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Bone Reports
journal homepage: www.elsevier.com/locate/bonr
Unique anabolic action of stem cell gene therapy overexpressing PDGFB-
DSS6 fusion protein in OVX osteoporosis mouse model
Wanqiu Chen
a,1
, Samiksha Wasnik
a,⁎,1
, Yawen Fu
a
, Leslie Aranda
a
, Charles H. Rundle
a,b
,
Kin-Hing William Lau
a,b
, David J. Baylink
a
, Xiaobing Zhang
a
a
Department of Medicine, Division of Regenerative Medicine, Loma Linda University, Loma Linda, California, USA
b
Musculoskeletal Disease Center, Jerry L. Pettis Memorial Veterans Affairs Medical Center, Loma Linda, CA, USA
ARTICLE INFO
Keywords:
Osteoporosis
OVX
PDGFB
DSS6
Fusion protein
Stem cell therapy
ABSTRACT
In the present study we sought to improve the efficacy and safety of our Sca1
+
PDGFB stem cell gene therapy for
osteoporosis in ovariectomized (OVX) mouse model. This therapy is administered by marrow transplantation.
We established the promise of this approach by previously showing that this therapy in normal mice increase
bone density, increased endosteal cortical and trabecular bone formation, caused de novo trabecular bone
formation, increased cortical thickness and improve bone strength. In the current study we produced a fusion
gene, PDGFB-DSS6. We reasoned that the DSS6, calcium binding protein would trap the PDGFB at the bone
surface and thereby limit the amount of PDGFB required to produce an optimal bone formation response, i.e.
efficacy with a lower engraftment. The result shows that indeed with a very low level of engraftment we
achieved a large increase in bone formation in the OVX model of bone loss. Serum analysis for biochemical
marker of new bone formation showed an approximate 75% increase in alkaline phosphatase levels in
Sca1
+
PDGFB-DSS6 group as compared to other groups. Quantitative analysis of bone by microCT showed a
massive increase in trabecular bone density and trabecular connectivity of the femur in the metaphysis in Sca1
+
PDGFB-DSS6 group. The increased cortical porosity produced by OVX was replaced by the Sca1
+
PDGFB-DSS6
therapy but not by the positive control Sca1
+
PDGFB. Additionally, an increase in the femur bone strength was
also observed specifically in Sca1
+
PDGFB-DSS6 as compared to other treatment groups, emphasizing the
functional significance of the observed anabolic action is on bone formation. In future work we will focus on
nontoxic preconditioning of our marrow transplantation procedure and also on transcriptional control of ther-
apeutic gene expression to avoid excess bone formation.
1. Introduction
In the US alone, there are 10 million non-traumatic fragility frac-
tures annually (Osteoporosis: Fragility Fracture Risk: Osteoporosis:
Assessing the Risk of Fragility Fracture, 2012). The most common osteo-
porosis-associated fracture is a vertebral compression fracture, which is
associated with substantial morbidity (Kanis, 1994). Antiresorptive
therapy has been shown to decrease vertebral fractures by 50%. Hip
fracture is much less common, but has 14–58% mortality within one
year of fracture (Schnell et al., 2010;Panula et al., 2011). Anti-
resorptive therapy only decreases non-vertebral fractures by 20–30%
(Kawai et al., 2011). Interestingly, anabolic therapies have been shown
to reduce non-vertebral and vertebral fractures by 35–60% (Minisola
et al., 2017;Rubin and Bilezikian, 2002;Neer et al., 2001). In addition,
current anabolic therapies are expensive, require strict compliance, and
lose their effectiveness in 1–2 years (Kawai et al., 2011). Therefore, a
stronger anabolic agent with a prolonged activity is considered to be a
major unmet therapeutic need in the treatment of osteoporosis.
One of the most important recent and novel findings on the pa-
thogenesis of osteoporosis is the identification of senescent bone cells,
mostly osteocytes but also osteoprogenitor cells (Farr et al., 2016).
Senescent cells are nonproliferating cells that secrete products detri-
mental to surrounding cells in tissue microenvironment. One might
expect that the accumulation of senescent cells would inhibit the action
of anabolic agents. However, this does not seem to be the case because
of the universal success of parathyroid hormone (PTH)-like medications
https://doi.org/10.1016/j.bonr.2019.100236
Received 8 May 2019; Received in revised form 6 December 2019; Accepted 9 December 2019
⁎
Corresponding author at: Department of Medicine, Division of Regenerative Medicine, Loma Linda University, Loma Linda, CA 92354, USA.
