Bone 81 (2015) 715

Contents lists available at ScienceDirect

Bone
journal homepage: www.elsevier.com/locate/bone

Review

Interactions between osteopontin and vascular endothelial growth

factor: Implications for skeletal disorders
Divya Ramchandani, Georg F. Weber
James L. Winkle College of Pharmacy, University of Cincinnati, USA

a r t i c l e

i n f o

Article history:
Received 27 November 2014
Revised 9 February 2015
Accepted 8 May 2015
Available online 27 June 2015
Keywords:
Bone
Healing
Remodeling
Microgravity
Kidney
Angiogenesis
Hypoxia
Apoptosis

a b s t r a c t
Osteopontin (OPN) and vascular endothelial growth factor (VEGF) are characterized by a convergence in function
for maintaining the homeostasis of the skeletal and renal systems (the bonerenalvascular axis regulates bone
metabolism). The two cytokines contribute to bone remodeling, dental healing, kidney function, and the adjustment to microgravity. Often, they are co-expressed or one molecule induces the other, however, in some settings
OPN-associated pathways and VEGF-associated pathways are distinct. In bone remodeling, OPN and VEGF
are regulated under the inuence of growth factors and hormones, hypoxia and inammation, the microenvironment, and various physical forces. Their abundance can be affected by drug treatment. OPN and VEGF
are variably associated with kidney disease. Their balanced levels are critical for restoring endothelial cell function and ameliorating the adverse effects of microgravity. Here, we review the relevant 83 papers of 257 articles
published, and listed in PubMed under the key words OPN and VEGF.
2015 Elsevier Inc. All rights reserved.

Contents
1.
2.

Introduction . . . . . . . . . . . . . . . . . . .
Skeletal healing and remodeling . . . . . . . . . .
2.1.
Growth factors and hormones . . . . . . .
2.2.
Hypoxia and Inammation . . . . . . . . .
2.3.
Micro-environment . . . . . . . . . . . .
2.3.1.
Three-dimensional scaffolds . . . .
2.3.2.
Porous scaffolds . . . . . . . . . .
2.3.3.
Inorganic scaffolds . . . . . . . .
2.3.4.
Biodegradable scaffolds . . . . . .
2.3.5.
Growth hormone-coated scaffolds .
2.4.
Mechanical, thermal or electromagnetic forces
2.5.
Drug treatment . . . . . . . . . . . . . .
3.
Dental remodeling . . . . . . . . . . . . . . . .
4.
Kidney disease . . . . . . . . . . . . . . . . . .
5.
Adjustment to microgravity . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction

Corresponding author at: College of Pharmacy, University of Cincinnati, 3225 Eden

Avenue, Cincinnati, OH 45267-0004, USA.
E-mail address: georg.weber@uc.edu (G.F. Weber).

http://dx.doi.org/10.1016/j.bone.2015.05.047
8756-3282/ 2015 Elsevier Inc. All rights reserved.

For this comprehensive review, 257 articles with the key words
osteopontin or OPN and vascular endothelial growth factor or
VEGF in PubMed were screened (time frame from year 1996 to year
2014) (Fig. 1). After the exclusion of 37 papers, which did not focus on

D. Ramchandani, G.F. Weber / Bone 81 (2015) 715

40
35

Publications

30
25
20
15
10

2014

2013

2012

2011

2010

2009

2008

2007

2006

2005

2004

2003

2002

2001

2000

1999

1998

1997

1996

Year
Fig. 1. Publication statistics. Papers on OPN and VEGF by publication year.

the interactions between these molecules, 220 articles were selected.

Publications pertaining to cancer are covered elsewhere [1]. Here, we
discuss the references relevant to the skeletal and renal systems.
Together, our two reviews cover the currently available literature on
the subject.
2. Skeletal healing and remodeling
Throughout the processes of osteoblast differentiation, matrix mineralization, tissue repair and bone regeneration, the expression and
levels of OPN and VEGF vary (Table 1) [216].
During bone development, these cytokines contribute to osteogenic
differentiation and matrix mineralization [17]. VEGF stimulates
osteoblastic differentiation. OPN is present as one of the important
non-collagenous components of the extracellular matrix and is generated by osteoblasts prior to matrix mineralization.
OPN and VEGF contribute to tissue repair. Most relevant growth factors and extracellular matrix-associated gene products are elevated
in the repair phase during intra-membranous bone regeneration.
OPN and VEGF levels are up-regulated in the rst few weeks after
long bone fractures, accompanied by severe endothelial dysfunction.
While OPN levels fall back to normal in parallel to the fracture
healing process, VEGF remains up-regulated for a month during
the remodeling phase, even after full recovery, thus establishing its
possible role in osteoclastic activity [18,19]. Stress or fatigue fractures are incomplete, non-displaced fractures that occur following
repetitive loading, rather than a single traumatic event. Stress fracture healing leads to a signicant up-regulation of OPN, VEGF, osteoprotegerin, cyclooxygenase-2, and RANK ligand over unloaded
healing, suggesting roles for these molecules in the remodeling process [20].
2.1. Growth factors and hormones
Various growth factors and other proteins that are important for
bone formation affect the expression levels of OPN and VEGF. Bone
morphogenic proteins are of critical importance. During osteoblast progenitor cell differentiation in bone marrow stromal cells, exogenous
bone morphogenetic protein (possibly provided by native bone marrow
stem cells transduced with BMP-2 and VEGF [21]) increases timedependently the mRNA levels of both VEGF and OPN along with other
bone matrix proteins and osteoblast-related genes (type I collagen,

