and the ability to induce mineralization
and bioactivity [27-29]. Tricalcium phosphate
presents itself as a biocompatible and bioactive
bioceramic; the zinc oxide has bactericidal
properties and the zirconia, besides
biocompatibility, presents good resistance to
corrosion, wear and compression [29,30].
Regenerative medicine and tissue
engineering with stem cell therapy hold promise
for bone regeneration [31,32]. They have
great potential for bone regeneration because
they exhibit a high capacity for regeneration,
proliferation and cellular differentiation, playing
na important role in the elds of medicine and
dentistry [33,34]. Adult bone marrow-derived
stem cells are multipotent and have the potential
to repair damaged tissues [35-37].
The increased interest in cell therapy is due
to the potential of mesenchymal cells to multiply,
self-renew and differentiate, both in vitro and in
(n=5); e foram eutanasiados 21 dias após o procedimento cirúrgico. Resultados: Na análise dos
scaffolds
por
microscopia eletrônica de varredura foram vericadas estruturas com poros abertos e interconectados, além
de adesão celular em todos os grupos. Na análise histológica, foi observado que todos os grupos apresentaram
trabéculas ósseas neoformadas, entremeadas por células da medula óssea e tecido conjuntivo. Conclusão: Na
histomorfometria, para os
scaffolds
não incorporados com MSC, observou-se que o grupo ZIRC apresentou maior
neoformação óssea e nos
scaffolds
incorporados com MSC, o grupo FOSF demonstrou melhores resultados em
comparação com os demais grupos incorporados com células mesenquimais, ambos exibindo diferença estatística
para os demais grupos (p<0,05).
PALAVRAS-CHAVE
Materiais biocompatíveis; Cimentos ósseos; Regeneração óssea; Tecido ósseo; Células mesenquimais.
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Bronze CSO et al.
A new approach in bone tissue regeneration: in vivo study of the impact of calcium aluminate cement scaffolds incorporated with mesenchymal cells
Bronze CSO et al. A new approach in bone tissue regeneration: in vivo study of
the impact of calcium aluminate cement scaffolds incorporated
with mesenchymal cells
vivo, into cells of different lineages, presenting
potential to improve repair or regeneration
of damaged tissues [32,38]. Studies combine
synthetic biomaterials with mesenchymal cells
to enhance differentiation and bone growth in
bone defects [7,8].
Preclinical trials have also demonstrated
that the association of mesenchymal cells
with biomaterials increases osteogenic
capacity [7,39,40]. Within this context, the aim
of this study was to verify whether the use of
scaffolds based on calcium aluminate cement
(CAC) and its blends (tricalcium phosphate,
zirc oxide and zirconia), incorporated or not
with mesenchymal cells can inuence, favor or
stimulate bone tissue regeneration, producing
important information regarding the potential
use of these biomaterials in cell therapy in tissue
regeneration.
MATERIAL AND METHODS
Scaffolds samples
Forty-eight scaffolds were produced by
foam replica method technique developed by
Schwartzwalder and Somers (1963) [41], that
consists in the impregnation of polyurethane
foam with ceramic solution followed by thermal
treatment to burn the organic part of the
foam [28]. The scaffolds were produced from 3M
Scotch Brite polymeric foams containing 49 pores
per linear inch. These were impregnated with
aqueous ceramic suspensions containing 60%-p
solid content of calcium aluminate cement (CAC),
followed by heat treatment at 1300°C. To form
the blends, 4% by weight additives (tricalcium
phosphate, zinc oxide or zirconia) were added
to the calcium aluminate cement. All scaffolds
were 4.0 mm in diameter and 4.0 mm in length.