E-mail addresses: WChen@llu.edu (W. Chen), Swasnik@llu.edu (S. Wasnik), YFu1@llu.edu (Y. Fu), Charles.Rundle@va.gov (C.H. Rundle),
William.Lau@va.gov (K.-H.W. Lau), DBaylink@llu.edu (D.J. Baylink), XZhang@llu.edu (X. Zhang).
1
These authors equally contribute to this work
Bone Reports 12 (2020) 100236
Available online 11 December 2019
2352-1872/ © 2019 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/BY-NC-ND/4.0/).
T
(even in the elderly) to stimulate bone formation and reduce fracture
risk (Ascenzi et al., 2012;Papapoulos, 2015). Moreover, sodium
fluoride, which is a bone cell mitogen, has been shown to produce a
marked and prolonged stimulation of bone formation in elderly osteo-
porotic subjects (Rubin et al., 2001;Kleerekoper and Balena, 1991).
Despite its bone anabolic action, sodium fluoride impairs bone quality
and therefore is not considered an effective anabolic agent (Kleerekoper
et al., 1991;Gutteridge et al., 2002). These observations emphasize the
responsiveness of the aged osteoporotic skeleton to regeneration.
With respect to the development of stronger anabolic agents, we
have recently developed an anabolic agent, platelet-derived growth
factor-B (PDGFB) that cause a massive, prolonged increase in new en-
dosteal and trabecular bone formation in normal mice, which was at-
tended by a marked increase in bone strength (Chen et al., 2015). Re-
garding safety, PDGFB has been approved by the FDA for promotion of
wound healing (Fang and Galiano, 2008). Also in our previous study we
found no safety issues regarding PDGF administration (Chen et al.,
2015). The main concern about our approach is that toxic effect of
preconditioning needed for marrow transplantation. Marrow trans-
plantation was utilized in order to target the PDGFB to sites of bone loss
in humans (Chen et al., 2015); namely, the cortical endosteum, the
cortical canals, and trabecular bone (Seeman, 2013). The targeting
mechanism that we used was bone marrow transplantation, in which
we engineered hematopoietic stem cells (Sca1
+
cells) to overexpress
PDGFB. Total body radiation increases in the bone niche, SDF 1, which
is a chemokine for the CXCR4 receptor and expressed by Sca-1 cells.
Consequently, the Sca 1 cells localize to the hematopoietic stem cell
niche which is a site where bone is lost (Chanda et al., 2010). These Sca-
1
+
cells do not respond by proliferation to PDGFB in vitro, as men-
tioned earlier paper (Chen et al., 2015)
Total body irradiation or chemotherapy as preconditioning man-
euvers is not acceptable for a nonlethal disease, such as osteoporosis.
Therefore, we sought to further target PDGFB to the bone surface in an
attempt to reduce the Sca1
+
-PDGFB engraftment required for bone
Fig. 1. Schematics of the experimental design. Two weeks after OVX or sham surgery animals undergo irradiation and transplantation with Sca1
+
cells that were
transduced with lenti -GFP or Sca1
+
-PDGFB or Sca1
+
-PDGFB-DSS6, and bone tissues were analyzed 10 weeks later.
Fig. 2. DSS6 endows a bone surface binding ability to GFP. As mentioned in the method section, the bone slices were incubated for 1 h with 50 ng/ml of green
fluorescent protein (GFP) or GFP-DSS6. The bone slices were then rinsed extensively with PBS. Representative fluorescence images of the GFP-DSS6-bone slices
conjugation are shown here.
W. Chen, et al. Bone Reports 12 (2020) 100236
2
formation. This would be expected to decrease the dose of the pre-
conditioning agent. We reasoned that a calcium-binding peptide at-
tached to the PDGFB might concentrate the PDGFB at the bone surface.
In this regard, DSS6 peptide (six repetitive sequences of aspartate,
serine, and serine), is a calcium-binding peptide and has been shown to
target the bone surface (Yarbrough et al., 2010;Zhang et al., 2012). Our
proposed strategy takes advantage of the high affinity of a unique DSS6
peptide for calcium and employs this peptide as a novel targeting ve-
hicle to deliver and retain an osteogenic growth factor, PDGFB (in a
fusion protein form with DSS6), at the site of bone loss. The goal of the
present study was to perform a preliminary experiment to test our hy-
pothesis that marrow transplantation with Sca1
+
PDGFB-DSS6 would
exhibit greater bone formation than with Sca1
+
PDGFB. We chose the
head of the femur for our sampling site. We sought to evaluate efficacy
before proceeding with elaborate studies of the safety.
