Extract
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Introduction
Enhanced bone formation is often required to treat bone
loss associated with trauma, revision joint arthroplasty, and
tumor resection. Autograft is currently the gold standard for
inducing bone repair. However, only a limited amount of
autogenous bone graft is available, and bone graft harvest
can involve substantial donor site
morbidity[1,2]. Therefore, there is great interest in identifying bone graft substitutes
that can stimulate bone repair. Bone morphogenetic
proteins (BMP) are known to possess strong osteo-inductive
properties and BMP gene therapy plays an important role in
modulating bone regeneration[3]. BMP are members of the
TGF-β (transforming growth factor-beta) superfamily of
growth factors[4,5]. Recombinant BMP stimulate bone
formation and repair in a variety of preclinical animal
models[6_8]. Recently, BMP have received FDA (Food and Drug
Admini-stration,USA )approval to treat recalcitrant tibial non-unions
[OP-1 (osteogenic protein-1)or BMP-7]. However, since very
large doses (ie milligrams) of BMP are necessary to induce a
biological effect in humans, more efficient and safer delivery
vectors must be obtained before clinical trials can be carried
out successfully. To date, viruses are the most efficient
vectors for gene delivery. Previous studies
have demonstrated that the adenoviral-mediated delivery of BMP can
stimulate new bone formation and healing in a critical-sized
femoral defect [9_11]. However, the application of adenoviral
vectors in clinical situations is limited by the lack of persistent
target gene delivery and the pronounced immune response in
immunocompetent animals and
humans[12,13].
Recombinant adeno-associated virus (AAV) may be an
ideal vector for delivering therapeutic factors. The vector is
non-pathogenic and elicits no inflammatory
response[14,15]. Furthermore, AAV often leads to efficient long-term
expression of secreted proteins both in vivo
and in vitro[16]. These advantages have been manifest in the considered use of
AAV in clinical trials[17]. However, only a few studies have
addressed the use of AAV vectors carrying BMP to induce
bone healing[18_20]. Thus, it is not known whether the
transfer of the BMP-7 gene into human adipose-derived
mesenchymal stem cells (ADMS) cells using a BMP-7-harboring
AAV could mirror the consistent success of these earlier
studies. This avenue is worth exploring, since committed
osteoblastic differentiation of ADMS cells would be
important prerequisite for AAV2_BMP-7 in vivo
gene therapy for bone healing.
Currently, there are 2 general types of pluripotent stem
cells that are potentially useful for gene therapy and tissue
engineering: embryonic mesenchymal and autologous
mesenchymal stem cells. Autologous mesenchymal stem cells
are more promising because they are immunocompatible and
there are fewer ethical issues to consider. Recent studies
have indicated that ADMS cells are capable of
differentiating along multiple mesenchymal cell lineages (osteoblasts,
adipocytes, chondrocytes, and
myoblasts)[21_25]. Under
osteogenic culture conditions, these cells can differentiate
into osteoblasts[24]. Large numbers of autologous ADMS
cells can easily be obtained from
fat[22]. Hence, these cells are a promising substrate for the clinical application of bone
tissue engineering. However, whether ADMS cells could be
effectively used in an ex vivo system (requiring harvesting,
manipulation, and re-implantation) for osteo-inductive
regional gene therapy requires further investigation.
The purpose of this study was to: (i) determine whether
human ADMS cells could be successfully transduced with
an AAV vector carrying the BMP-7 gene and express
the BMP-7 protein; (ii) observe whether these
BMP-7-expressing cells could display the differentiated osteoblastic
phenotype in vitro; and (iii) investigate whether implantation of
collagen-wrapped BMP-7-expressing cells could induce new
bone formation in vivo.
Materials and methods
Cells BHK-21 cells (ATCC, Manassas, VA, USA) were
cultured in Dulbecco's modified Eagle's medium (DMEM)
(Gibco BRL, Grand Island, NY, USA) containing 10% fetal
bovine serum (FBS, Gibco BRL, USA) and penicillin (100
U/mL)/streptomycin (100 mg/mL) (Sigma, St Louis, MO,
USA) at 37 oC in humidified atmosphere with 5%
CO2.
