to the PDF file. - Romanian Journal of Legal Medicine

Rom J Leg Med [23] 61-68 [2015]
DOI: 10.4323/rjlm.2015.61
© 2015 Romanian Society of Legal Medicine Anatomic phenotyping of osseous progenitor cells. Implications in tissue
engineering and bone remodeling
Petru Razvan Melinte1,*, Gheorghe S. Dragoi2, Rodica Croitoru1
_________________________________________________________________________________________
Abstract: The bone remodeling process is achieved by the combined action of osteoblast and osteoclasts forming a
team called bone multicellular units, and it is a systematic process that renews preexistent bone tissue. The authors proposed
themselves to achieve a microanatomic study of remodeling process inside bone compacta and to describe the morphologic
and functional features of the osseous progenitor cells. For the micro anatomic study we used pieces of bone harvested from
cortical tibia diaphysis of adult individuals and fetus that were processed and analyzed by means of histochemical (van Gieson,
Gömöri, Mac Mannus) and histophysics methods (interferential and polarized light). Mesenchymal Stem cells harvested from
bone marrow aspirate were used to obtain osteoblasts by differentiation in the osteogenic medium; furthermore, the authors
characterized the derived osseous progenitor cells. The implications in bone remodeling as well as in designing new biomaterials
useful in orthopedic surgery are discussed.
Key Words: bone remodeling, osseous progenitor cell, mesenchymal stem cell, osteoblast, osteoclast.
O
steon permanent renewal, as remodeling
process, is a phenomenon that still raises
many questions and issues. For an anatomic and
topographic assessment of the "remodeling” process,
many studies have been performed: Podenphant and
Engel [1] have described variations in regional bone
remodeling; there is also a variation of the remodeling
activity at different bone surfaces [2, 3]. Besides, the
remodeling activity of cortical bone part is 5-10 times
lower than the one in cancellous bone structure.
The remodeling activity is not yet fully elucidated.
The most important consequences of the process [4, 5]
are those of offering a way for accessing the minerals and
grow factors deposited inside bone tissue allowing their
utilization in other regions and secondly of replacing the
mechanically compromised bone tissue. So, remodeling
activity prevents the accumulation of micro defects
that could cause future fractures. But the remodeling
intensity in different areas of the skeleton is not justified
by any of these processes. The spongy bone is considered
to possess a higher turnover by remodeling. Parfitt [5, 6]
had recently studied and debated this concept, describing
the cortical endosteal surface with the highest rate of
remodeling and inside the thickest trabecula of the
spongy bone tissue, the lowest [5, 6].
Osteoblast and osteoclasts action together in
order to achieve bone remodeling, by forming a team
called either Bone Multicellular Units (BMU) [3, 7],
either bone remodeling units [2, 3].
In conclusion, preexistent bone tissue renewal is
achieved by the systematic process of bone remodeling.
The balance between the amount of resorbed bone tissue
and the replaced one by new formation leads to gain or
loss of bone tissue located at different levels; of course,
1) University of Medicine and Pharmacy of Craiova, Departament of Anatomy, Romania
* Corresponding author: Email: [email protected]
2) Romanian Academy of Medical Sciences, Bucharest, Romania
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Melinte P.R. et al. Anatomic phenotyping of osseous progenitor cells. Implications in tissue engineering and bone remodeling
during lifetime, this processes record many differences
[2, 5].
Progresses made regarding the knowledge of
compact and cancelous bone structure and function
using immune histochemistry and histomorphometry
methods led to the redefinition and reevaluation of
classical concepts for a better understanding of the genesis
and time-space differentiation of morph and functional
bone units. The purpose of our study was to perform a
microanatomic study regarding the remodeling process
within bone compacta and to describe the osseous
progenitor cells by their functional and morphologic
characteristics.
MATERIALS AND METHODS
For the micro anatomic study we used pieces
of bone harvested from cortical tibia diaphysis of adult
bodies (15 cases) and foetuses (7 cases) in the Department
of Human Anatomy in the University of Medicine and
Pharmacy of Craiova. The fragments harvested from
these bones were fixed in formaldehyde 10% solution
at a 7,4 ph (Lili formula). Decalcification was done in
a 10% solution of EDTA. We used transverse serried
sections through the cortical of the tibia diaphysis in
order to analyze the topography of the haversian and non
haversian bone lamella systems. We visualized the lamella
fascicles by means of histochemic (van Gieson, Gömöri,
Mac Mannus) and histophysics methods (interferential
and polarized light).