2. Material and methods
2.1. Animal study
Female C57BL/6J mice of 6 to 8 weeks of age were purchased from
the Jackson laboratory. All experimental protocols were approved by
the Institutional Animal Care and Use Committee at Loma Linda
University and the Animal Care and Use Review Office of the United
States Department of the Army. In conducting research using animals,
the investigators adhered to the Animal Welfare Act Regulations and
other Federal statutes relating to animals and experiments involving
animals and the principles set forth in the current version of the Guide
for Care and Use of Laboratory Animals, National Research Council.
2.2. Ovariectomy surgery
Ovariectomy (OVX) surgery was conducted on 2-month old C57BL/
6 J female mice. Mice were anesthetized by an intraperitoneal injection
of 105 mg/kg ketamine and 21 mg/kg xylazine (in a total of ~0.1 ml
volume). Body temperature was maintained by a 37 °C recirculating-
water heating pad. The back and sides of the mice were shaved and
cleaned with 70% ethanol and Betadine. Under aseptic conditions, the
pair of ovaries was removed from the mice by dorsal incision into the
region between the dorsal hump and the base of the tail. Removal of the
fimbrial end of the fallopian tube was done to ensure completeness of
the ovariectomy. The muscle incision was closed with 6–0 silk sutures,
and skin incisions closed with 3–0 silk sutures. Post-operative analgesic
(0.060 mg/kg in 0.05 ml buprenorphine, subcutaneously) was ad-
ministered for each mouse. After surgery, animals were treated for two
days, twice a day, with buprenorphine, and monitored closely there-
after. The animals were observed during recovery until alert and mo-
bile. The surgical procedure for control, sham-operated mice was the
same, except that the ovaries were not removed (Thompson et al.,
1995).
2.3. Bone marrow (BM) Sca1
+
cell isolation
Bone marrow Sca1
+
cell isolation was performed as previously
described (Chen et al., 2015). Briefly, bone marrow cells were har-
vested from mice femurs and tibias, and Sca 1
+
cells were purified
using Sca1
+
MACS magnetic beads (MiltenyiBiotec, cat no 130-106-
641). Before viral transduction, cells were cultured for 48 h. in Iscove's
modified Dulbecco's medium (IMDM, Invitrogen) containing 10% FBS
(Invitrogen) and 100 ng/mL each of human TPO, mouse SCF, human
Flt3L, human IL-3, and human G-CSF (Chen et al., 2015).
2.4. Lentiviral vectors and transduction of Sca1
+
cells
Human PDGFB ORF was subcloned into a lentiviral vector and fused
with DSS6 peptide under the control of the PGK promoter. Lentiviral
packaging was performed as previous described (Chen et al., 2015). SIN
lentiviral vectors were produced by transient transfection of the vector
plasmid in human 293T cells along with helper plasmid (CMVdR8.74)
and envelope plasmid (MD.G). Sca 1
+
cells were transduced once at a
MOI of 2 for 6 h. in six- well nontissue culture-treated plates precoated
with RetroNectin (Takara). From here onwards the lenti Sca1
+
-GFP-
PGK-PDGFB construct will be mentioned as Sca1
+
-PDGFB and Sca1
+
-
GFP-PGK-PDGFB-DSS6 as Sca1
+
-PDGB-DSS6 throughout the article.
2.5. Transplantation
Two weeks after ovariectomy surgery, OVX mice were irradiated
with a
60
Co source (Eldorado model, Atomic Energy of Canada) at a
single dose of 8 Gy (0.543 Gy/min) in the Department of Radiation
Medicine of the Loma Linda University. Twenty-four hours later,
1×10
6
lentiviral transduced Sca1
+
cells were resuspended in 200 μL
Fig. 3. Bone marrow transplantation and % engraftment. Two weeks after the
OVX surgery, the C57/BL6 mice were divided into 2 groups, and transplanted
with Sca1
+
cells that were transduced with, 1) Lenti GFP-PDGFB (wild-type),
and 2) Lenti GFP-PDGFB-DSS6. OVX sham surgery animals were served as
control, and transplanted with Sca1+ cells that were transduced with Lenti
GFP. To ensure engraftment of hematopoietic stem/progenitor cells, mice were
myeloablated by irradiation at 8 Gy before transplantation. Ten weeks after
transplantation, the engraftment level was evaluated by analyzing the bone
marrow cells for % GFP
+
cells by FACS. Data are means ± SEM **P< .01,
ns = not significant.