alkaline phosphatase, osterix, osteocalcin, and parathyroid hormone

receptor), thus enhancing osteo-inductivity, bone matrix formation,
and mineralization. VEGF, secreted by osteoblasts in response to bone
morphogenetic protein, is involved in coupling angiogenesis to bone
formation [17]. The bone morphogenic protein BMP-2 also enhances
ectopic bone formation by recruiting circulating bone marrow-derived
osteoblast progenitor cells via CXCR4/SDF-1 interaction at the site of
BMP-2 secretion. These circulating progenitor cells express CD44, a receptor of OPN, and their interaction likely contributes to the process
(of note, OPN may upregulate CXCR4/SDF-1 signaling [22]). Simultaneously, the VEGF levels in muscular tissue surrounding the source of
BMP-2 are elevated, thus suggesting a possible contribution by VEGF
to angiogenesis at the site of bone regeneration [23]. TGF- signaling
is important for ossication and chondrocyte differentiation. In the absence of its type II receptor, the expression of OPN and VEGF is lowered
and terminal differentiation is delayed [24]. The effects of osteoinductive growth factors (BMP-2 and TGF-1) may be enhanced by
thermal stress preconditioning. Together they elevate the expression
of OPN and VEGF along with osteocalcin, osteoprotegerin and cyclooxygenase 2 (COX-2), thereby speeding up the process of bone regeneration and healing as compared to heating or growth factor addition
alone [25]. Matrix vesicles, small molecules and plasma components
may stimulate osteogenesis. Their actions involve bone morphogenic
proteins as critical mediators. Matrix vesicles, present at the initiation
site of calcication in all skeletal tissues, carry morphogenic information
to nearby osteoblasts and chondrocytes in the form of osteogenesis promoting agents, including bone morphogenetic proteins (BMPs)-1
through -7 and angiogenesis stimulating proteins like VEGF and OPN
(which also recruits osteoclasts during the bone resorption process).
Other non-collagenous matrix proteins, including bone sialoprotein,
osteonectin as well as osteocalcin, contribute, thus promoting skeletal
cell differentiation and bone formation [26]. To accelerate bone healing,
BMP-2 (maybe in conjunction with VEGF or TGF-1) may be given
directly [17,27], after thermal stress preconditioning [25], in vesicles
[26], through the transduction of stem cells [21], or by implant coating
[28].
Precursor cells contribute importantly to osteogenesis. Cord blood
cells have mesenchymal multi-potency, and under lineage-specic stimulation may differentiate into osteoblasts, chondroblasts and adipoblasts.
They are thus an attractive source for the treatment of musculoskeletal
defects in tissue engineering. Upon stimulation of cord blood stem
cells with an osteogenic environment (consisting of dexamethasone,
-glycerophosphate and ascorbic acid) on a collagen I/III scaffold for
three weeks, the expression of osteogenic markers including OPN,
osteonectin, bone sialoprotein, collagen I and alkaline phosphatase
increases along with the angiogenic marker VEGF [29]. The same mixture
induces in embryonic stem cells a gene expression pattern similar to osteoblastic progenitors [27]. The intravenous transplantation of allogeneic
mesenchymal stem cells increases OPN and VEGF mRNA expression and
improves bone regeneration and angiogenesis in avascular necrosis of the
femoral head [30]. Upon stimulation with platelet-rich plasma, the expression of both VEGF and OPN along with other bone matrix proteins
increases in bone marrow cells, thus enhancing osteogenesis and angiogenesis during the wound healing process [31].
The SIBLING protein (small integrin-binding ligand, N-linked glycoprotein) matrix extracellular phosphoglycoprotein (MEPE) alters the
expression levels of both OPN and VEGF via its effects on the bone
renal axis and on bone metabolism. Its overexpression is correlated
with a reduction in circulating and urinary OPN and urinary calciumphosphate levels, thus decreasing diet-induced renal calcication. It is
also responsible for bonerenal neovascularization via an increase in
VEGF expression coupled with increased aldosterone levels [32].
Platelet-derived growth factor (PDGF) is a critical factor involved in
bone formation and an important regulator of signal transduction in
mesenchymal cells. While its function is commonly linked to OPN, its
interactions with VEGF are much more limited. Platelet-derived growth

D. Ramchandani, G.F. Weber / Bone 81 (2015) 715

Table 1
Functions of OPN and VEGF in bone. OPN and VEGF individually induce specic functions in various bone cell types.
Bone cell type

Function

Association with VEGF

Association with OPN

Osteoprogenitor cells

Develop into osteoblasts

Osteoblasts

Bone formation

Osteocytes

Maintenance of matrix
mineralization

Promotes osteogenic differentiation by vascularization [2]

and proliferation of pre-osteoblastic cells [3]
i. Produced by osteoblasts [5,6]
ii. Express VEGFR-1,2,3 and VEGF-A treatment induces
increases chemotactic migration and proliferation of
osteoblasts [7]
i. Osteocyte-secreted VEGF interacts with VEGFR-2 on
endothelial cells, activates MAPKERK signaling to
mediate angiogenesis [10]
ii. Apoptotic osteocytes secrete more VEGF than
non-apoptotic osteocytes [11]
VEGF promotes osteoclastic bone resorption and survival
of mature osteoclasts by the activation of intracellular
tyrosine kinase through the auto-phosphorylation of one
of its receptors (both KDR/Flk-1 and Flt-1 are expressed by
osteoclasts) [14]