The pore size distribution and total porosity
of the scaffolds were evaluated previously to
this study, by mercury intrusion porosimetry -
all of them showed porosity between 50-60%
and pore distribution with peaks in diameters:
0.015; 30 (micropores) and mainly, 200 µm
(macropores) [28]. These scaffolds were also
previously analyzed for bioactivity and behavior
in cell culture (cell adhesion, cell viability, protein
production, differentiation into bone cells and
mineralization nodule formation) presenting
positive results [28]. To analyze the surface
topography of the samples, a scanning electron
microscope (SEM) (EVO/MA10) from the Central
Multiuser Analytical Laboratory of the Research
and Development Institute of Universidade do
Vale do Paraíba (UNIVAP) was used. The samples
were positioned on an aluminum platform
(stub), aided by a double-sided carbon tape
(3M, Sumaré SP, Brazil) and metallized with a
thin gold layer by sputtering in the metallizing
machine (EMITECH K550X, Sputter Coater,
Qriorum Technologies), for 130s. This study was
developed by our research group [28].
Ethics committee
This study was approved by the Research
Ethics Committee, Brazil (CEUA, protocol
012/2019), of São José dos Campos Institute
of Science and Technology - UNESP, and was
conducted according to the ethical principles
for animal experimentation, adopted by the
Brazilian College of Animal Experimentation
(CONCEA). This work also followed the guidelines
recommended by ARRIVE (Animal Research
Reporting of In Vivo Experiments) [38].
Isolation of mesenchymal cells
Mesenchymal cells were obtained from the
bone marrow of the femurs of 3 Wistar male rats,
at 3 months old, weighing about 350 g [42].
Initially, the animals were euthanized with an
overdose of anesthetic using a combination of
the drugs Xylazine hydrochloride (Anasedan®
- Vetbrands, Jacareí - Brazil) and Ketamine
hydrochloride (DopalenⓇ - Vetbrands, Jacarei -
Brazil). Three times the recommended dose for
the animal’s weight was applied intramuscularly,
and after conrmation of anesthesia, decapitation
was performed. Subsequently, 6 femurs were
removed and placed in a 50 mL falcon tube
containing the transport solution composed of
95% filtered MEM alpha (minimum essential
medium) and 5% gentamicin. After transport
to laminar flow cabinet and cleaning of the
femurs with 0.12% chlorhexidine, bone marrow
cells were isolated and inserted into 250 mL
and 75 cm2 cell culture asks with alpha MEM
culture medium (Gibco) supplemented with
10% Bovine Fetal Serum (SBF) and gentamicin
(500 µg/mL) (Gibco). Next, the flasks were
incubated in an incubator at 37°C temperature
with atmospheric humidity containing 5% CO2.
The culture medium was changed every three
days and the progression of the culture was
evaluated by inverted phase microscopy (Carl
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Bronze CSO et al.
A new approach in bone tissue regeneration: in vivo study of the impact of calcium aluminate cement scaffolds incorporated with mesenchymal cells
Bronze CSO et al. A new approach in bone tissue regeneration: in vivo study of
the impact of calcium aluminate cement scaffolds incorporated
with mesenchymal cells
Zeiss Microscope - Axiovert 40C, Germany).
After conuence of the cells (seven days after
isolation) they were enzymatically released and
plated at a density of 2x10^4 cells in each well
of the 96-well microplate (Kasvi) containing the
scaffolds, which were previously sterilized under
ultraviolet (UV) light for 15 minutes.
Surgical procedure
In the in vivo assays of this study, bone
defects were made in the right and left femurs
of 20 adult male rats (Rattus norvegicus,
albinus, Wistar), with approximately 90 days
old, weighing about 350g. Initially, the animals
were weighed and anesthetized according to their
weight by intramuscular injection of Xylazine
hydrochloride (Anasedan® - Vetbrands, Jacareí
- Brazil) and Ketamine hydrochloride (DopalenⓇ
- Vetbrands, Jacarei - Brazil). Then in the medial
region of the femurs trichotomy and antisepsis
with iodized alcohol solution were performed.