Animals Eight- to 12-week-old male severe combined
immune-deficient (SCID) mice were purchased from the
Animal Administration Center of Sun Yat-Sen University
(Guangzhou, China). All animal experimental protocols were
approved by the Animal Care and Use Committee of Sun
Yat-Sen University (China).
Plasmids The pcDNA1.1/AMP(ampicillin)-hBMP-7
plasmid was provided by Pu-yi SHENG of the First Affiliated
Hospital, Sun Yat-Sen University (China). The 1.3 kb BMP-7
coding sequence was amplified by a PCR system
(Invitro-gen, Carlsbad, CA, USA) from the pcDNA1.1(+) plasmid
containing the human BMP-7 cDNA. The primer sequence
for the PCR was as follows: upstream primer:
5'-GTG GTA CCG ATG CAC GTG CGC TCA CTG-3', and down stream
primer: 5'-AGA AGA TCT CTC GGA GGA GCT AGT GGC
AG-3', with the introduced Kpn I and
Sal I restriction sites under-lined. After purification, the gene fragment was cloned into
plasmid pUC18 (a vector for DNA sequencing)(Invitrogen,
USA) and the resulting recombinant plasmid was designated
pUC18-hBMP-7. pUC18-hBMP-7 was digested with Kpn
I and Sal I and further ligated to pSNAV(a plasmid shuttle for
packaged AAV) (AGTC Gene Technology, Beijing, China).
The resultant plasmid (pSNAV-hBMP-7) was transformed
into Escherichia coli DH5α, and positive colonies were
screened by PCR and restriction enzyme digestion.
Packaging, purification, and titration of the AAV
vector BHK-21cells were transfected with the purified
pSNAV-BMP-7 plasmid according to a standard calcium
phosphate precipitation method. The cells were then cultured in
selection medium containing 800 µg/mL G418 (Gibco BRL,
USA). G418-resistant BHK-21 cell clones were isolated and
the integrity of the hBMP-7 gene was determined by PCR
using the primers detailed previously. To package the virus,
stably-transfected BHK-21 cells were infected with
recombinant herpes simplex virus type 1 (rHSV-1), which can express
the AAV2 rep and cap genes of wild-type AAV. For
large-scale AAV production and purification, BHK-21 cells were
cultured in 6 roller bottles (110 mm×480 mm, Wheaton,
Millville, NJ, USA) at 37 oC at 1 roll/min. Confluent cells in
10 mL medium were infected with helper virus rHSV-1 at a
MOI (multiplicities of infection) of 0.1 and incubated for 2 h.
The collected cells were treated with chloroform,
PEG8000(Polyethylene Glycol 8000)/NaCl for precipitation, and
chloroform extraction for purification, sequentially. The viral
titer was quantitatively determined using a DNA dot
blot[26], and the purity was examined by SDS-PAGE. The titers
averaged approximately 4×1011 vector genomes (vg) per mL and
the purity was >95%. AAV-enhanced green fluorescence
protein (EGFP) was also constructed using the same
procedure. The pEGFP-C1 plasmid (TaKaRa, Shiga, Japan)
was used for the amplification of the EGFP-sequence.
Preparation of human ADMS cells from adult human
fat Human raw lipoaspirates from patients undergoing
selective suction-assisted lipectomy were collected after
obtaining informed consent from the patients according to
procedures approved by the Ethics Committee at the First
Affiliated Hospital of Sun Yat-Sen University (China). The
procedure described by Zuk et
al[27] was used with some modifications. Briefly, the raw liposuctioned aspirate was
extensively washed with D-Hanks' solution to remove
contaminating blood cells and local anesthetics. The
extracellular matrix was digested with 0.2% collagenase II (Sigma, USA)
at 37 oC for 30 min to release the cellular fractions. The cells
were washed twice, then plated in T-75 tissue culture flasks
at a density of 2×106/mL and cultured in DMEM/F-12
medium (Gibco BRL, USA) containing 10% FBS (Gibco BRL,
USA), 100 U/mL penicillin, and 100 µg/mL streptomycin
(Gibco BRL, USA) at 37 oC in a humidified atmosphere with
5% CO2. Once the adherent cells were more than 80%, they
were detached with 0.125% trypsin and 0.01% EDTA (Life
Technologies, Gaithersburg, MD, USA), and replated at a
1:3 dilution under the same culture conditions. All the
experiments were done with cells at the fifth and tenth
passages, and the results presented here are all based on
the fifth passage cell clones.