A late-stage osteogenic differentiation marker, induced by

Wnt/-catenin signaling [4]
i. Expressed by osteoblasts [8]
ii. Upon OPN overexpression, there is enhanced nodule
formation, expression of differentiation markers (ALP and
OCN) and ALP activity by osteoblasts [9]
i. Expressed in osteocytes [12]

Osteoclasts

Bone resorption and

remodeling

ii. Hypoxia-induced osteocyte OPN mediates

disuse-induced bone resorption [13]
i. Expressed by osteoclasts [12]

ii. OPN mediates attachment of osteoclasts to bone mineral

to initiate bone resorption and remodeling [15]. Also, OPN
enhances osteoclast survival by a Ca2+-NFAT-dependent
pathway [16]

factor specically enhances OPN expression in marrow stromal cells

without affecting the expressions of other osteoblastic differentiation
markers (specically ALP and collagen type I). Thus it is not a differentiating factor [33]. In receptor turnover, PDGF-BB regulates the recycling
of the OPN receptor integrin V3 after internalization back to the cell
surface for the formation of focal complexes that are necessary for cell
adhesion in osteoblasts [34]. The presence of PDGF-BB potentiates the
FGF-2-mediated VEGF expression via the MAP kinase and SAPK/JNK
pathways [33].

2.2. Hypoxia and Inammation

In bone healing, damage to the vasculature at the defect site can
create a low oxygen environment compared with healthy bone. The
transcription factor core binding factor l (Cbfa1) enhances the osteogenic differentiation of mesenchymal stem cells under hypoxia by increasing the expression levels of both OPN and VEGF along with bone
morphogenic protein [35]. A combination of hypoxia and bonederived scaffolds aid tissue engineering via increasing the expression
of both osteogenic and angiogenic markers (OPN, VEGF, RUNX-2 and
BMP-2/4), thus enabling the differentiation of bone marrow-derived
mesenchymal stromal cells [36]. The temporary exposure of mesenchymal cells to hypoxia may provide a favorable means of treatment
for avascular bone injuries. Mesenchymal stromal cells can be used
in biocompatible scaffolds, such as mesoporous bioactive glass, for
restoring bone function. Although bone morphogenetic proteins and
VEGF could stimulate these cells, their direct delivery is compromised
due to size constraints. A temporary exposure to hypoxia can be
accomplished with the cell-permeable prolyl hydroxylase inhibitor
dimethyloxallyl glycine. It leads to a decrease in osteoblastic marker
expression but effectively induces a sustained up-regulation of OPN
and an up to 2-fold increase in VEGF levels through a HIF-1 mediated
pathway [37,38]. In adipose tissue-derived stem cells, hypoxia causes a
HIF-1-independent decrease in the expression of osteogenic markers
(including OPN, alkaline phosphatase, and osteonectin) and a HIF-1dependent increase in VEGF secretion [39].
Up-regulation of OPN and VEGF plays important roles in hypoxic
environments created as a result of inammation or tissue injury.
Osteoblasts from the sclerotic zones of osteoarthritic sub-chondral
bone express higher levels of VEGF and OPN than osteoblasts from
non-sclerotic regions. Whereas VEGF gene expression remains rather
constant, OPN protein expression increases [40]. The levels of OPN
along with tissue transglutaminase increase in calcic tendinopathy

without any change in the levels of VEGF in the calcied areas. This
increase does not match other types of lesions [41].
2.3. Micro-environment
Skeletal remodeling or regeneration is dependent on microenvironmental factors. Those include a 3-dimensional scaffold and perforations in the resident bone bed. The material used in tissue engineering is important and the performance of inorganic scaffolds needs to be
compared to biodegradable ones. Finally, the enhancement of healing
by growth factor coating of implant materials may provide substantial
support by combining structural and hormonal stimuli.
2.3.1. Three-dimensional scaffolds
Although conventional culture conditions grow cells on smooth
surfaces, the in situ micro-environment is 3-dimensional. Differential
responses between these settings, related to OPN and VEGF expression,
occur in articular chondrocytes [42], bone marrow stromal cells [43,44],
liposuction-derived stem cells [45] and osteo-progenitor cells [46].
Bone marrow stromal cells, when seeded onto collagen scaffolds in osteogenic media, undergo differentiation to form cells of osteogenic
and vascular lineages. In 3-dimensional scaffolds, they display a high expression of both OPN and VEGF, even without BMP-2. The VEGF receptor (VEGFR-2) is continuously expressed. On the other hand, when
stromal cells are cultured in 2D lms, mRNA expression of VEGF
becomes evident after a week, while OPN levels gradually increase
over 12 days, and may require treatment with BMP-2 [43,44]. Genetic
angiogenesis markers are enhanced prior to osteogenesis markers in
fat-derived stem cells in 3D scaffolds. VEGF is expressed as early as 1 h
after initiation of 3D culture and remains up-regulated by almost 7fold compared to 2D culture. Similarly, OPN shows a high baseline expression followed by a peak at day 10 in 3D, and continuously remains
up-regulated compared to 2D cultures. Thus, VEGF promotes early angiogenesis in 3D scaffolds followed by osteoblast differentiation [45].
Conventional monolayer culture of articular chondrocytes leads to a
loss of their chondrogenic phenotype and to their de-differentiation
into a broblast-like phenotype. Markers for hypertrophy, including
VEGF and OPN, are highly induced in monolayer and pellet cultures,
but have lower expression in 3D cultures. Conversely, chondrogenic
potential is present mostly in 3D cultures [42].
2.3.2. Porous scaffolds
The presence or absence of perforations in the resident bone bed affects vascularization and remodeling. Physical scaffold parameters, such