The incision was made with a no. 15 scalpel blade
and the ap was detached to access the bone
tissue. A bone defect was made with a 4.0 mm
diameter spherical drill bit in both femurs, under
abundant irrigation with 0.9% sodium chloride,
in order to avoid heating due to the friction of
the bur with the bone. Upon arrival at the local
animal facility, the animals were randomly
separated by the technician without any specic
criteria, ensuring randomization. On the day of
surgery, one animal from each cage was selected,
completing the randomization process. The four
groups were dened according to the bone defect
lling material: CAC for the control group, CAC
with a tricalcium phosphate blend (FOSF), CAC
with a zirconia blend (ZIRC), and CAC with a
zinc oxide blend (ZNO). The bone defect site
of the right femur was lled with the scaffold
without embedded cells, while in the left femur,
the lling was with the scaffold embedded with
mesenchymal cells. The scaffolds inserted in
the right and left femurs of the rats were made
of the same material, in order to avoid any
systemic effect that the material might present.
In all animals, the flap was repositioned and
sutured with silk thread no. 4 (Ethicon/Johnson
& Johnson). It is important to emphasize that the
surgeries, the intervention process, and the future
histomorphometric evaluations were carried out
by the same investigators.
For 5 days, analgesia was provided by
Tramadol, which doesn’t exhibit anti-inammatory
effect [43], at a dose of 8mg/kg, administered
every 12 hours. The animals were put back in cages
containing 05 animals, and have been monitored
for 21 days. Then, were euthanized with an
overdose of the combined solution of the drugs
Xylazine hydrochloride (AnasedanⓇ Vetbrands,
Jacarei - Brazil) and Ketamine hydrochloride
(Dopalen® - Vetbrands, Jacareí - Brazil) and
decapitated. The femurs were removed and placed
in 10% formalin for at least 48 hours, and later
submitted to the histological processing for further
histological and histomorphometric analyses.
Incorporarion of mesenchymal cells to scaf-
folds
After obtaining the mesenchymal cells and
scaffolds, two scaffolds from each group were
cultured with the cells for 5 days. After this
period, the cells were fixed and the scaffolds
were evaluated by micrographs obtained by
SEM. For xation, a dehydration protocol with
increasing concentrations of alcohol was used:
10%, 25%, 50%, 75%, and 100%. The scaffolds
with cells remained in each stage for 20 minutes.
After these stages, the material was dried in an
oven at 37°C for 16 hours.
HISTOLOGICAL AND HISTOMORPHO-
METRIC ANALYSIS
After fixation with formaldehyde, the
femurs with bone defects were CUT into smaller
fragments and submitted to decalcicarion using
the demineralization technique at the Bone Tissue
Laboratory of ICT/Unesp, using 20% formic acid
for approximately 90 days. Subsequently, the
pices were trasversely sectioned in the center of
the scaffold insertion region and the fragments
were included in paraffin blocks using tissue
processor (LEICA TO 1020, USA). The pieces
were embedded in paraffin and submitted
to the routine laboratory technique for the
preparation of histological slides. Five slices were
prepared for each bone fragment and stained
with hematoxylin and eosin. In the histological
analysis, aspects of the development of bone
repair, formation of granulation tissue, new bone
formation, the arrangement of bone trabeculae
and bone maturation until nal remodeling were
observed. For the histomorphometric analysis, the
histological sections were photographed with a
Zeiss Axioskop 40 light microscope (Carl Zeiss
Brasil), with a digital câmera coupled to Canon,
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Bronze CSO et al.
A new approach in bone tissue regeneration: in vivo study of the impact of calcium aluminate cement scaffolds incorporated with mesenchymal cells
Bronze CSO et al. A new approach in bone tissue regeneration: in vivo study of
the impact of calcium aluminate cement scaffolds incorporated
with mesenchymal cells
model Power Shot A640. Digital images (JPEG
format) were obtained with 2x magnication
in the region of the defect. These images were
analyzed usign the Image J software (National
Institutes of Health, Bethesda, MD), which makes
it possible to quantify the newly formed bone in
the scaffold insertion region. The average area of
the regions corresponding to the newly formed
bone repair tissue was calculated for each group.
Statistical analysis
As per previous study [44], the number of
animals was estimated by a statistical calculation
from simple group analysis considering the
reliability estimate (Log ẞ), sample error estimate
(Log p) and the margin of loss of animals during
the experiment.