Fig. 4. High serum ALP levels were observed in the Sca1
+
PDGFB-DSS6 treated
animals, but not in the Sca1
+
PDGFB treated animals. The measurement was
conducted 10 weeks after transplantation. Data are means ± SEM. **P < .01,
***P< .001.
W. Chen, et al. Bone Reports 12 (2020) 100236
3
IMDM and transplanted into each recipient mouse via tail vein injection
under anesthesia. The mice were restrained in the mouse restrainer
during tail vein injection, and then properly returned to their cages
(Chen et al., 2015).
2.6. μCT analysis
AμCT analysis of the femoral bone was performed using a Scanco
VivaCT 40 instrument (Scanco Medical). Femurs were scanned at an
isotropic voxel size of 10.4 μm3, and energies of 55 keV and 70 keV
were used to scan the distal metaphysis and the midshaft, respectively.
For metaphyseal analysis, the region of interest included the in-
tramedullary space inside the endosteal surface at a distance proximal
to the condylar growth plate that was normalized for femur length. The
midshaft analysis region of interest used two contours to include the
cortex and exclude the intramedullary space, and was also normalized
for any variations in bone length. Within their respective regions of
interest, trabecular bone was segmented at a density of > 220 mg/cm
3
and the cortical bone segmented at a density of > 260 mg/cm
3
.
Trabecular and cortical partial bone volume (BV/TV) was determined.
Trabecular number, thickness and spacing were also examined, as was
the trabecular connectivity density. Cortical porosity was calculated as
1−BV/TV (midshaft) from the two-contour examination of the fe-
moral midshaft cortex (Chen et al., 2015).
2.7. Bone strength analysis
The mechanical strength of the femurs was evaluated at midshaft by
the three-point bending test, using an Instron DynaMight 8841 servo-
hydraulic tester (Instron). Bones were stored frozen in saline-soaked
gauze, thawed, and rehydrated in saline before testing. The femur was
positioned on the tester with the anterior aspect upwards on supports
that were 2 mm in diameter and 7 mm apart. The bone was preloaded
to 1 N at the midshaft and then loaded to failure using a blade excursion
rate of 5.0 mm/s (Chen et al., 2015).
2.8. Serum Alkaline phosphatase measurement
Serum ALP activity was measured by the QuantiChrom ALP kit
(BioAssay Systems) (Chen et al., 2015).
2.9. Effect of PDGFB-DSS6 IV injections on trabecular bone formation
At 1 month after ovariectomy, mice were received with PBS or
PDGFB-DSS6 (0.5 mg/kg or 5 mg/kg) i.v. thrice per week for 4 weeks.
Representative von Kossa staining images from L3 vertebrae showed
increased trabecular bone formation following PDGFB-DSS6 treatment.
2.10. GFP-DSS6 binding to bone chips
We choose DSS6, a six repeating sequence of AspSerSer (Fang and
Galiano, 2008;Seeman, 2013), as a fusion partner of PDGFB to allow
PDGFB to target mineralized bone surface. We first sought to confirm
that DSS6 could efficiently bind and localize DSS6 fusion protein at the
bone surface in vitro. Bone chips were first incubated with green
fluorescent protein (GFP) or GFP-DSS6 (50 ng/ml) for 1 h, and then
washed twice with 1× PBS. All images were captured with an Olympus
BX51 microscope system (Olympus).
2.11. Statistical analysis
All data were presented as mean ± SEM. OVX Sham and OVX
treated groups were compared by Student t-test or one way ANOVA. In
other parts two-way and one-way ANOVA were used. A P-value < .05
were considered statistically significant.
3. Results
3.1. Experimental strategy
The goal of this study was to study the bone anabolic efficacy of our
Fig. 5. Representative X-ray pictures of femurs harvested from mice received Sca1
+
GFP control, Sca1
+
PDGFB or Sca1
+
PDGFB-DSS6 cells. In each group, 6–7 mice
underwent OVX to induce osteopenia, followed by hematopoietic stem/progenitor cell transplantation 2 weeks later. Animals were analyzed at 10 weeks after
transplantation.
W. Chen, et al. Bone Reports 12 (2020) 100236
4
stem cell gene therapy in OVX mice. We compared Sca1
+
cells that
were transduced with PDGFB (control) vs. PDGFB-DSS6 (fusion protein
with bone specific targeting) to determine whether PDGFB-DSS6 was
superior to PDGFB alone in anabolic action. The primary end-point was
the amount and distribution of the new bone formed in response to our
stem cell gene therapy. Secondary end-points include x-ray imaging of
long bones, and serum alkaline phosphatase. Animals were divided into
4 groups: OVX sham, OVX GFP (untreated), OVX Sca1
+
-PDGFB and
OVX Sca1
+
- PDGFB-DSS6 (bone targeting). Fig. 1 shows the general
experimental design using the lenti transduced Sca1
+
cells.