The study of morphology and phenotypic
characteristics of osseous progenitor cells differentiated
from Stem cells represented an intermediate stage of the
research project CEEX entitled "Molecular mechanisms
of adhesion of the osteoblasts differentiated from stem
cells and bone tissue explants at orthopaedic biomaterials"
leaded by Ştefana Petrescu at Biochemistry Institute in
Bucharest, where the authors participated, representing
the University of Medicine and Pharmacy of Craiova [8,
9]. Bone marrow sampling was possible during some
orthopaedic surgeries such as total or partial artroplasty
of the hip.
Mesenchimal Stem cells isolation from
bone marrow. Isolation of mesenchimal Stem cells
was performed having as biological source bone
marrow aspiration from human patients with hip
artroplasty. For isolating Mesenchymal Stem Cells
(MSC) from the bone marrow aspirate, three different
methods were tested. Bone marrow was divided into
three fractions from which stem cells were isolated
in different ways. The first method consisted in
growing cell mass from bone marrow in the culture
medium, the second method involved bone aspiration
dilutions with phosphate buffered solution (PBS),
62
followed by cell centrifugation and sediment cultivation
and the third method consisted in the separation of bone
marrow cell suspension on ficol for a concentration of
mononuclear cells and their separation from other cell
populations in the sample taken. The cells were grown
and passed twice for expansion. At the end of this range,
they were characterized by flow citometry in order to
evaluate their phenotype and after its confirmation,
they were differentiated in osteogenic medium. During
differentiation, samples were taken at intervals of 1 day, 7
and 14 days for characterization, using specific markers.
In conclusion, bone marrow taken from patients
was separated on Ficoll (Amersham) for isolating the
fraction that contains mesenchymal Stem cells. The
obtained cells ring was then grown in αMEM medium
(Sigma), supplemented with 15% inactivated fetal
bovine serum, 50 units /ml penicillin (Gibco), 50 mg /
ml streptomycin (Gibco), 1% L-Glutamine (Gibco) and
maintained at 37oC in the atmosphere with 5% CO2. After
10 days, mesenchymal stem cell accessed to the culture
vessel surface were mixed for expansion and cultivated in
αMEM medium, supplemented with 10% fetal serum in
density of 5000 cells/cm2.
Mesenchymal Stem cells immune typing by
in flow cytometry. After another 10 days after the
first passage, the cells were harvested for expansion
(mixture 2) and some of them were kept for immune
phenotyping with in flow cytometer. There are a series
of surface markers expressed by mesenchymal stem cells
that differentiates them from other cell populations in
bone marrow. Although there still isn’t an universally
recognized markers set as being specifically expressed by
them only, most researchers consider CD14, 34, and 45
negative markers and CD13, 29 and 90 as positive ones
for the characteristic phenotype of mesenchymal stem
cells. We used these ones also, together with a negative,
unmarked control with fluorofori coupled antibodies in
order to validate the isolation method used.
Methods for the characterization of osseous
progenitor cells. Differentiation and confirmation
of osteoblast phenotype with specific markers - the
principle of the method. Mesenchymal Stem cells located
in the second passage were used to obtain osteoblasts by
differentiation in the osteogenic medium. After 28 days
of cultivation in medium with dexamethasone, the cells
were tested for alkaline phosphatase activity, enzyme
expressed at elevated levels in osteoblasts and fewer
in MSC. We also tested the mineralization formation
centers in confluent cellular monolayer. Experiments
were performed in parallel on mesenchymal Stem cells,
differentiated osteoblasts and dermal fibroblasts as
negative control, because they do not belong to the same
cell line as the studied cells.
