An ectopic study of tissue-engineered bone with Nell-1 gene modified rat bone marrow stromal cells in nude mice.

Abstract

Tissue engineering techniques combined with gene therapy have been recently used to improve osteogenesis. NEL-like molecule-1 (Nell-1), a novel growth factor, has been reported to have specificity for osteochondral lineage. The study assessed the osteogenic differentiation of rat bone marrow stromal cells (bMSCs) after Nell-1 gene modification and examined its ectopic bone formation ability in a nude mice model with tissue engineering technique.
bMSCs obtained from Fischer 344 rats were transduced with either AdNell-1 (Nell-1 group) or Ad-beta-galactosidase (AdLacZ, LacZ group) or left untransduced (untransduced group). The expression of Nell-1 protein was determined by Western blotting and transfer efficiency was assessed. mRNA expressions of osteopontin (OP), bone sialoprotein (BSP) and osteocalcin (OC) were assessed by real-time PCR 0, 3, 7, 14, and 21 days after gene transfer. Alkaline phosphatase (ALP) activity was measured and von Kossa test was also conducted. Finally, with a tissue engineering technique, gene transduced bMSCs, combining with beta-tricalcium phosphate (beta-TCP) at a concentration of 2 x 10(7) cells/ml, were implanted at subcutaneous sites on the back of nude mice. Four weeks after surgery, the implants were evaluated with histological staining and computerized analysis of new bone formation.
Under current transduction conditions, gene transfer efficiency reached (57.9 +/- 6.8)%. Nell-1 protein was detected in Nell-1 group but not in untransduced group and LacZ group. Induced by Nell-1, BSP and OP expression were increased at intermediate stage and OC expression was increased at later stage. ALP activity and the number of calcium nodules were highest in Nell-1 group. Four weeks after implanted into nude mice subcutaneously, the percentage of new bone area in Nell-1 group was (18.1 +/- 5.0)%, significantly higher than those of untransduced group (11.3 +/- 3.2)% and LacZ group (12.3 +/- 3.1)% (P < 0.05).
This study has demonstrated the ability of Nell-1 to induce osteogenic differentiation of rat bMSCs in vitro and to enhance bone formation with a tissue engineering technique. The results suggest that Nell-1 may be a potential osteogenic gene to be used in bone tissue engineering.

Full text

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Chin Med J 2009;122(8):972-979
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Original article

An ectopic study of tissue-engineered bone with Nell-1 gene
modified rat bone marrow stromal cells in nude mice

HU Jing-zhou, ZHANG Zhi-yuan, ZHAO Jun, ZHANG Xiu-li, LIU Gen-tao and JIANG Xin-quan

Keywords: bone marrow stromal cells; Nel-like protein type 1 gene; tissue engineering

Background Tissue engineering techniques combined with gene therapy have been recently used to improve
osteogenesis. NEL-like molecule-1 (Nell-1), a novel growth factor, has been reported to have specificity for osteochondral
lineage. The study assessed the osteogenic differentiation of rat bone marrow stromal cells (bMSCs) after Nell-1 gene
modification and examined its ectopic bone formation ability in a nude mice model with tissue engineering technique.
Methods bMSCs obtained from Fischer 344 rats were transduced with either AdNell-1 (Nell-1 group) or
Ad-β-galactosidase (AdLacZ, LacZ group) or left untransduced (untransduced group). The expression of Nell-1 protein
was determined by Western blotting and transfer efficiency was assessed. mRNA expressions of osteopontin (OP), bone
sialoprotein (BSP) and osteocalcin (OC) were assessed by real-time PCR 0, 3, 7, 14, and 21 days after gene transfer.
Alkaline phosphatase (ALP) activity was measured and von Kossa test was also conducted. Finally, with a tissue
engineering technique, gene transduced bMSCs, combining with β-tricalcium phosphate (β-TCP) at a concentration of
2×107 cells/ml, were implanted at subcutaneous sites on the back of nude mice. Four weeks after surgery, the implants
were evaluated with histological staining and computerized analysis of new bone formation.
Results Under current transduction conditions, gene transfer efficiency reached (57.9±6.8)%. Nell-1 protein was
detected in Nell-1 group but not in untransduced group and LacZ group. Induced by Nell-1, BSP and OP expression were
increased at intermediate stage and OC expression was increased at later stage. ALP activity and the number of calcium
nodules were highest in Nell-1 group. Four weeks after implanted into nude mice subcutaneously, the percentage of new
bone area in Nell-1 group was (18.1±5.0)%, significantly higher than those of untransduced group (11.3±3.2)% and LacZ
group (12.3±3.1)% (P <0.05).
Conclusions This study has demonstrated the ability of Nell-1 to induce osteogenic differentiation of rat bMSCs in vitro
and to enhance bone formation with a tissue engineering technique. The results suggest that Nell-1 may be a potential
osteogenic gene to be used in bone tissue engineering.
Chin Med J 2009;122(8):972-979

