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 signicant
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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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
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 inuence
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 vericadas 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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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
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 inuence, 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 conrmation 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 conuence 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 specic
criteria, ensuring randomization. On the day of
surgery, one animal from each cage was selected,
completing the randomization process. The four
groups were dened 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-inammatory
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 decalcicarion 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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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
model Power Shot A640. Digital images (JPEG
format) were obtained with 2x magnication
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 conrmed, 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%
signicance 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 veried that all of them presented highly
porous structures with open and dened 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 decalcication 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 inammatory 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.