Immunophenotype analysis The medium was removed
from the flasks, and the cell layers were detached and washed
with phosphate-buffered saline (PBS, Gibco BRL, USA)
containing 0.5% bovine serum albumin (BSA, Sigma, USA), and
incubated with primary antibodies for 30 min at 4
oC. To detect intracellular antigens, we fixed the cells in 2%
paraformaldehyde for 15 min at 4 oC and then permeabilized
them with 0.1% saponin (Sigma, USA) for 1 h at room
tem-perature. Working concentrations for primary antibodies
against human CD29, CD31, CD34, CD44, CD45, CD105,
CD106, CD166, CD184, and HLA-ABC (BD Biosciences, San
Jose, CA, USA) were 10_20 ng/mL, respectively. We used
same-species, same-isotype-irrelevant antibody as the
negative control. After washing with PBS containing 0.5% BSA,
the cells were incubated with fluorescein isothiocyanate and
phycoerythrin-conjugated secondary antibodies for 30 min
at 4 oC. After three washes, the cells were resuspended
in PBS and analyzed by flow cytometry using a
FACSCalibur flow cytometer and CellQuest Pro software (BD Biosciences,
CA USA ).
AAV vector transduction The cells recovered after 2
passages were cultured as a monolayer in 6-well plates at a
density of 2×105 cells per well in DMEM/F-12 containing
10% FBS. Subconfluent cells were incubated with either
AAV2-BMP-7 or AAV2-EGFP at a MOI of
1×105 in a total volume of 500 µL serum-free medium for 1 h at 37
oC. The medium was then aspirated and 1 mL fresh growth medium
(FBS-DMEM/F-12 containing 30 mmol/L sodium butyrate)
was added. A MOI of 1×105 was chosen as a result of pilot
studies demonstrating that the MOI of
1×105 would produce the highest level of transgenic expression. Morphological
changes were monitored with a phase contrast microscope.
All experiments were carried out in triplicate. EGFP
expression was detected by fluorescent microscope or flow
cyto-metry.
Flow cytometry EGFP expression was detected in
AAV2-EGFP-transduced ADMS cells 1 week after transduction by
FACScan flow cytometry. One million harvested cells were
analyzed by flow cytometry, and the rest of the cells were
maintained in culture again for a subsequent analysis. The
percentage of EGFP-positive cells and the expression of the
mean fluorescence intensity (MFI) and EGFP were analyzed
using CellQuest Pro software (BD Biosciences, USA). The
EGFP expression of the cells was also detected by
fluorescence microscopy.
Determination of BMP-7 production To quantify the
expression level of BMP-7, ADMS cells culture medium was
collected from d 2 to d 56 after transduction for ELISA
analysis (ADL, San Antonio, TX, USA). ELISA was carried out
according to the manufacturer's recommendations. Briefly,
standards and culture medium were incubated at room
temperature with sample buffer in 96-well plates for 90 min and
then with biotin-labeled anti-human BMP-7 detection
antibody for 60 min. Finally, a streptavidin-horseradish
peroxidase conjugated antibody was added at room temperature
and incubated for 30 min. Bound BMP-7 was detected by
adding tetramethylbenzidine substrate solution for 15 min
and the plates were read at 450 nm.
For the Western blot analysis, human ADMS cells were
transduced with either AAV2-BMP-7 or AAV2-EGFP, or left
non-transduced. The harvested medium was collected on
d 28 and d 56 post transduction. Proteins were separated by
SDS-PAGE and transferred to the nitrocellulose membrane.