GFP-DSS6 binds to bone slices.
We first determined the bone-targeting ability of DSS6. Incubation
of green fluorescent protein (GFP)-DSS6 fusion protein with bone slices
produced an intense green fluorescence as compared with GFP alone,
demonstrating that DSS6 fusion protein can target and bind to the bone
(Fig. 2).
3.2. Bone marrow transplantation
After total body irradiation each mouse in the stem cell therapy
group was injected with 1 million transduced Sca1
+
cells in-
travenously. To evaluate the level of engraftment of the Sca1
+
cells,
GFP
+
cells in peripheral blood was measured at 1 and 2 months after
transplantation by FACS analysis. In our Sca1
+
GFP-OVX control mice,
engraftment was about 18%, which is similar to what we have seen in
the past (Chen et al., 2015)(Fig. 3). However, in the two OVX groups
(PDGFB and PDGFB–DSS6) engraftment was only ~8% (Fig. 3). These
results raise the possibility that OVX reduces the engraftment of Sca1
+
cells.
3.3. Serum ALP at 10 weeks post therapy
At 10 weeks after the stem cell gene therapy, we determined the
serum ALP levels, which is a serum biomarker of bone formation.
OVX
Sham
Sca1-
GFP
Sca1-
PDGFB
Sca1-
PDGFB-
DSS6
0.000
0.005
0.010
0.015
0.020
0.4
0.5
0.6
0.7
0.8
BV/TV%
****
0
5
10
15
Tb.N (mm-1)
****
0.00
0.02
0.04
0.06
0.08
TB-Th(mm)
*
*
****
OVX Sham
Sca1-GFP
Sca1-PDGFB
Sca1-PDGFB DSS6
0
5
10
15
20
25
600
800
1000
Conn. Den (mm-3)
****
0
2
4
6
8
Cortical Porosity (%)
ns
****
****
***
A.
B.
OVX Sham
Sca1-GFP
Sca1-PDGFB
Sca1-PDGFB DSS6
Fig. 6. A) Representative MicroCT 3D pictures
of femurs harvested from mice received GFP
control, PDGFB or PDGFB-DSS6 overexpressing
Sca 1
+
cells. In each group, OVX was performed
in 6–7 mice to induce osteopenia, followed by
hematopoietic stem/progenitor cell transplan-
tation 2 weeks later. Animals were analyzed at
10 weeks after transplantation. MicroCT three
dimensional bone structure analysis of femurs
from OVX osteoporosis mouse after treatment
with Sca1
+
-PDGFB or Sca1
+
-PDGFB-DSS6.
PDGFB-DSS6 is a fusion protein of PDGFB and
bone-surface binding peptide DSS6. B)
Specimens were analyzed by microCT at
10 weeks after transplantation (n=4–7). The
following parameters of new bone formation
from the microCT analysis are shown: trabe-
cular partial bone volume (BV/TV); con-
nectivity density (Conn. Density); trabecular
number (Tb.N); trabecular thickness (Tb.Th),
and cortical porosity. Data are means ± SEM.
****P< .0001.
W. Chen, et al. Bone Reports 12 (2020) 100236
5
Compared to the OVX sham group, there was no significant increase in
the Sca1
+
group or the Sca1
+
-PDGFB group. However, the serum ALP
was 75% increase in the Sca1
+
-PDGFB-DSS6 group compared to the
Sca1
+
-GFP control group (Fig. 4).
3.4. X-ray of the femur long bones
We first determined bone mineral density of femurs at 10 weeks
after therapy by X-ray imaging. X-rays showed increased trabecular
bone, particularly in the metaphysis and femoral trochanter, and cor-
tical thickening in the Sca 1
+
-PDGFB-DSS6 compared to the Sca1
+
-
PDGFB and Sca1
+
groups (Fig. 5).