The formula used was:
n = logβ log p × 1.2 ∴ = log 0.05
(1)
log 0.5 × 1.2 = 5,18 ≈ 5 animals
After the calculation, we obtained an
estimated number of 5 animals per group.
The data collected were initially submitted to
the Shapiro-Wilk normality test. Once the normal
distribution of data was conrmed, they were
submitted to analysis of variance (ANOVA) for
intergroup comparison and complemented by
the Tukey test, when necessary, to verify the
statistical differences between the means of
the groups. For intragroup analysis (scaffold
incorporated with cells and not incorporated with
cells), the t-test was used to verify the differences.
GraphPad Prism 9 statistical software (GraphPad
Software, San Diego, CA, USA) was indispensable
to perform the tests. For all statistical tests a 5%
signicance level was adopted.
RESULTS
Scaffolds characterization
To evaluate the surface topography of the
CAC scaffold samples and their blends, a scanning
electron microscope (SEM EVO/MA10) was
used. The three-dimensional (3D) appearance
characteristic of the scaffolds was observed and
it was veried that all of them presented highly
porous structures with open and dened pores.
These pores were interconnected with
different sizes in the magnifications, as
shown in Figure 1; showing that the scaffolds
design is suitable for its use as a biomaterial
substitute, for bone tissue - since it mimicked
an extracellular matrix in 3D, enabling cell
migration and multiplication inside this network
of interconnected pores.
Evaluation of incorporarion of mesenchymal
cells to scaffolds
Two scaffolds from each group were plated
together with the cells for 5 days. After this
period, the cells were fixed and the scaffolds
were evaluated by micrographs obtained by
SEM (Figure 2). It was noted cell adhesion in all
scaffolds used in this study, irrespective of their
composition.
Histological analysis
In all groups, the histological sections
revealed neoformed bone tissue formed by
trabeculae covered by osteoblasts, containing
numerous osteocytes inside. These trabeculae
were thin and widely spaced and sometimes
thicker and more continuous, interspersed
with bone marrow cells and sometimes brous
connective tissue. The scaffolds were dissolved
in the process of decalcication with formic acid
and therefore, only residues of these could be
visualized as areas of brownish pigmentation
in the histological sections of all groups. There
were no inammatory processes or foreign body
reaction in any group. Sometimes a bone bridge
was observed between the ends of the pré-
existing cortical bone, in the region of insertion of
the scaffolds. It was found that this neoformation
invaginated into the medullary region of the
femur, occupying part of this region.
In the CAC group incorporated with
mesenchymal cells, the newly formed bone
tissue developed mainly in the lower region of
the scaffold and proliferated towards the bone
marrow of the femur (Figure 3). The bone
trabeculae in this group were thicker compared
to the CAC group without cells (Figure 4).
In the histological sections of the FOSF group
without incorporation with mesenchymal cells, it
was possible to observe that the newly formed bone
tissue occurred mainly in the region underlying
the anterior region occupied by the scaffolds
(Figure 5). Large amounts of bone marrow cells
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Bronze CSO et al.
A new approach in bone tissue regeneration: in vivo study of the impact of calcium aluminate cement scaffolds incorporated with mesenchymal cells
Bronze CSO et al. A new approach in bone tissue regeneration: in vivo study of
the impact of calcium aluminate cement scaffolds incorporated
with mesenchymal cells
were visualized interspersing the bone trabeculae.
When incorporated with mesenchymal cells,
however, it was possible to observe new bone
formation starting from the sides of the pré-existing
cortical bone towards the Center of the scaffold
insertion region (Figure 5). A bone bridge with
tinner áreas between the córtices can be seen.
In the group with ZNO scaffolds incorporated
and not incorporated with mesenchymal cells, the
presence of thick bone trabecular was observed in
the region of the scaffolds, without bone bridge
formation (Figure 6). Figure 7 shows the ZNO
group incorporated with mesenchymal cells.