10

D. Ramchandani, G.F. Weber / Bone 81 (2015) 715

as pore size and pore geometry, determine their surface hydrophilicity

and permeability, thus affecting diffusion, oxygen tension and nutrient
exchange for bone marrow stem cells. Larger pore sizes with higher
diethyl fumarate incorporation in poly-propylene fumarate scaffolds
lead to a substantial increase in the expression of BMP-2, broblast
growth factor 2, TGF-1 (tumor growth factor-1), VEGF, the transcription factor RUNX2 and osteocalcin over scaffolds with smaller pore
sizes. Further, a more controlled pore geometry, obtained via stereolithographic scaffold design, enhances the expression levels of these factors, as well as alkaline phosphatase and OPN, over random pore architecture scaffolds with irregular pore architecture, thus promoting an
earlier osteoblastic differentiation and matrix maturation [47]. During
bone deposition and grafting, perforations in the receptor bed accelerate the incorporation process because they lead to increased revascularization and stimulated levels of bone deposition with signicantly less bone resorption in the perforated than the non-perforated
environment. Over several days, grafts on perforated beds show an earlier re-vascularization and higher bone density when compared with
non-perforated beds. There is a presence of higher levels of angiogenesis
and osteogenesis proteins (VEGF, OPN, tartrate-resistant acid phosphatase and alkaline phosphatase). OPN is continuously expressed postoperatively in both perforated and non-perforated settings, whereas
VEGF is initially expressed only in a perforated micro-environment
but peaks at day 60 in both settings. This early VEGF expression and
hence re-vascularization, accelerates the remodeling process and leads
to a higher bone deposition [48,49]. In bone tissue engineering, perfusion culture of osteo-progenitor cells within porous scaffolds elevates
the deposition of extracellular matrix via mechano-transductive autocrine signaling pathways that are responsible for higher levels of ERK,
P38, prostaglandin E2, cytosolic calcium and cyclooxygenase 2. Both
steady and intermittent perfusion regimens increase the levels of cyclooxygenase 2 within hours, VEGF after 24 h and OPN, osteocalcin, bone
sialoprotein and collagen-11 after two weeks of perfusion over static
culture [46]. A combination of hypoxia and highly porous bonederived scaffolds aid in tissue engineering via increasing the expression
of both osteogenic and angiogenic markers (OPN, VEGF, RUNX-2 and
BMP-2/4), thus enabling the differentiation of bone marrow-derived
mesenchymal stromal cells [36].

2.3.3. Inorganic scaffolds

Scaffold design in bone tissue engineering affects the expression of
osteogenic and angiogenic markers in bone marrow stem cells, with
suitable material leading to enhanced osteoblast differentiation and
faster tissue regeneration (Table 2). Titanium dioxide (TiO2) scaffolds
induce higher expressions of OPN, VEGF, IL-6 and osteoprotegerin
over silicon dioxide (SiO2) and calcium monophosphide (CaP) coatings,
they thus show better osteoblast differentiation capability [50]. Amorphous calcium phosphate decorated polyester scaffolds are promising
tools for bone tissue engineering. Specically, zirconia-hybridized
amorphous calcium phosphate elevates the expression levels of OPN
and VEGF along with bone sialoprotein, BMP-2, BMP-4 and osteocalcin
over zinc-hybridized amorphous calcium phosphate, which displays
higher alkaline phosphatase activity, prostaglandin E2 and increased
cell number and is associated with a sustained and longer release
of ions [51]. Perfusion ow in calcium phosphate scaffolds downregulates the expression of hypoxia-related HIF-1, OPN and VEGF,
while up-regulating osteogenic alkaline phosphatase and osteocalcin
in osteoblasts [52]. Copper-containing mesoporous bioactive glass
scaffolds promote OPN and VEGF expression along with HIF-1, alkaline phosphatase and osteocalcin, enhancing angiogenesis and osteogenesis for the treatment of bone defects [53].
2.3.4. Biodegradable scaffolds
Self-cell therapy is a bone tissue engineering technique, wherein
osteoblasts are cultured in biodegradable scaffolds to generate bone
implants for the treatment of critical size post-traumatic bone defects.
Three distinct scaffolds, demineralized cancellous (trabecular) bone,
autoclaved cancellous bone, and hydroxyapatite ceramics, are nontoxic, do not alter the proliferation of osteoblasts and can be used to
generate bone implants of clinically suitable sizes within less than
three weeks. Among these tissue carrier systems, VEGF mRNA levels
are similar. However, the expression of osteogenic differentiation
markers, like OPN along with osteocalcin, BMP-2A and alkaline phosphatase, is higher in demineralized cancellous bone than in autoclaved
cancellous bone or in hydroxyapatite scaffolds [54]. Biologically meaningful gradients in material properties can be obtained by changing
the composition of scaffolds. Among hydroxyapatite and poly(lactic-

Table 2
Tissue engineering in bone healing. The composition of the scaffold provided profoundly impacts the process of bone healing. IL-6 = interleukin-6, BSP = bone sialoprotein, BMP = bone
morphogenic protein, AP = alkaline phosphatase, PGE2 = prostaglandin E2, OCN = osteocalcin, OPG = osteoprotegerin, FN = bronectin.
Scaffold