3.5. μCT of femur
In the Sca1
+
PDGFB-DSS6 group there was a massive increase in
trabecular bone density of the femur, particularly in the metaphysis
compared to the Sca1
+
GFP control and the Sca1
+
- PDGFB. No dif-
ference was seen in any of the μCT parameters between the Sca1
+
GFP
control and the Sca1
+
PDGFB (Fig. 6A).Nodifferences in bone density
were observed between the sham OVX control and the Sca1
+
GFP
control. However we saw highly significant increases in cortical por-
osity in the Sca1
+
GFP group and in the Sca1
+
-PDGFB group compared
to the non-OVX group. Accordingly, in this study statistically significant
differences in microCT bone parameters in response to OVX were ob-
served only in cortical porosity (Fig. 6B).
Strikingly, we observed a > 20-fold increase in BV/TV in the Sca1
+
PDGFB-DSS6 compared to the Sca1
+
PDGFB or the Sca 1-GFP controls.
In addition, trabecular number, trabecular thickness and trabecular
activity were all much greater in the Sca1
+
PDGFB-DSS6 that in the
Sca1
+
PDGFB or Sca1
+
GFP (Fig. 6B).
The connectivity density after Sca1
+
-PDGFB-DSS6 treatment
was > 50-fold higher than the Sca1
+
- PDGFB group, suggesting that
our therapy has a strong ability to cause de novo bone formation; i.e.,
trabecular bone formation where there was no bone before. We found
increased cortical porosity in Sca1
+
-GFP or Sca1
+
-PDGFB treated mice,
likely due to OVX-induced bone loss. However, Sca1
+
-PDGFB-DSS6
treatment completely replenished the lost cortical bone. (Fig. 6B).
These data demonstrate that the PDGFB-DSS6 therapy induces de novo
bone formation in OVX marrow cavity and corrects cortical bone loss
due to OVX.
3.6. Bone strength analysis
We then asked whether the increased trabecular connectivity and
decreased cortical porosity would translate into increased strength.
Bone strength was measured by the 3-point bending of the right femurs.
As expected, the Sca1
+
PDGFB-DSS6 group showed a significant in-
crease in bone strength compared to the other 3 test groups, including
the OVX sham (Fig. 7).
3.7. PDGF-DSS6 protein enhances trabecular bone formation
The striking results of PDGF-DSS6 gene therapy in OVX mice en-
couraged us to develop the PDGF-DSS6 protein to avoid marrow
transplantation. To this purpose, we have conducted preliminary stu-
dies. IV injection of PDGFB-DSS6 not only increased bone formation in
femurs (not shown), but also in lumbar vertebrae (Fig. 8) in a dose-
dependent manner. These results demonstrate that injection of PDGFB-
DSS6 protein is efficacious.
4. Discussion
The most salient finding in the present study was huge amount of
bone formation produced by our Sca1
+
PDGB-DSS6 stem cell gene
therapy, which targeted the PDGFB therapy to the bone surface by
virtue of DSS6, which is a calcium binding peptide. Here the Sca1 cells
are the source for the abundant production of PDGFB-DSS6. This ana-
bolic action of PDGF with the PDGF-DSS6 construct was achieved at a
much lower Sca1
+
cell engraftment compared to our earlier studies
(Chen et al., 2015), strongly suggesting a substantial reduction in the
ratio of PDGFB produced per unit of the amount of bone formed. The
future significance of this lower required anabolic dose is that it should
reduce the preconditioning dose required for an optimal anabolic ac-
tion. Moreover, the overall lower synthesis of PDGFB should reduce off
target effects with HSC PDGFB-DSS6 stem cell gene therapy.
In the PDGFB-DSS6 group, there was a large increase in trabecular
bone formation. This was associated with an enormous increase in
trabecular connectivity. Also there was new trabecular bone formation
in sites where trabecular bone had not been present in the past, de novo
bone formation. Another advantage of this therapy is to decrease in
cortical porosity, which is known to be important for the preservation
of bone strength (Augat and Schorlemmer, 2006). Parathyroid hormone
like therapeutic agents are also known to produce increased trabecular
bone formation (Calvi et al., 2001); whereas, Romosozumab apparently
can form new bone, in cortical as well as trabecular regions (McClung
et al., 2018;McClung et al., 2014;Graeffet al., 2015) however, the
robust anabolic effect of treatment is limited to the first few months of
therapy(McClung, 2018). In our previous work we found that PDGFB
stem cell therapy increases bone formation in vivo in red and in fatty
marrow(Chen et al., 2015). In our earlier study we found evidence for
differentiation capacity from the treatment of Sca1
+
cells over-
expressing PDGFB. We found an increase in BMP 2 expression, which
presented a conundrum because PDGFB alone does not cause bone cell
Fig. 7. The Sca1
+
-PDGFB-DSS6 treatment increases bone strength.