Following incubation with a goat polyclonal antibody against
BMP-7 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at
a dilution of 1:250, the membrane was incubated with a rat
anti-goat IgG-horseradish peroxidase (Zhongshan
Bio-chemical, Beijing, China) at a dilution of 1:1000.
Immunoreactivity was determined using the ECL chemiluminescence
reaction (Amersham, Arlington Heights, IL, USA).
ALP staining (Ca-Co technique) Cells that began to grow
on the cover glass 14 d after transduction were extensively
washed with PBS, fixed with cool acetone for 10 min, flushed
with distilled water, dipped into 5 mL buffer [consisting of
20 g/L β-phosphoric acid glycerin natrium (10 mL), 20 g/L
barbital sodium (20 mL), 20 g/L addex-magnesium (1
mL), and 5 mL distilled water] (pH =9.2, Zhongshan Biochemical,
China), and incubated at 37 oC for 6 h. The samples were
then washed with distilled water and dipped into 20 g/L
aqueous solution of cobaltous nitrate for 5 min. After another
wash with distilled water, the samples were dipped into
aqueous solution of sulphon ammonium for 3 min. Following a
final wash, each sample was dehydrated routinely, mounted,
and examined microscopically.
Chinalizarin staining Fourteen days after transduction,
Chinalizarin staining was performed. The culture medium in
the petri dish was withdrawn, and the cells were washed
with distilled water and fixed with 40 g/L formaldehyde. The
samples were stained with Chinalizarin S liquid for 5 min.
After washing with acetone, a mixture of acetone,
dimethyl-benzene, and distilled water, the samples were
microscopically examined.
Scanning electron microscopy
Fourteen days after trans-fection, the cells grown on the cover glass were fixed with 25
g/L glutaral for 24 h. After washing with PBS, the cells were
dehydrated with gradient ethanol, permutated with isoamyl
acetate, dehydrated at the critical point, and vacuumized with
spurted metal. Treated samples were observed with
JSM-T300 scanning electron microscopy (SEM, JEOL, Tokyo,
Japan).
Transmission electron microscopy
analysis Fourteen days after transfection, digestive juices, including
pancreatin-ethylene dinitrilotetraacetic acid, was fallen after
digestion, and a little DMEM culture medium including 10%
FBS was added to stop digestion. The suspension was put
in a centrifuge tube and centrifuged at
1000×g for 8 min. After being fixed with 25 g/L glutaral at room temperature for
12 h, the samples were put into 10 g/L osmium tetroxide and
treated for 24 h at room temperature. After being dehydrated
with gradient ethanol and permutated with acetone, the
samples were embedded with epoxy resin and cut into
ultra-thin sections. The cut sections were stained with acetic acid
double uranium and lead acetate, and its ultrastructure was
observed with JEM21200EX TEM (JEOL Company, Japan).
Osteocalcin production Osteocalcin secreted into the
culture medium was determined by a radioimmunoassay
using a human osteocalcin assay kit following the
manufac-turer's recommendations (Dongya Immune Technique
Institute, Beijing, China).
Implantation of BMP-2-transduced cells into SCID
mice Eight- to twelve-week-old male SCID mice were used in
this study. Animals were anesthetized with an intramuscular
injection of ketamine (1.5 mg) and xylazine (0.3 mg) and were
prepared for aseptic surgery. A 1 cm incision was made on
the lateral aspect of the left thigh. The quadriceps
musculature was identified in which a 1 cm slit was made. Two and
half million human ADMS cells transduced with
AAV2-BMP-7 at a MOI of 1×105 were harvested and resuspended with 50
µL PBS, and then placed onto a collagen type I matrix (Sangon
Biochemical, Shanghai, China) that was cut in the shape of a
5×3×2 mm3 rectangular block. Finally, these carriers were
implanted into a muscle pouch in the quadriceps portion of
the hind limb and the wound was sutured immediately. After
surgery, the animals were allowed ad
libitum activity. Human ADMS cells were transduced by infection with the
AAV2-BMP-7 vector at a MOI of
1×105 for 24 h, 7 d prior to implantation. Non-transduced and AAV2_EGFP-transduced
(at a MOI of 1×105) human ADAS cells served as the controls.