OPN

VEGF

Others

Comment

Reference

Inorganic
Titanium oxide, silicon dioxide, calcium monophosphide

Increase

Increase

IL-6, osteoprotegerin

[50]

Amorphous, calcium phosphate decorated polyester

Increase

Increase

Copper-containing mesoporous bioactive glass scaffold

Increase

Increase

BSP, BMPs (zirconia),

AP, PGE2 (zinc)
HIF-1, AP, OCN

TiO2 N SiO2, CaP resulting in better osteoblast

differentiation
Zirconia-hybridized N zinc-hybridized,
perfusion ow suppresses OPN, VEGF, HIF-1

Coated
ECM-coated titanium
Biomimetically-coated implant
Simvastatin-coated scaffolds
Osteostatin-coated mesoporous ceramics

Increase
Increase
Increase
Increase

Increase
Increase

OCN
OPG, OCN
PCNA, RUNX-2

Biodegradable
Hydroxyapatite/collagen

Increase

Increase

FN

Hydroxyapatite/poly(lactic-co-glycolytic acid)

Increase

Increase

AP

Hydroxyapatite ceramics
Autoclaved cancellous bone
Demineralized cancellous bone

(Increase)
(Increase)
Increase

Increase
Increase
Increase

Amino acid nanobers

Increase
(sustained)

Increase
(transient)

OCN, BMP-2, AP

[51]
[53]

Increased bone remodeling after fracture

Suitable agents are BMP-2, VEGF, or both
Promotes osteogenic differentiation
Treatment of osteoporosis

[58]
[28]
[59]
[62]

Higher HA content increases matrix

deposition
Higher HA content (2.5:15:1) is more
efcacious for osteogenic differentiation,
vessel density

[56]

More effective than autoclaved cancellous

bone or hydroxyapatite scaffolds
Osteogenic differentiation

[55]

[54]
[54]
[54]
[57]

D. Ramchandani, G.F. Weber / Bone 81 (2015) 715

co-glycolic acid) (PLG) composite scaffolds, those with higher hydroxyapatite (HA) content (HA:PLG ratio 2.5:1 or 5:1) are more efcacious
than scaffolds with lower HA:PLG ratios (1:1 or 0:1) in promoting osteogenic differentiation and bone tissue repair. As the percentage of
hydroxyapatite increases, there is more widespread vessel density
throughout the scaffolds, and mesenchymal stem cells form more mineralized tissue with more functional shape and size. This is matched by
increased VEGF secretion and an increase in early and late markers of
osteogenic differentiation, including alkaline phosphatase and OPN
[55]. An increasing apatite content on collagen substrates leads to a proportional increase in the deposition of VEGF-A, OPN and bronectin.
This matrix deposition is responsible for cell survival via VEGF and improved cell adhesion via OPN. Ultimately, a uniform matrix deposition
leads to increased cell migration on collagen-mineralized surfaces
[56]. Simple repeating units of amino acids assemble into nanober
scaffolds, such as RAD16, a biomaterial with potential for applications
in tissue engineering. RAD16 has osteogenic differentiation properties.
It increases OPN and VEGF levels in bone marrow cells within two
weeks. Although VEGF expression goes down thereafter, OPN expression continues to increase until week four [57].
2.3.5. Growth hormone-coated scaffolds
The interactions between cells and scaffolds constitute a very
important component in tissue engineering, which can be enhanced
by growth factor coating of the scaffold materials. An early presence of
OPN around titanium implant surfaces increases bone remodeling
after a fracture. Bone healing around the implant can be improved by
coating it with extracellular matrix (generated from cultured cells) or
growth factors such as BMP-2 or VEGF, either alone or in combination.
Bio-mimetically coated implant surfaces display increasing levels of
OPN and osteocalcin (expressed before matrix mineralization) over
two weeks. Therefore, matrix or BMP-2 plus VEGF improve the effectiveness of osteoblast differentiation and matrix mineralization via angiogenesis [28,58]. An alternative coating agent on scaffolds for tissue
engineering may be simvastatin, which promotes osteogenic differentiation via enhancing the gene expression and secretion of both VEGF-A
and OPN along with other osteoblastic markers including osteoprotegerin and osteocalcin [59,60]. According to genome wide association
studies, OPN and VEGF-A are among the top genes associated with osteoporosis and modications of the jawbone [61] (the jaw is the secondary target of osteoporosis), and therefore are likely to play a role in its
pathogenesis. Osteostatin-loaded mesoporous ceramics lead to an
increase in OPN, PCNA, VEGF and RUNX2, thus improving bone regeneration in osteoporosis [62].
2.4. Mechanical, thermal or electromagnetic forces
Processes such as distraction osteogenesis, hydrodynamic shear
stress, ultrasound, pulsed laser deposition, thermal stress, and electromagnetic elds impose forces that alter the rate of bone formation
and the expression of various proteins and growth factors associated
with the regeneration process.
Distraction osteogenesis is a surgical procedure used to reconstruct
skeletal deformities and elongate the long bones. Uniaxial mechanical
strain, the underlying force, acts on osteoblasts to stimulate cellular
and molecular factors that aid in decreasing the treatment time. During
the phase of active distraction, there is a higher expression of angiogenic
factors (VEGF-A and -D, VEGFR-2 and neuropilin, ANG1 and ANG2, and
both TIE receptors), along with the extracellular matrix protein OPN and
bone morphogenic proteins. The marker levels for angiogenesis and
osteogenesis correlate with each other, reecting a major role for
blood vessel formation in the process. The up-regulation of mRNA for
VEGF and OPN, along with broblast growth factor 2 and collagen I,
occurs via gradual distraction, which leads to complete bony union during healing, whereas acute lengthening leads to a brous non-union.
VEGF and OPN levels show an early up-regulation, peaking under