Representative loading force displacement graph presented. Three-point
bending test was used to measure bone strength at the midshaft of the femur.
Maximum load-to-failure on Sca1
+
-PDGFB-DSS6 treated femurs was sig-
nificantly greater than that of the other groups. Data are means ± SEM.
**P< .01, ***P< .001, ****P < .0001.
Fig. 8. PDGFB-DSS6 protein promotes bone formation in lumbar vertebrae in
OVX mice. At 1 month after ovariectomy, mice were received with PBS or
PDGFB-DSS6 (0.5 mg/kg or 5 mg/kg) i.v. thrice per week for 4 weeks.
Representative von Kossa staining images from L3 vertebrae showed increased
trabecular bone formation following PDGFB-DSS6 treatment.
W. Chen, et al. Bone Reports 12 (2020) 100236
6
differentiation in vitro. We concluded that PDGFB had some type of
secondary action in the marrow space which led to an increase in BMP
2. It requires the marrow space for PDGFB to have this action because
when PDGFB was given to rats it did not increase periosteal bone for-
mation (Mitlak et al., 1996). In our previous study of the treatment of
normal mice with PDGFB via overexpressing Sca1 cells we measured
both bone formation and resorption markers. We saw evidence of a
modest increase in bone resorption but a much greater increase in bone
formation (Chen et al., 2015).
We also found that PDGFB stem cell therapy was prolonged, to the
extent that the new trabecular bone takes a considerable portion of the
marrow cavity. This is not a disadvantage, because by using tran-
scriptional genetic control, it should be possible to utilize this stem cell
gene therapy according to the monitored skeletal needs of the patient;
viz, bone density and serum bone formation markers.
In the present study and in our previous study we found that the
increase in bone formation was attended by an increase in bone
strength. Because of the effect of our therapy to increase endosteal and
trabecular bone, and because our therapy increases bone formation in
both red marrow and fatty marrow sites, it is anticipated that endosteal
and trabecular bone formation would be increased throughout the
skeleton with this novel therapeutic regimen.
There were 2 unexpected findings in our study. First, in the 2 groups
that were OVX, the engraftment of our Sca1
+
cells was somewhat less
than we have seen previously in non-OVX mice (Chen et al., 2015;Hall
et al., 2007). In this regard, there are several studies suggesting that
estradiol has an impact on hematopoietic stem cells. Accordingly, in
mice estradiol increases the retention of hematopoietic stem cells in the
vascular niche in the bone marrow (Kim et al., 2017). Also estrogen
receptor alpha promotes mouse hematopoietic stem cell regeneration
(Chapple et al., 2018). Estrogen also promotes hematopoietic stem cell
renewal (Nakada et al., 2014). Probably relevant to the current study,
radiation significantly decreased marrow cellularity and increased the
amount of marrow adipose tissue in OVX mice compared to intact mice
(Hui et al., 2012). In aggregate, these findings suggest that OVX may
have modestly impaired engraftment of our Sca 1 cells. Consequently,
in the 2 groups of mice with OVX and PDGFB therapy there was a
significant decrease in engraftment. This unexpected finding revealed
the greater potency for increases in bone density in the PDGFB-DSS6
group over the PDGFB group.
Second, we did not detect a drop in total or cortical bone density in
response to OVX control mice. We have no definitive explanation for
these negative results. This could be related to the fact that there was a
simultaneously decrease in ovarian estrogen production and a bone
repletion formation process following radiation preconditioning, which
causes bone loss (Hu et al., 2010;Georgiou et al., 2012). In any case, we
did see a statistically significant increase in cortical porosity in the
Sca1
+
GFP in the Sca1
+
PDGFB groups, compared to the non-OVX
group. It seems likely that this is an OVX consequence. Noteworthy is
that the Sca1
+
PDGFB-DSS6 group completely eliminated the increased
porosity attributable to OVX. Thus our therapy mainly counteracts the
effects of OVX also the well-established effects of radiation bone da-
mage (Hu et al., 2010;Georgiou et al., 2012). Once marrow trans-
plantation is conducted with nontoxic preconditioning (see below) the
anabolic effects of our stem cell gene therapy may be even more sub-
stantial. One interesting potential of our stem cell gene therapy is a
possibility that mechanical loading at specific sites of the skeleton
would act to determine the distribution of new bone formation, al-
lowing for fortification of bone strength at specific sites considered to
be a risk for fracture. Considerable future work needs to be accom-
plished to optimize our stem cell gene therapy.