Romanian Journal of Legal Medicine RESULTS
A. Micro anatomic analysis of the remodeling
process inside bone compacta
From the analysis of serial cross sections
through decalcified fragments of tibia bone examined in
polarized light, we observed the existence of three micro
topographic areas: an area with internal circumferential
bone lamellas, an area of interstitial bone lamellas and an
area with external circumferential bone lamellas. All areas
are dotted with osteons. The highest density of osteons
was noted in the intermediate zone. When examining
with 4, 10 and 20 objectives, we found heterogeneity in
shape and structure of osteons, induced by the variability
in spatial distribution of bone lamellas and osteon beams
(Fig. 1).The examination in polarized light through the
decalcified bone sections allows us only to guess osteon
borders without providing the certainty limit of an
osteon circumference (Fig. 1). Processing these sections
through histochemistry methods such as - PAS, Gomori
and Van Gieson- provides rigor in visualization osteon
borders. This border has been nominated as "cementalis
linea”. Certainty osteon border (line cementalis) used
as a reference in the morphometric determinations is
provided by examining the sections in interferential light
or standard image processing in the 3D system using
EMBOSS method. The study of cross and longitudinal
sections of tibia fetus bone (8 months antepartum)
allowed us to observe and record the following anatomic
and topographic aspects (Fig. 1): neovasculogenesis
associated with differentiation of collagen beam with
perivascular distribution; cutting tunnels with basis
targeted towards the periosteum; longitudinal routes of
vascular structures; centripetal differentiation of collagen
beam from osteon wall structure.
B. Morphologic and phenotype analysis of
osseous progenitor cells
In order to highlight active alkaline phosphatase,
NBT - BCIP (Nitro Blue Tetrazolium/5 -Bromo -4Chloro -3- Indolyl Phosphate) was used as specific
substrate of the enzyme, providing a color type reaction.
Thus, cells expressing active alkaline phosphatase
(ALP) color in dark purple. Differentiated cells are
highly colored with NBT - BCIP, MSC presents a basal
expression of ALP and dermal fibroblasts none. By MSC
differentiation, a confluent cellular monolayer with
mineralization properties is obtained. We tested the
behavior of differentiated cells by Alizarin Red S staining,
a colorant that specifically binds tissues calcium. This
colorant colors obviously better the differentiated cells
than the stem cells from which we started. This fact
demonstrates that the obtained osteoblasts are able to
form a mineralized bone tissue-like structure (Fig. 2).
Mineralization was assessed in cells stained with
Alizarin Red S, a red colorant that specifically binds
Vol. XXIII, No 1(2015)
calcium ions (Fig. 2). The differentiated cells were fixed for
20 minutes with a 4% p-formaldehyde solution, at room
temperature. The samples were then washed with PBS and
incubated with a 0.5 % solution of Alizarin Red / PBS for
10 minutes, at room temperature. Finally, the cells were
washed and examined under optical microscope. Next, we
performed cell viability tests; we studied the morphology
and adhesion of osseous progenitor cells after their
attachment to orthopedic surgery materials: bio glass
films deposited on titanium by pulsed laser deposition
(PLD) and hydroxyapatite films (HA) deposited on
quartz by radio-frequency magnetron sputtering (MS).
Due to these studies, the different effects of osseous
progenitor cells interactions with two types of materials
were revealed. Estimation of cell differentiation degree
requires numerous markers: core binding factor alpha 1
(Cbfa1 / Runx2) - the main transcription factor involved
in osteogenesis, collagen type I, osteopontin, osteonectin,
osteocalcin, bone sialoprotein and alkaline phosphatase
which can modify the mineralization process (Fig. 3).
Osteoblasts mineralization capacity can be revealed in
vitro, by Alizarin red staining. In vitro differentiation of
mesenchymal stem cells towards osteoblasts requires the
apposition of some factors which in vivo are released by
cells located in osteoblasts vicinity. Among these factors,
ascorbic acid and 1α, 25-OH vitamin D3 are mandatory
for extracellular matrix deposition and mineralization.
Dexamethasone is also used as differentiating factor
[9, 10] and enhancer of mineralization [11]. Therefore,
in the first stage, we evaluated, in vitro, mesenchymal
stem cell differentiation (MSC) into primary osteoblasts,
according to the protocol developed during the research
project conducted in collaboration with the Institute
of Biochemistry in Bucharest and we investigated their
specific molecular markers: alkaline phosphatase,
collagen, BSP, osteocalcin, osteonectin, CD29 (Fig. 2
and 3).