11

distraction periods, more during gradual distraction than during acute

lengthening. The expression of VEGF and broblast growth factor-2 is
present at the leading edge of the distraction gap (albeit at a slightly
lower level compared to fracture healing) [6365].
Perfusion culture that applies hydrodynamic shear stress to bone
marrow stromal cells promotes the formation of extracellular matrix
more than static cultures. In bone tissue engineering, perfusion culture
of osteo-progenitor cells within porous scaffolds elevates the deposition
of extracellular matrix via mechano-transductive autocrine signaling
pathways that are responsible for higher levels of ERK, P38, prostaglandin E2, cytosolic calcium and cyclooxygenase 2. Steady or intermittent
perfusion regimens increase the levels of cyclooxygenase 2 within
hours, VEGF after 24 h, and OPN, osteocalcin, bone sialoprotein and
collagen-11 after two weeks of perfusion over static culture. An intermittent ow regimen, however, signicantly increases the levels of
prostaglandin E2 and enhances cellular retention over steady ow and
thus may prove to be important for mechano-transductive signaling
[46]. Under continuous or pulsatile ow, the expression of OPN and
VEGF mRNA is higher than that under static conditions. The secretion of
OPN protein, by contrast, is increased under pulsatile ow at a higher frequency over both continuous ow and static environments [66]. On the
other hand, perfusion ow in 3D calcium phosphate scaffolds downregulates the expression of hypoxia-related HIF-1, OPN and VEGF,
while up-regulating osteogenic alkaline phosphatase and osteocalcin in
osteoblasts [52].
Low intensity pulsed ultrasound, used to stimulate bone proliferation and differentiation of stem cells into osteoblasts, increases the expression of VEGF and OPN along with interleukin-8, broblast growth
factor 2, interleukin-1 and alkaline phosphatase [67].
Pulsed laser deposition, improves the physicochemical properties
of degradable and non-degradable polymers, used in bone replacement
or regeneration, by enhancing the homogeneity of contained bioactive
inorganic particles, thus increasing the angiogenic and osteogenic abilities of osteoblast precursor cells via up-regulating the expression of
VEGF, OPN, alkaline phosphatase, osteocalcin and collagen I [68].
In pre-osteoblastic cells, thermal stress preconditioning together
with tensile stress [69] or with osteo-inductive growth factors (BMP-2
and TGF-1) [25] elevates the expression of OPN and VEGF, and possibly
osteocalcin, osteoprotegerin and cyclooxygenase 2, thereby speeding up
the process of bone regeneration and healing as compared to heating or
growth factors alone.
Low-frequency electromagnetic elds or biphasic electric currents
may modify cell biochemistry in a way that is applicable to tissue regeneration. Calcium ion cyclotron resonance at dened frequency can aid in
the differentiation of cardiac stem cells and bone marrow derived mesenchymal stem cells while inhibiting vasculogenesis and without affecting
cell proliferation. It does so by increasing the expression of markers of
osteoblast differentiation, including alkaline phosphatase, osteocalcin,
and OPN, while reducing the expression of VEGF [70]. In contrast, the
stimulation of osteoblasts with a biphasic electric current increases cell
proliferation and expression of VEGF without affecting the levels of
osteogenesis-related genes, like OPN, alkaline phosphatase and collagen
I, or growth factors for osteoblast differentiation (BMP-2, -4, IGF-2 and
TGF-1) [71].
2.5. Drug treatment
Bone remodeling involves osteoblast differentiation, matrix mineralization and the expression of various proteins related to osteogenesis
and angiogenesis. The treatment with drugs, which possess various
mechanisms of action, has emerged as one of the options to accelerate
this process. Many of the agents act directly or indirectly on OPN and
VEGF. Acemannan or puerariae radix increase the expression of VEGF
and OPN, responsible for new capillary formation and osteoblast differentiation, leading to accelerated bone regeneration consecutive to tooth
extraction or wounding. Puerariae radix also induces the osteogenesis

12

D. Ramchandani, G.F. Weber / Bone 81 (2015) 715

markers collagen I, alkaline phosphatase and osteocalcin [72,73].