Six animals were allocated to each of the treatment groups.
Evaluations for bone formation Eighteen SCID mice were
sacrificed 3 weeks after implantation in a muscle pouch, and
radiographic examination was performed. The newly-formed
bone tissues were harvested from the hind limbs of the SCID
mice and fixed in buffered 10% formalin, and then decalcified
with 10% EDTA. The specimens were then dehydrated and
embedded in paraffin. The tissues were cut into 5
μm sections and stained with HE.
Statistical analysis For each experiment, multiple
samples (n=3) were taken, and data were reported as mean±
SD. Data were analyzed using the two-tailed Student's
t-test with a level of significance of
P<0.05. The SPSS 11.0 software package (Chicago, IL,USA) was used for the
statistical analysis.
Results
Morphology and phenotypes of cultured human ADMS
cells The adherent-cultured ADMS cells assumed a
fibroblast-like morphology when observed under a light
microscope. The morphology was maintained through
repeated subcultures under non-stimulating conditions. To
characterize the phenotypes of adherent adipose-derived
cells at the fifth passage, flow cytometry was performed.
The results showed that these cultured cells were positive
for CD29, CD44, CD105, CD166, and HLA-ABC. In addition,
no expression of the hematopoietic and endothelial lineage
markers (CD31, CD34, CD45, CD106, and CD184) was
observed (Figure 1). The phenotype was similar to those we
isolated from the bone marrow, except that the latter was
positive for CD106. It was also similar to that reported in
previous studies[27_29].
EGFP expression in vitro The expression of EGFP in
human ADMS cells, initiated 12 h after transduction with
AAV2-EGFP as the MFI increased gradually.
AAV2-EGFP-transduced ADMS cells demonstrated the highest MFI
(values were 1040) at d 7, and lasted for 8 weeks
[P<0.05 at each time point (different sample at each time point); Figure
2]. The flow cytometry analysis revealed that the expression
of EGFP in ADMS cells occurred in approximately 98.9% of
the cell population when infected at a MOI of
1×105. However, more than 60% of the cell population died when the MOI
was 1×106. More than 95% of the cell population died when
the MOI reached1×107 (Figure 3).
Phenotypic changes of rAAV2-BMP-7 transduced
cells Seven days after infection of human ADMS cells with
AAV2-BMP-7 or AAV2-EGFP at a MOI of
1×105, obvious cellular phenotypic changes were observed. Uninfected cells
or cells transduced with AAV2-EGFP displayed significant
fibroblast-like differentiation. However, ADMS cells
transduced with AAV-BMP-7 did not fuse and displayed
mononuclear ellipse-like or polygonal cell morphology, similar to
osteoblastic cells.
Expression of BMP-7 in ADMS cells The
in vitro
release kinetics of BMP-7 from AAV2-BMP-7-infected ADMS
cells was evaluated over the course of 56 d using ELISA. No
detectable BMP-7 was produced by the uninfected and
AAV2-EGFP-treated cells (t=14.34, P<0.05). However, cells
transduced with AAV2-BMP-7 produced low levels of
BMP-7 by d 2 (69.14±3.21
ng/106 cells), followed by an
increase in production with a mean of 146.45±10.60
ng/106 cells from d 6 to d 56 (Figure 4). Furthermore, the secreted
BMP-7 protein in the harvested medium of
AAV2-BMP-7-transduced human ADMS cells was confirmed by Western
blot analysis (Figure 5).
ALP and Chinalizarin staining ALP staining revealed a
small number of tiny, brown-black granules in the plasma.
The positive stain rate in the 500 cells evaluated was 85% in
the hBMP-7 group (Figure 6D), while no such granules were
evident in the non-treated and AAV2-EGFP-treated ADMS
cells. Fourteen days after transduction, the aggregation of
ADMS cells was obvious, and grew to form a calcium nod.
Chinalizarin staining produced a red color (Figure 6C),
indicative of Chinalizarin and calcium salts. No red
compound was found in the non-treated and AAV2-EGFP-treated
cells (Figure 6A,6B).