In the ZIRC group without incorporation
with the mesenchymal cells, it was observed
that the newly formed bone tissue occurred
from the sides of the pré-existing bone córtices
towards the center of the scaffold insertion
region, with formation of a bone brisge between
the pré-existing bone cortices. In the ZIRC group
Figure 1 - Scanning electron micrographs of scaffolds prepared from sponge impregnation in aqueous suspensions of calcium aluminate
cement and its blends. Legends: 1) Calcium aluminate cement and its blends containing 4%-w of: 2) FOSF group, 3) ZIRC group, 4) ZNO group.
Magnificarions: A: 25x; B: 40x; C: 80x.
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Bronze CSO et al.
A new approach in bone tissue regeneration: in vivo study of the impact of calcium aluminate cement scaffolds incorporated with mesenchymal cells
Bronze CSO et al. A new approach in bone tissue regeneration: in vivo study of
the impact of calcium aluminate cement scaffolds incorporated
with mesenchymal cells
incorporated with mesenchymal cells, there
was formation of bone tissue at the interface
with the region previously lled by the scaffold
(Figures 8).
Histomorphometric analysis
The histomorphometric analysis was
performed using images of histological sections
Figure 3 - Histological section observed in the: a) CAC/MSC group original magnification 20x; b) CAC/MSC group original magnification 100x.
A) Newly formed bone trabeculae; B) Connective tissue between bone trabeculae; C) Residue of the scaffold; D) Bone marrow cells. The arrows
indicate newly formed bone trabecular at the inferior interface of the scaffold.
Figure 2 - Cell adhesion after 5 days on scaffolds, seen by scanning electron microscopy (SEM). Legends: In green, cells are highlighted. A)
CAC - Magnification: 3.900x; B) FOSF - Magnification: 708x; C) ZNO - Magnification: 679x; D) ZIRC - Magnification: 508x.
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A new approach in bone tissue regeneration: in vivo study of the impact of calcium aluminate cement scaffolds incorporated with mesenchymal cells
Bronze CSO et al. A new approach in bone tissue regeneration: in vivo study of
the impact of calcium aluminate cement scaffolds incorporated
with mesenchymal cells
performed in the scaffol insertion region. Image J
software (National Institutes of Health, Bethesda,
MD) was used to quantify bone tissue proliferation
at the interface with the scaffold. Regarding
the scaffolds that were not incorporated with
mesenchymal cells, it was observed that the
zirconia blend showed greater bone formation,
showing a statistical difference when compared
Figure 4 - Histological section observed in the: a) CAC/MSC group original magnification 20x; b) CAC/MSC group original magnification 100x.
Legends: A) Residue of the scaffold; B) Connective tissue; C) Newly formed bone trabeculae. The arrows indicate newly formed bone trabecular
at the inferior interface of the scaffold.
Figure 5 - Histological section observed in the FOSF group: a) FOSF group original magnification 20x; b) FOSF group original magnification
100x; c) FOSF/MSC group original magnification 20x; d) FOSF/MSC group original magnification 100x. Legends: A) Bone marrow cells; B)
Neoformed bone trabécula; C) Connective tissue; D) Medullary tissue interspersed with bone trabecular; E) Connective tissue containing
scaffold residue. The arrows indicate newly formed bone trabecular at the inferior interface of the scaffold.
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A new approach in bone tissue regeneration: in vivo study of the impact of calcium aluminate cement scaffolds incorporated with mesenchymal cells
Bronze CSO et al. A new approach in bone tissue regeneration: in vivo study of
the impact of calcium aluminate cement scaffolds incorporated
with mesenchymal cells
Figure 6 - Histological section observed in the ZNO Group: a) ZNO group original magnification 20x; b) ZNO group original magnification
100x; Legends: A) Represent newly formed bone trabeculae; B)Represent connective tissue; C) Represent residue from the scaffold. The arrows
indicate newly formed bone trabecular at the inferior interface of the scaffold.
Figure 7 - a) ZNO/MSC group original magnification 20x; b) ZNO/MSC group original magnification 100x. Legends: A) Connective tissue; B)
Newly formed bone trabecula; C) Fibrous connective tissue; D) Scaffold remnants. The arrows indicate newly formed bone trabecular at the
inferior interface of the scaffold.