T
he replacement of bone is a major clinical issue to
deal with. Tissue engineering has emerged as a
possible alternative strategy to regenerate bone. Three
components of tissue engineering essential to promote
bone regeneration are as follows: isolation and expansion
of osteoprogenitors or mesenchymal stem cells, provision
of appropriate osteoinductive factors, and an appro-
priately designed scaffold that mimics the structural
environment.1

It was reported that bone marrow stromal cells (bMSCs)
have osteogenetic differentiation potential in vitro and in
vivo,2,3 therefore bMSCs may play an important role in
the process of bone regeneration. Several studies have
shown that tissue-engineered bones constructed by
combining bMSCs with different biomatrices can
enhance the bone regeneration in vivo.4-6 To maximize the
capacity of bMSCs to regenerate bone, the applications of
exogenous growth factors such as bone morphogenetic
proteins (BMP)-2 or BMP-4, by protein release or by
gene transfer method have been shown to promote their
differentiation into osteoblasts and new bone formation in
vivo.7-10

BMPs, the commercially available recombinant growth
factors, are multi-functional. Alongside osteoinductivity
and bone formation efficacy, they also play important
roles in embryonic development and cellular functions in
postnatal and adult animals.11 However, the osteo-
inductivity of BMPs is not specific for bone cells and this
may give rise to overdose exerting, which in turn brings
unpredictable side effects locally and systematically.12
DOI: 10.3760/cma.j.issn.0366-6999.2009.08.018
Department of Oral and Maxillofacial Surgery (Hu JZ, Zhang ZY
and Zhao J), Shanghai Research Institute of Stomatology, Shanghai
Key Lab of Stomatology (Zhang XL and Jiang XQ), Ninth
People′s Hospital, School of Medicine, Shanghai Jiao Tong
University, Shanghai 200011, China
Department of Neurosurgery, Cedars-Sinai Medical Center, Los
Angeles, CA 90048, USA (Liu GT)
Correspondence to: Dr. JIANG Xin-quan, Shanghai Research
Institute of Stomatology, Shanghai Key Lab of Stomatology, Ninth
People′s Hospital, School of Medicine, Shanghai Jiao Tong
University, Shanghai 200011, China (Tel: 86-21-63135412. Fax:
86-21-63135412. Email: xinquanj@yahoo.com.cn)
This study was supported by grants from National Natural Science
Foundation of China (No. 30400502 and 30772431), Program for
New Century Excellent Talents in University (NCET-08-0353),
Science and Technology Commission of Shanghai Municipality
(No. 07DZ22007, 08410706400, 08JC1414400, and 08QH1401700),
Shanghai Rising-star Program (No. 05QMX1426), and Shanghai
Education Committee (No. 07SG19).

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Thus, it should be advantageous to explore and apply an
alternative growth factor that would provide a more
targeted and controlled rate of bone growth in certain
situations.

NEL (a protein strongly expressed in neural tissue
encoding epidermal growth factor-like domain)-like
molecule-1 (Nell-1) is a novel growth factor believed to
specifically target cells committed to the osteochondral
lineage.13,14 Previous studies have reported Nell-1 gene
up-regulation accelerates osteogenic differentiation and
bone formation in committed osteoblasts or goat
bMSCs.14-19 Alkaline phosphatase (ALP) staining was
reported to be more pronounced in goat bMSCs
transduced with
AdNell-1
Ad-β-galactosidase (AdLacZ), and the von Kossa staining
two weeks after gene transfer revealed a significant
increase in calcium nodules in AdNell-1-transduced goat
bMSCs as compared with the AdLacZ group.15 However,
osteogenic differentiation of bMSCs induced by Nell-1
gene thus far has not been extensively investigated in
vitro, and there is no
tissue-engineered bones with Nell-1 gene enhanced
bMSCs and 3-D biomatrics, by an ex vivo method.