The phytoestrogen formononetin promotes fracture healing via enhancing VEGF and VEGFR2 levels specically at the fracture site, without affecting their expression at healthy sites. It also induces mesenchymal
differentiation by increasing the gene expression of OPN along with
osteocalcin, collagen I and alkaline phosphatase over two weeks [74].
Fracture healing can be supported by the local delivery of long-acting
insulin, which enhances the expression of the early osteogenic markers
collagen-12 and OPN, angiogenesis (through increased VEGF-C expression) and mineralized tissue formation [75]. Simvastatin coating
on titanium dioxide scaffolds promotes osteogenic differentiation via
enhancing the gene expression and secretion of both VEGF-A and OPN
along with other osteoblastic markers including osteoprotegerin and
osteocalcin [59].
3. Dental remodeling
Both OPN and VEGF are expressed in the developing teeth [76].
Dental pulp cells may differentiate into osteogenic or odontogenic
cells after tooth injury, such as crown fracture, because their expression
of VEGF and OPN, along with osteocalcin and HSP27, is increased [77].
Mechanical stress on the teeth may cause periods of hypoxia with ensuing apoptosis in cementoblasts. Hypoxia, created due to mechanical
stress during orthodontal treatment, decreases cell proliferation and increases apoptosis, associated with the elevated production of HIF-1,
VEGF and OPN. In this setting, the expression pattern of OPN mRNA is
correlated to the expression pattern of HIF-1. By contrast, long-term
hypoxia inhibits cementoblastic function, manifested by a decreased expression of OPN, alkaline phosphatase, osteocalcin, bone sialoprotein,
and osteoprotegerin [78]. N-acetylcysteine prevents the adverse effects
of resin-based dental restorations by increasing cell differentiation and
decreasing cell death in dental pulp stromal cells. The agent protects
dental pulp stromal cells from the deadhesion-induced decrease in
VEGF secretion and increase in apoptosis, partly by the induction and mobilization of NF-B to the nucleus which is then responsible for regulating
various differentiation and anti-apoptotic genes. N-acetylcysteine elevates the expression of OPN, osteocalcin and dental sialoprotein in a stepwise manner during cell differentiation [79] (Fig. 2).
The periodontal ligament contains abundant blood vessel networks,
providing the neighboring cells with an oxygen enriched microenvironment. During orthodontic treatment, the mechanical stress
applied to the tooth is transmitted to the root and alveolar bone by
the ligament, and leads to periodontal remodeling. Malassez's epithelial
rest cells respond to stretching forces with an up-regulation of HSP-70,
OPN and VEGF mRNA [80]. Growth factors, including epidermal growth
factor and nerve growth factor play important roles in the maintenance
and homeostasis of periodontal ligaments by Malassez's epithelial rest
cells. Nerve growth factor inhibits periodontal ligament calcication. It
decreases the levels of OPN, VEGF and BMP-2 when compared with
resting cells or cells exposed to epidermal growth factor. By contrast,

autocrine
survival
signal

VEGF
OPN

VEGF
OPN

X
mechanical stress
through
orthodontics

(reversal
of block)

NAC

apoptosis

(hypoxia)
blood
vessel

stromal pulp cell

or cementoblast

Fig. 2. Protection of dental cells by OPN and VEGF. Mechanical stress exerted by orthodontics may cause hypoxia and lead to cell death. The secretion of OPN and VEGF by pulp cells
or stromal cells protects from cell death in an autocrine fashion. N-acetylcysteine (NAC)
may restore OPN and VEGF production and anti-apoptosis under mechanical stress and
hypoxia.

Table 3
OPN and VEGF effects on the periodontal ligament. Various growth factors have distinct
effects on the secretion of OPN and VEGF, and they induce diverse functions.
VEGF

OPN

Biology

Reference

BDNF
EGF

Up
Up

Up
Unchanged

[82]
[81]

NGF

Down

Down

Periodontal tissue regeneration

Maintenance of periodontal ligament
homeostasis
Inhibition of periodontal ligament
calcication

[81]

epidermal growth factor alone increases the secretion of VEGF by

Malassez's epithelial rest cells without affecting OPN expression levels
[81]. Brain-derived neurotrophic factor is a secreted protein that improves periodontal tissue regeneration by its effects on the periodontal
ligament and endothelial cells, inducing proliferation and angiogenesis.
It time-dependently increases the mRNA expression levels of VEGF, and
dose-dependently increases OPN synthesis in periodontal ligament
cells. The induced OPN is localized at the dentin surface, where the
new cementum is formed, the induced VEGF enhances endothelial cell
proliferation and neo-vascularization [82] (Table 3).
When dental pulp stem cells and primary osteoblasts are cultured
on biocoral scaffolds, VEGF and OPN expression is elevated in both cell
types compared to no scaffold. OPN is expressed much earlier in osteoblasts than in dental pulp stem cells [83]. Enamel matrix derivative
in combination with natural bone mineral is clinically important in periodontal and oral regenerative surgery as it promotes the expression
of OPN and VEGF along with several other osteoblast differentiation
markers and growth factors in primary osteoblasts [84]. In demineralized
dentin tubes, OPN expression is elevated similarly by both Emdogain and
propylene glycol alginate over three weeks. VEGF mRNA expression is
elevated two weeks after propylene glycol alginate injection. Upon injection of Emdogain, by contrast, an increase in VEGF mRNA arises only after
three weeks [85].
4. Kidney disease
The skeletal and renal systems closely collaborate in maintaining the
homeostasis of electrolytes. OPN and VEGF are variably associated with
renal pathologies, including acute injury, nephrogenic systemic brosis,
renal lymph-angiogenesis, nephrotoxicity (OPN and VEGF are increased),
progressive renal failure (VEGF inhibits OPN expression), focal segmental
glomerulosclerosis (OPN is increased, VEGF is decreased), diabetes
(OPN and VEGF are reduced), and the treatment of chronic kidney disease (increased VEGF, decreased OPN).
VEGF and OPN are up-regulated in acute kidney injury [86]. In
nephrogenic systemic brosis (induced by gadodiamide), OPN and
VEGF are persistently elevated. Specically, OPN is involved in the
regulation of dystrophic calcication and contributes to inammatory
pathways as a chemo-attractant for macrophages, dendritic cells
and T-lymphocytes [87]. Chronic proteinuria-derived renal lymphangiogenesis is accompanied by tubular expression of OPN and VEGFC and precedes interstitial brosis [88]. In progressive renal failure,
VEGF treatment decreases brosis by reducing the OPN expression
levels in the renal tubules [89]. Podocyte injury in focal segmental
glomerulosclerosis leads to a down-regulation of VEGF in the diseased
glomeruli along with nephrin, -actinin-4, dendrin, FAT atypical
cadherin 1, and Wilms tumor protein 1. OPN, on the other hand,
displays substantial up-regulation and may contribute to sclerosis progression [90]. Short term exposure of kidney endothelial cells to high glucose in diabetes decreases the levels of OPN and VEGF leading to a
reduction in cell migration and capillary morphogenesis, thus setting
the stage for vascular complications at the later stages of the disease
[91]. HIF activation by prolyl hydroxylase inhibitors like dimethyloxalylglycine may be a strategy for the treatment of chronic kidney disease.
Activation of HIF-1 induces the expression of VEGF, which acts as a survival factor in podocytes. Dimethyloxalylglycine treatment also decreases