Figure 8 - Histological section observed in the ZIRC groups: a) ZIRC group original magnification 20x; b) ZIRC group original magnification 100x.
Legends: A) Connective tissue; B) Newly formed bone trabecula; C) Fibrous connective tissue containing blood vessels (granulation tissue). The
arrows indicate newly formed bone trabecular at the inferior interface of the scaffold.
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A new approach in bone tissue regeneration: in vivo study of the impact of calcium aluminate cement scaffolds incorporated with mesenchymal cells
Bronze CSO et al. A new approach in bone tissue regeneration: in vivo study of
the impact of calcium aluminate cement scaffolds incorporated
with mesenchymal cells
to all other groups (p<0.05). The ZNO group
was the second group with the highest bone
formation, followed by the CAC and FOSF group.
However, there was no statistical signicance for
new bone formation in these groups (ZNO, CAC
and FOSF) (p>0.05). Regarding the scaffolds
that were incorporated with mesenchymal cells,
greater bone neoformation was observed in the
FOSF/MSC group, which showed a statistical
difference when compared to all other groups
(p<0.05). The ZNO group was the second group
with the highest new bone formation and also
showed statistical difference when compared to
the other groups (p>0.05). For the CAC group,
the results obtained revealed that the bone
neoformation values were similiar when these
materials were incorporated with mesenchymal
cells, with no statistical difference (p>0.05)
being observed between them. The results
obtained for the FOSF group revealed that new
bone formation was greater when this material
was incorporated with mesenchymal cells, with
a statistical difference being observed (p<0.05).
For the ZNO group, the results revealed that
new bone formation was greater when this
material was incorporated with mesenchymal
cells, with a statistical difference (p<0.05) when
compared to the scaffolds without cells. For the
ZIRC group, the results revealed that new bone
formation was greater when this material was
not incorporated with mesenchymal cells, with
a statistical difference being observed (p<0.05).
The results shown in Figure 9 refer to the
comparison of new bone formation obtained
at the scaffolds, in which all materials were
compared, when they were incorporated and not
incoporated with mesenchymal cells.
In the comparison between all groups of
scaffolds of different material, incorporated or
not with mesenchymal cells, it was veried that
the FOSF groups incorporated with cells and the
ZIRC not incorporated with mesenchymal cells
presented the best results, with greater bone
formation at the interface with the scaffolds, with
no statistical difference between them (p>0.05).
DISCUSSION
In the present study, bone formation was
evaluated in defects in rat femurs, which were lled
with biomaterials based on calcium aluminate
cement (CAC) and its blends of tricalcium
phosphate (FOSF), zinc oxide (ZNO) and
zirconia (ZIRC). In the conguration of scaffolds,
associated or not associated with mesenchymal
cells. Biomaterials are used to restore or replace
some tissue, organ or function of the body [12].
In this study, biomaterials (scaffolds based on
calcium aluminate cement) were used with the
functions of lling and restoring the volume of
lost bone tissue, providing a local mechanical
function besides serving as a support for cell
proliferation and stimulating bone neoformation.
The aim was to contribute to the innovation of
new biomaterials for the medical and dental
area, through the analysis of the potential in
vivo use of these scaffolds, in which important
information will be obtained regarding their
use in tissue regeneration associated with cell
therapy. The products based on SCC come from
a new generation of biomaterials that have been
developed in order to perform their functions in
bone tissue regeneration following the increase
of life expectancy of the population [45].
The CAC possesses relevant properties for its use
as biomaterial, especially in the repair of bone
defects, due to its advantages of biocompatibility
and high mechanical resistance when subjected
to compression [45]. CAC has relevant properties
for its use as a biomaterial, especially in the
repair of bone defects, due to its advantages
of biocompatibility and high mechanical
strength when submitted to compression [45].
Furthermore, there is the advantage with regard
Figure 9 - Comparison between all evaluated groups. Legends:
Graph of new bone formation at the interface of the insertion region
of the CAC scaffolds and their blends: tricalcium phosphate(FOSF),
zinc oxide (Zno) and zircônia (Zirc), whitout cells (sc) and with
incorporation of cells (cc). Different letters indicate statistical
difference.