The defining marker of osteoblastic lineage is
the activity of alkaline phosphatase. Thus, after culturing
bone cells on biomaterials surface, we have fixed the
samples and incubated them with an alkaline phosphatase
substrate solution ELF 97, that becomes fluorescent
after cleavage activity of enzyme phosphate group. The
result confirms enzymatic activity in biomaterials and
standard cases. Also, one can notice the mitotic activity
of cells. Osteoblasts are cells that secrete collagen type
I and express proteins like osteocalcin, osteonectin,
osteopontin and BSP (bone sialoprotein). In our study,
we tested the expression and localization of some of them
within primary osteoblasts differentiated from MSC
(Fig. 3). In vitro osteoblasts express osteocalcin,
extracellular matrix protein that has the ability to
bind calcium and to participate in mineralized matrix
formation. They also express osteonectin and bone
sialoprotein, located mainly intra-cytoplasmic. These
experiments demonstrate that obtained bone cells
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Melinte P.R. et al. Anatomic phenotyping of osseous progenitor cells. Implications in tissue engineering and bone remodeling
I. Cavitas
Medullaris
II. Area with circumferential lamellas and
osteons
III. Area with osteons separated by interstitial lamellas
A
B
C
Figure 1. A: Distribution of osteons in relation to interstitial and circumferential osseous lamellas; transverse section through
tibia – one can observe circumfrential and interstitial osseous lamellas and osteons with concentric lamellas. Non stained section
examined in polarized light; 2D reconstruction, Oc. 7, Ob. 4. B: Central canal of osteon opens into medullary cavity; linea
cementalis is clearly visible at the periphery; in polarized light, osteon lamellas appear circumferential and elliptical; Gomori
argentic impregnation, Oc. 7, Ob. 20. C: Newly formed osteon inside fetus tibia, examined in polarized light; Hematoxiline Eosine
stain, Oc. 7, Ob. 20.
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Romanian Journal of Legal Medicine Vol. XXIII, No 1(2015)
A
B
C
D
E
F
G
H
I
J
K
L
Figure 2. A, B, C: Activity of alkaline phosphatase in osteoblasts differentiated from stem cells; A – derm fibroblast cells; B –
mesenchimal stem cells; C – osteoblasts differentiated from MSC and marked by NBT-BCIP. D, E, F: Mineralization centers
in osteoblasts differentiated from MSC; D – derm fibroblast cells; E – mesenchimal stem cells; F – osteoblasts differentiated
from MSC after the second passage were marked by Alizarin red that spots Ca2+ ions. G, H, I: Activity of alkaline phosphatase
expressed by bone cells after adhesion to tested and standard materials; G – standard probe; H – titanium and hidroxiapatite
biomaterial; I – hidroxiapatite biomaterial. J, K, L: Testing of specific osteoblast markers by immunefluorescence; J – osteocalcin;
K – osseous sialoprotein; L – osteonectine.
are functional and possess the expected phenotype.
Mineralized bone tissue formation was observed in
culture after one month differentiation and after reaching
the total confluence of cellular monolayer.
DISCUSSION
The 3D geometry of the osteons is generally
different inside compact or spongy bone, but the intimate
cellular processes are quite similar. Activation is the first
event of bone remodeling process and it is accomplished
six times a minute inside a normal adult skeleton [5, 12].
During the activation process, precursors of mononuclear
osteoblasts that derive from circulating monocytes,
gather together near the surface of the bone and fuse into,
leading to the formation of multinuclear osteoclasts. It is
not fully known at the moment, why some bone surface
are preferred rather than others, but a possible theory
appeared in the beginning of 1900 [5, 13-17] stating that
the osteocyte acts as a mechanical sensor, detecting the
flaws of mechanical properties of the surrounding bone
matrix and it communicates them to the bone surface
through a canaliculi network [5, 13]. Once formed, the
osteoclasts destroy the organic and inorganic parts of the
bone matrix. The osteoclasts move while digging inside
bone tissue, in spongy bone tissue and on the cortical
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Melinte P.R. et al. Anatomic phenotyping of osseous progenitor cells. Implications in tissue engineering and bone remodeling
A
B
C
D
E
F
G
H
I
J
K
L
Figure 3. Osteoblast cells observed in fluorescence microscopy; osteoblasts differentiated from stem cells after second passage on
hydroxyapatite. A, D, G – after 1 day; B, E, H – after 4 days; C, F, I – after 7 days; J – expression of osteocalcine; K – expression of
collagen type I; L – expression of osteonectine.
periosteal and endosteal surfaces. At cortical level, they
dig cylindrical canals that are quite parallel to the bone
longitudinal axis. Although multinuclear osteoclasts
initiate this process, there is evidence that says that the
final phase is achieved by mononuclear osteoblasts
derived from multinuclear cells [18]. The resorption
phase is followed by the reversal phase [19]. The
mononuclear cells implicated in the resorption process
disappear, probably by apoptosis and they are replaced
by osteoblasts while the cycle evolves to the formation
phase [5]. Osteoblasts teams follow the osteoclasts as they
cross the bone surface or dig tunnels inside bone cortical.