D. Ramchandani, G.F. Weber / Bone 81 (2015) 715

the levels of OPN and collagen IV, thus reducing macrophage inltration
and brosis to provide benecial effects [92].
Acute renal allograft rejection leads to an increased expression of
both OPN and VEGF. The perfusion of transplanted kidneys with a
monoclonal antibody against CD47 reduces ischemia-reperfusion injury
and lowers the levels of acute kidney injury biomarkers, including OPN,
VEGF, cystatin C, and TIMP-1. It thus improves transplantation outcomes [93].
Both OPN and VEGF may be markers of acute drug-induced or chronic nephrotoxicity. Various agents have distinct effects.
Gentamicin, ochratoxin A, sevourane, cisplatin, vancomycin and
bacitracin induce increased OPN expression [94].
Tacrolimus induces increased VEGF levels. Functionally, the induction
of VEGF by tacrolimus needs to be distinguished from VEGF increases
caused by lupus nephritis, kidney injury, or micro-albuminuric and
proteinuric diabetes [94].
Cyclosporin-mediated nephrotoxicity after transplantation leads to
an increased expression of both OPN and VEGF [94]. OPN levels rise
while VEGF levels are reduced. VEGF may have benet in the treatment of post-cyclosporine hypertension and nephropathy, because
it decreases OPN expression and reduces macrophage inltration
and collagen III deposition. Uric acid-lowering agents like the xanthine oxidase inhibitors allopurinol and benzbromarone decrease
OPN while restoring VEGF levels, thus limiting the severity of the
renal disease [95,96].
OPN, not VEGF, levels increase time- and dose-dependently in
aristolochic acid-induced renal injury, thus serving as an injury biomarker [97].
Long-term peritoneal dialysis may be employed in end-stage renal
disease. It can lead to encapsulating peritoneal sclerosis. The pathogenesis
of this complication is characterized by inammation, neoangiogenesis,
epithelialmesenchymal transition, and brosis. Matrix metalloproteinase 2 (MMP-2), which degrades type IV collagen, plays an important
role in the pathogenesis. The levels of the pro-angiogenic cytokines
OPN and VEGF, as well as transforming growth factor and monocyte
chemotactic protein 1, are correlated to MMP-2 [98].
5. Adjustment to microgravity
Extended space missions are associated with a prolonged exposure
to weightlessness (microgravity, very low g-forces). Their medical effects include muscle atrophy, osteopenia, slowing of the cardiovascular
system, anemia, balance disorders, and a weakening of the immune system, which in their sum lead to adverse health outcomes for astronauts.
Microgravity may have severe effects on various cellular features and
gene expression patterns by endothelial cells, which affect tissue remodeling. Balanced levels of OPN and VEGF are critical for restoring endothelial cell function and ameliorating the adverse effects of microgravity.
Endothelial cells are highly sensitive to conditions of weightlessness.
Their expression of various genes, including OPN, is up-regulated within
minutes of exposure to low g-forces and declines with time. Within a
week of microgravity, the endothelial cells display a delay in the formation of tubular structures of vascular intima, accompanied by a reduced
secretion of OPN, VEGF, broblast growth factor 2, soluble TNFRSF5,
TNFSF5, intercellular adhesion molecule-1 (ICAM-1), tumor necrosis
factor receptor 2 (TNFR-2), interleukin-18, complement C3, and von
Willebrand factor. There is an increase in necrotic cells. Consecutively,
the OPN protein levels slightly increase again over the ensuing week
and are higher than the OPN levels from cells under normal gravity.
By contrast, the levels of secreted VEGF are consistently lower in microgravity conditions than under normal gravity [99,100]. The introduction
of VEGF into endothelial cells in microgravity reduces cell death by suppressing the levels of FAS, PARP-116, PARP-85, NF-B, BAX and activated caspase-3, and it also counterbalances the increased levels of OPN

13

[101,102]. Although externally added VEGF and/or broblast growth

factor 2 do stimulate the formation of tubular structures by accumulation of secreted proteins, this increase is more profound under normal
gravity conditions than under microgravity [100]. The above observations led to the suggestion that improving the interactions of VEGF
and broblast growth factor 2 with endothelial cells under microgravity
conditions could lead to improved engineering of vascular intimas and
reduce the cardiovascular risks posed by prolonged space ights [100].
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