11
Braz Dent Sci 2025 Jan/Mar;28 (1): e4296
Bronze CSO et al.
A new approach in bone tissue regeneration: in vivo study of the impact of calcium aluminate cement scaffolds incorporated with mesenchymal cells
Bronze CSO et al. A new approach in bone tissue regeneration: in vivo study of
the impact of calcium aluminate cement scaffolds incorporated
with mesenchymal cells
to its price and of its derivatives, beyond national
production and less dependence on imports [45].
In vitro studies [28], CAC scaffolds with 60%
solids content added with 4% additive weight of
FOSF, ZNO and ZIRC were not cytotoxic, showing
adequate cell viability, since all compositions
showed values higher than 70% in the MTT
assays. However, the ZNO and ZIRC blends
showed the highest number of viable cells and
were the groups that presented the highest values
of alkaline phosphatase activity, indicating their
ability to induce mineralization [28].
The results of this study indicated, through
quantication and analysis of the newly formed
bone tissue at the interface of the biomaterials
insertion region, that the use of CAC scaffolds
and their blends (FOSF, ZNO and ZIRC) allowed
bone neoformation in this region, incorporated or
not with mesenchymal cells. The incorporation
of mesenchymal cells in the scaffolds was
benecial and had inuenced positively in bone
neoformation in all groups, except in the ZIRC
blends group. However, this group of scaffolds
produced with ZIRC blends not incorporated with
mesenchymal cells was the one that showed the
greatest bone neoformation at the interface with
the insertion region of the scaffold, suggesting
a better potential for bone regeneration when
not incorporated with mesenchymal cells. When
incorporated with mesenchymal cells, the group
of the blend FOSF stood out positively, forming a
greater amount of bone tissue, indicating that it
is a promising material to be used in cell therapy.
Comparing all groups, incorporated or not with
mesenchymal cells, the groups that showed the
best results were the TCP with cells and ZIRC
without cell groups.
Furthermore, regarding the pore distribution
of these scaffolds, verified by Mercury
porosimetry [28] demonstrated that these same
zirconia and zinc oxide blends had larger pores
compared to the other groups, which seems to
favor bone formation [19,21]. Corroborating
this information, in the results of the present
in vivo study, for the scaffolds not incorporated
with mesenchymal calls, it was verield that the
zirconia blend was the one that presented the
best results, showing greater bone neoformation
compared to the other groups, presenting a
signicant statistical difference (
p
<0.05) with the
other groups, followed by the ZNO blend, shower
no statistical difference for CAC and FOSF.
Therefore, these in vivo results are consistent with
the in vitro results. Zirconia-based biomaterials
have gained attention as a biomaterial for
hard tissue reconstruction due to theis good
machanical, chemical and biological properties.
They have a low corrosion rate, low toxicity and
low bacterial adherence [46-48], proving to be
relevant for tissue engineering applications [49].
The combinarion of CAC and ZIRC in the
stabilization of vertebral compression fractures
resulted in high compressive strength values,
similar to PMMA [50]. In the present study, the
zirconia blend scaffolds presented the best results
and showed a higher rato of bone neoformation
when not incorporated with mesenchymal cells.
The association of mesenchymal cells with
scaffolds and other biomaterials offers a strategy
to improve bone differentiation and growth
compared to scaffolds whithout these cells [51].
This strategy has shown to be promising, showing
positive results in terms of stimulating the bone
regenerative process [8,39]. Preclinical tests
by [7,40] Also showed that the association of
mesenchymal cells with scaffolds increases the
osteogenic capacity. In the present study, this
premise was true and corroborates these results
for all groups, except for the zirconia blend
group, since in the intragroup results, comparing
scaffolds incorporated or not with mesenchymal
cells, it was veried that the incorporated scaffolds
presented better results, due to greater bone
formation. Only the ZIRC blend group presented
results with lower values when the scaffolds were
associated with mesenchymal cells (p<0.05).