The osteoblasts fill the grooves with lamellar bone tissue
66
organized as osteons, on the bone surfaces. Inside bone
cortical, the osteoblasts create concentric bony lamella
disposed in a concentric manner in order to refill the
bony tunnels. The traverse sections through cortical
osteons are consequently branchy ramifications that form
in a similar manner except the fact that the bony lamella
“grow” predominantly to the center.
Bone remodeling, in adults, realizes continuously
renewal of bone matrix at the level of small regions of
bone called "remodeling units”. In this case, in a rigorous
coordination, osteoclasts and osteoblasts carry out their
work in the same place. Although there are numerous
data regarding the osteoclasts central role in the genesis
Romanian Journal of Legal Medicine and regulation of bone mass structures, there is little
information on its role in bone remodeling [3]. Many
questions remained unanswered about the osteoclasts
ability to recognize places where they will act and about
the selection of these places. One of the biggest enigma
of bone biology is finding the factors that determine cell
sequence inside the "bone multicellular unit” - osteoclasts
and osteoblasts. Little is known about the nature of
well-established interrelations between osteoclasts and
osteoblasts [3, 20, 21].
Bone development involves three basic
mechanisms: longitudinal growth, modeling and
remodeling. During growth, a continuous adaptation of
macroscopic bone shape is performed [3, 5, 22, 23]. Bone
structures surfaces suffer modeling processes by emerge
of continuous resorbtion in some areas and continuous
formation in other areas. In this way is achieved the bone
form sculpture, by adding bone material in some areas
and its removal in others. In remodeling process, bone
structures are permanently renewing in the bone during
lifetime. Remodeling is a phenomenon that takes place
on all bone structures surfaces. It is easily remarked the
anatomical difference between modeling and remodeling
[3, 24-26]. Modeling of bone structures is achieved by
osteoclastic and osteoblastic activity, located on different
anatomical structures. Remodeling takes place through
successive osteoclastic and osteoblastic activity, located
on the same anatomical structure of the bone [3, 8, 27].
CONCLUSION
Compact bone part is a heterogeneous structure
through the variability of the form of central canal and
of the osteon wall structure. Morphological balance of
compact bone is provided by osteoclast and osteoblast
cells activity as part of remodeling unit. In the absence of
specific markers for osteoclasts, we take into account that
the evaluation of osteons in the remodeling cycle may
be achieved by using classical methods of examination
Vol. XXIII, No 1(2015)
in direct light, polarized light, phase contrast, dark
field and / or processing these images using the Emboss
method in interferential lines. Linea cementalis must be
regarded as the border of mature osteon; resorption line
is a morphological evidence for the beginning of a new
cycle of remodeling of mature osteon and of regenesis for
a new osteon. Our morphologic observations not only do
they have major implications in biomechanical and bio
pathological studies, but also in medico-legal expertise of
bone fragments.
Following the analysis of our observations, we
found that osteoblast and adult stem cells have got close
to each other at the level of plasma membranes and this
fact could be explained in two ways: cells tend to switch
portions of membrane at the tangent area between them,
or we are witnesses of a cell fusion phenomenon between
two cells, forming a hybrid one.
On the first day of cultivation, cells from stemprimary osteoblast cultures have mainly round form
supported by the radial distribution and in concentric
circles of the actin filaments of cytoskeleton; at 4 days
it is observed that stem cells acquire the characteristic
fusiform morphology and osteoblasts have polygonal
shape; interaction between them is evident at 4 days,
sometimes leading to fusion/co-localization of membrane
markers.
Immunofluorescence experiments showed that
osteoblasts differentiated in vitro express characteristic
markers for the bone line; osteocalcin is massively
secreted by bone cells and it can be seen intracellular
but especially around the cell where it tends to form
mineralized bone matrix; type I collagen may be
observed intracellular during trafficking or towards cell
membrane where it forms extracellular matrix fibers; BSP
is a protein with perinuclear localization, osteonectin
appears in all cytoplasm and in the dendritic extensions
of the membrane. The study of osseous progenitor cells
morphology and specific markers could be useful for
designing biomaterial used in orthopedic surgery.
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