Observation under SEM and transmission electron
microscopy Calcium nod of cells were visualized as
high-density, irregularly-shaped, dense granules, the central
density of which was highest, decreasing gradually towards the
circumference (Figure 7). Collagen fibers were observed in
the stroma and were lined as a band shape with bubbles of
dense corpuscle, which was similar to matrix vesicles; part of
matrix vesicle started having deposition of calcium salts
(Figure 8).
Osteocalcin assay Osteocalcin production was detected
at d 14 (30.0±5.0 ng/mL) in the AAV-BMP-7-treated human
ADMS cells (Figure 9), but was undetectable in the untreated
and AAV-EGFP-treated human ADMS cells (t=23.97,
P<0.05).
Osteoinductive activity of transduced human ADMS
cells We transduced human ADMS cells with
AAV2-BMP-7 at a MOI of 1×105, and implanted them into the hind limb of
SCID mice to determine the biological activity of
genetically-modified cells in terms of ectopic bone formation.
Radiographic examination revealed ectopic bone formation in all
mice implanted with AAV2-BMP-7-transduced human ADMS
cells at 3 weeks after implantation (Figure 10C). A
histological examination revealed woven bone with reconstitution of
the bone marrow cavity in the muscle pouch 3 weeks after
implantation (Figure 11C). No ectopic bone formation was
seen in the control mice implanted with the naive ADMS
cells (Figures 10A,11A) or AAV2-EGFP-transduced cells
(Figures 10B, 11B).
Discussion
The present study demonstrates that the abundant and
easily obtained human adipose tissue is an ideal source of
autologous mesenchymal stem cells for gene therapy
application. Furthermore, AAV2-BMP-7 infects and efficiently
induces human ADMS cells to display the differentiated
osteoblast phenotype and ectopic bone formation in SCID
mice. To the best of our knowledge, this is the first report in
the field of AAV2-based BMP-7 gene transfer using human
ADMS cells. Ex vivo transduction of human ADMS cells
with AAV2-BMP-7 was associated with long-term transgene
expression in vitro and the induction of new bone formation
in vivo.
Protein therapies are hampered by high manufacturing
costs, unpredictable side effects, and the lack of an ideal
matrix to deliver proteins in a continuous manner over
time[30]. Therefore, both viral- and non-viral-based gene therapies
are currently being developed to enhance bone repair by
both in vivo and ex vivo
strategies[8,11,31_34]. Our goal has
been to develop regional gene therapy as 1 aspect of a
comprehensive tissue engineering strategy to enhance bone
repair. Previous studies have demonstrated that adenoviral gene
therapy is an attractive vector for regional gene
therapy[35_37],
but there are several potential limitations of adenoviral
vectors in clinical situations. Although adenoviral vectors
infect dividing and non-dividing cells, there is no integration
into the host genome, and protein production is limited to 2
weeks in vitro[38]. In addition, there is a marked immune
response to the adenovirus by immunocompetent animals,
which makes its clinical utility somewhat limited. Recent
studies have demonstrated that the AAV vector is ideal for
the delivery of therapeutic
factors[6,13]. The vector is non-pathogenic, elicits no inflammatory response, can infect
dividing and non-dividing cells, and often leads to the
efficient, long-term expression of secreted proteins
in vivo and in
vitro[9,16]. However, whether rAAV2-BMP-7 induces
human ADMS cells to display the differentiated osteoblast
phenotype in vitro and bone formation in vivo
requires
further study.
The present results confirm that the AAV2-BMP-7-
infected human ADMS cells display the osteoblast
differentiated phenotype and can induce new bone formation
in vivo. It has been reported that human ADMS cells are pluripotent
mesenchymal precursor cells, which are capable of
differentiating into myoblasts, adipocytes, and osteoblasts under
appropriate stimulation conditions[25]. In this study, we
observed that human ADMS cells infected with
AAV2-BMP-7 gave rose to 1 terminally-differentiated cell type expressing
the markers of osteoblasts and new bone formation
in vivo.