Thisnsatisfactory result may have occurred due
to the zirconia íons had been solubilized during
the 5 days of impregnation with the cells in the
culture medium. Thus, the hypothesis would
be that CAC scaffold would remain without the
zirconia ions incorporated with the cells, ans
the performance wouldbe similar to the CAC
scaffolds without additives, which was exactly
the result found. The CAC and zirconia blend
scaffolds showed similar results when both were
incorporated with mesenchymal cells (p>0.05).
Authors evaluated blends of CAC with calcium
chloride (CaCl2) addociated with bismuth oxide
(Bi2O3) and zinc oxide (ZNO) as radiopaciers
in osteogenic cell cultures and concluded that
CAC with CaCl2 associated with ZNO promoted
a better survival rate and differentiation of
osteoblastic cells, suggesting greater potential
for bone repair of this blend in the contexto
of endodontic therapies [52]. Zinc oxide acts
12
Braz Dent Sci 2025 Jan/Mar;28 (1): e4296
Bronze CSO et al.
A new approach in bone tissue regeneration: in vivo study of the impact of calcium aluminate cement scaffolds incorporated with mesenchymal cells
Bronze CSO et al. A new approach in bone tissue regeneration: in vivo study of
the impact of calcium aluminate cement scaffolds incorporated
with mesenchymal cells
indirectly in bone repair by acting on enzymes
and hormones that are related to bone growth,
in addition to inhibiting osteoclasts, which are
responsible for bone resorption [33]. In this
present study, the ZNO blend scaffolds stood
out in the intergroup comparison, ranking 2nd
in bone neoformation, with statistical difference
fot the other groups (p<0.05). In the intragroup
comparison, when these were incorporated with
mesenchymal cells, they showed more promising
results compared to the scaffolds not incorporated
with these cells (p<0.05). The potential of
tricalcium phosphate in compositions for use
asa bone substitute has been reported in the
literature [29,53]. In a later study, results
were obtained that showed that β-tricalcium
phosphate presented a greater volume of new
bone formation [53]. In others, it showed greater
mineralizes matriz (mineralization nodules) in
the group of scaffolds of the tricalcium phosphate
blend, indicating this composition as promising
for tissue engineering [28]. In this present study,
the scaffolds of the tricalcium phosphate blend,
when associated with mesenchymal cells, was
the group with the greatest bone neoformation,
proving to be a potential material for bone
regeneration in the presence of cells.
CONCLUSION
When incorporated with mesenchymal cells,
the blend group containing tricalcium phosphate
stood out positively, forming greater amount
of bone tissue, indicating that it is a promising
material to be used in cell therapy. Comparing all
groups, embedded or not with cells mesenchymal,
the groups that demonstrated the best results
were the FOSF with cells and ZIRC without cells.
Acknowledgements
The authors are grateful to Coordination of
Superior Level Staff Improvement (CAPES) for
supporting this research.
Author’s Contributions
CSOB: Investigation, Resources, Formal
Analysis, Data Curation, Writing – Original
Draft Preparation. LADG: Validation, Formal
Analysis, Writing – Original Draft Preparation.
JCRA: Writing – Review & Editing, Visualization.
RSG: Data Curation. ILNGV: Conceptualization,
Formal Analysis, Investigation, Methodology,
Validation, Visualization. IRO: Conceptualization,
Methodology, Funding Acquisition. LMRV:
Conceptualization, Methodology, Supervision,
Project Administration.
Conict of Interest
The authors have no proprietary, nancial,
or other personal interest of any nature or kind
in any product, service, and/or company that is
presented in this article.
Funding
The authors declare that no nancial support
was received.
Regulatory Statement
This study was submitted and approved by
the Ethics Committee of Research in Animals of the
Institute of Science and Technology of Sao Jose dos
Campos/UNESP (012/2019-CEUA/ICT-UNESP).
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Letícia Adrielly Dias Grisante
(Corresponding address)
Universidade Estadual Paulista, Instituto de Ciência e Tecnologia, Patologia
Bucal, São José dos Campos, SP, Brazil.
Email: l.cruz@unesp.br
Date submitted: 2024 Mar 07
Accept submission: 2024 Sept 10