An alternative to our marrow transplant for targeting our anabolic
stem cell gene therapy to the sites of bone loss would be to utilize
PDGFB-DSS6 fusion protein in insulin pump type of administration or
by subcutaneous injection. However in both of these approaches there
would be a greater chance for extra skeletal effects of PDGFB than what
would occur with our Sca 1
+
PDGFB-DSS6 therapeutic. With regards to
the alternative strategy, our preliminary data with the direct IV injec-
tions of PDGFB-DSS6 protein increased the bone formation in in lumbar
vertebrae (Fig. 8) in a dose-dependent manner. These results demon-
strate that injection of PDGFB-DSS6 protein is efficacious.
In regard to safety in our previous study we saw no changes in
peripheral red blood count of white blood count and no changes in soft
tissue organs with the exception an enlarged spleen. We attributed the
increased spleen size to extra medullary hematopoiesis, because much
of the marrow cavity was overtaken by trabecular bone (Chen et al.,
2015). Regarding the adverse effects from preconditioning, there are
novel approaches in the process of development that could substantially
reduce the risk associated with preconditioning. Accordingly, an anti-
body technology to reduce the endogenous CD45 HSC, c-kit, is now in
clinical trials (Czechowicz et al., 2018). This therapy does not require
any toxic preconditioning and the only risk is a reduced white blood cell
count for approximately 2 weeks. Also, a valine deficient diet, which
has a temporary nutritionally toxic effect on hematopoietic stem cells,
has shown positive results for preconditioning in both mice and humans
(Taya et al., 2016). Therefore, in the future there is a possibility that the
adverse effects of preconditioning can be minimized to the extent that
the benefit to risk ratio would be sufficiently high to justify HSC-PDGFB
therapy for a subpopulation of severely osteoporotic patients. This
therapy would be expensive but current anabolic therapies are also
expensive (Martin, 2014).
As with any anabolic agent, there is a concern that PDGFB therapy
might cause oncogenic effects on extra skeletal bone or organ fibrosis.
That PDGFB does not produce heterotrophic bone formation was re-
cently demonstrated in the publication on four subjects with Kosaki
disease, which is caused by a gain of function through overexpression of
PDGFRB (Minatogawa et al., 2017;Gawlinski et al., 2018). These pa-
tients exhibited longitudinal skeletal overgrowth without heterotrophic
bone formation or evidence of oncogenesis. A soft tissue untoward ef-
fect was myofibromatosis. Nonetheless, for our therapy further onco-
genic studies of and fibrotic actions will be required in future studies.
Only limitation of our current study is the lack of tissue distribution
analysis of PDGFB-DSS6. Relevant to this issue is that when PDGFB was
given systemically to rats, there is an increase in bone formation but
this was attended by liver and kidney fibrosis(Mitlak et al., 1996),
which was most likely due to an elevated blood level of PDGFB.
However, in our previous study where we targeted the PDGFB with the
bone marrow transplant, we saw no increase in fibrosis of soft tissue
organs and there was no increase in serum PDGFB (Chen et al., 2015).
With our current study there would be even less likelihood increased
serum PDGFB or increased soft tissue fibrosis because of the targeting
action of DSS6. In any case, it will be important in future studies to
perform a thorough tissue distribution of PDGFB and of PDGFB-DSS6.
In conclusion, the results of this proof of principle study together
with those findings in our previous study (Chen et al., 2015) strongly
suggest that our robust anabolic agent coupled with successful skeletal
targeting mechanism has the potential to increase bone density and
strength at sites in the skeleton were bone is lost during osteoporosis.
For this therapy to be applied clinically, new strategies in bone marrow
transplant conditioning will need to be substituted for toxic pre-
conditioning (Hui et al., 2012).
Acknowledgements
Funding
This work was supported by the Telemedicine and Advanced
Technology Research Center (TATRC) at the US Army Medical Research
and Material Command (USAMRMC) under grant no. W81XWH-12-1-
0023 (DJB). The views, opinions and/or findings contained in this re-
port are those of the author(s) and should not be construed as an official
Department of the Army position, policy or decision unless so
W. Chen, et al. Bone Reports 12 (2020) 100236
7
designated by other documentation.
Author contributions
WC, SW, YF, LA, and CHR, performed the experiments. CHR, K-
HWL, DJB, and XZ interpreted the data. XZ and DJB conceived, di-
rected, and supervised the study. K-HWL provided critical reading of
the manuscript, all authors reviewed the manuscript.
Declaration of competing interests
The authors declare that they have no competing interests.
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