We report that the production of BMP-7 protein reaches
a peak as late as 1 week after infection, compared with
adenovirus-mediated gene delivery in human ADMS cells, in
which the desired BMP-7 protein is produced as early as 24
h after infection[39]. This may be due to the fact that the AAV
is a single-stranded DNA virus, and there is a rate-limiting
step of second-strand DNA synthesis in the nucleus of
infected cells. Some in vivo examinations have also
demonstrated the same delayed transgene
expression[40]. Further-more, we observed that the delayed BMP-7 protein
expression did not affect the osteogenic biological function of
BMP-7. From a clinical standpoint, it seems that the short
period of delayed osteoinductive protein production does
not hamper the treatment of relative longer-term cases of
fracture healing or spinal fusion. Delayed transgene
expression might protect secreted therapeutic proteins from
immunologic attack induced by destruction of the vascular barrier
at the time of virus injection[41].
Different types of clinical strategies will be necessary to
manage bone repair problems based on the extent of bone
loss and soft tissue injury. Recombinant proteins may be
suitable for small bone defects or primary bone repair
scenarios such as spinal fusion. Since AAV are associated with
transient BMP production, they may be more successful in
the treatment of small to medium-sized defects associated
with more adverse biological environments. Finally, the
potential for long-term protein expression associated with the
AAV vector may be better suited to use in more
sophisticated tissue engineering strategies that will be necessary to
treat massive bone defects often associated with tumor
resection, fracture nonunion, and revision total joint
arthro-plasty. Others have noted that transduced cells can
produce EGFP for several months. Since we do not have
satisfactory solutions for severe bone loss problems at this time,
the use of AAV gene therapy may be an effective strategy.
In addition, for some systemic and metabolic bone diseases
such as osteoporosis, the efficient long-term secretion of
BMP-7 proteins mediated by AAV is another outstanding
advantage. The advantages of direct gene therapy strategy
also include relatively simple technique requirements,
minimized invasion, and the potential for lower costs.
Direct in vivo application of the recombinant BMP-7
protein can induce the endochondral ossification
cascade[42], but its clinical use is severely limited by the lack of an
appropriate delivery system to achieve a sustained and localized
effect in vivo[30]. Our results demonstrate that a
genetically-engineered, cell-based protein delivery system using ADMS
cells can generate BMP-7 protein continuously until bone
nodules are formed in vitro, and that this sustained
BMP-7 delivery platform allows in vivo
bone formation. Although we found that heterotopic new bone was induced after
transplanting BMP-7-producing ADMS cells, we have no direct
evidence to demonstrate whether the implanted cells
underwent endochondral bone formation or just exerted their
effect as a vehicle delivering BMP-7, inducing new bone
formation in primitive pluripotent mesenchymal cells attracted
to the sites of implantation. In previous studies, Lee
et al[43] used Y-chromosome-specific FISH (fluorescence in situ
hybridization)to follow the fate of transplanted cells
in vivo. They demonstrated that only 5% of genetically-engineered,
original, muscle-derived cells differentiated into osteogenic
cells, while a large number of implanted cells enhanced bone
healing primarily by delivering BMP-2. Whether the ADMS
cells underwent the same fate in our study remains unknown.
Further investigation is being performed to determine the
early cellular events after transplantation.
One potential disadvantage of AAV vector use in this
setting is that prolonged BMP-7 production could lead to
heterotopic bone formation. This potential problem will have
to be evaluated when the AAV vector is tested in bone repair
models. One strategy to avoid continued protein expression
is to use a gene-regulated system such as a
tetracycline-regulated expression
vector[44]. A doxycycline-regulated system has been used to induce bone repair by regulated
the BMP expression in 2 different critical-sized defect
models[45,46]. In clinical situations when protein expression needs
to be tightly regulated, this type of regulated gene
expression system would be quite useful.
The mechanism of differentiation of human ADMS cells
into osteoblasts remains unclear. Nonetheless, we propose
that AAV2_BMP-7 gene therapy using human
adipose-derived mesenchymal stem cells represents a novel and
feasible approach for treating a variety of orthopedic problems.
Acknowledgement
We thank the AGTC Gene Technology Company for their
technical assistance in packaging the AAV vector.
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