In view of the potentially important role of Nell-1 gene,
we investigated the effect of Nell-1′s regulation of
osteogenic differentiation in rat bMSCs in vitro. Using an
ex vivo method, we constructed tissue-engineered bone by
combining
Nell-1 gene
β-tricalcium phosphate (β-TCP)
composites were implanted in nude mice subcutaneously.
We intended to assess whether more bone tissues could be
obtained after Nell-1 gene modification.

METHODS

Animal models
Six-week-old male Fischer 344 rats with a weight of
(125±15) g, and 6-week-old male athymic nude mice
with a weight of (20±2) g were enrolled in the
experiments. All procedures concerning animals were
approved by the Animal Research Committee of the
Ninth People′s Hospital, School of Medicine, Shanghai
Jiao Tong University (Shanghai, China).

bMSCs culture and gene transfer
Rat bMSCs were isolated and cultured according to the
protocol reported by Maniatopoulos et al.20 Briefly, both
ends of the femora were cut off at the epiphysis and the
marrow was flushed out using Dulbecco′s modified
Eagle′s medium (DMEM) (Gibco BRL, USA) with 10%
fetal bovine serum (Hyclone, USA) supplemented with
200 U/ml heparin (Sigma, USA). Cells were cultured in
DMEM containing 10% fetal bovine serum, 100 U/ml
penicillin, and 100 µg/ml streptomycin at 37°C in an
atmosphere of 5% CO2. The medium was changed after
24 hours to remove non-adherent cells and then was
renewed three times a week. When 90% confluence was
than in those with
report on fabricating
transduced bMSCs
scaffolds.
and
The
reached, bMSCs were released from the culture
substratum using trypsin/EDTA (0.25% w/v trypsin,
0.02% EDTA), and were moved to dishes (10 cm in
diameter) at 1.0×105 cell/ml in 10 ml. Culture medium
was further supplemented with 50 µg/ml ascorbic acid, 10
mmol/L β-glycerophosphate, and 10–8 mol/L dexame-
thasone after 2 passages. Cells at passage 3 were used for
the following gene transfer studies.

bMSCs were transduced with an adenovirus over-
expressing Nell-1 (AdNell-1) or LacZ (AdLacZ) at a
multiplicity of infection (MOI, pfu/cell) of the working
titre (50–80 pfu/cell) as previously described.14,21

Gene transfer efficiency was assessed according to the
protocol of Partridge et al.1 Using inverted phase contrast
microscopy (Leica DMRIRB, Heidelberg, Germany),
expression of β-galactosidase was visualized by staining
with X-gal for control AdLacZ cells 72 hours after gene
transduction. Cells were fixed with 4% paraformaldehyde
for 10 minutes at room temperature, then were stained for
3 hours at 37°C using a solution containing 20 mg/ml
X-gal, 5 mmol/L potassium ferricyanide, 5 mmol/L
potassium ferrocyanide, and 2 mmol/L magnesium
chloride in PBS. Gene transfer efficiency was determined
by calculating the percentage of LacZ-expressing cells
present in 10 randomly selected 40× fields, a quantitative
method described by Zhang et al.16

To determine the expression of Nell-1 protein, whole cell
extracts were prepared from transduced bMSCs. After
washing with ice-cold PBS, the cells were lysed using
protein extraction regent (Kangchen bio-tech, Shanghai,
China). Equal amount of protein samples were
fractionated by electrophoresis in 6% polyacrylamide gels
and transferred to polyvinylidene difluoride (PVDF)
membrane (Amersham Biosciences, USA). Membranes
were exposed to anti-Nell-1 (1:850 dilution) and
anti-β-actin antibodies (1:10 000 dilution, Sigma, USA).
Blots were exposed to secondary goat anti-rabbit for
Nell-1 and anti-mouse for β-actin immunoglobulin G
antiserum conjugated to horse radish peroxidase, and
developed with enhanced chemiluminescene (ECL) plus
chemiluminescence reagent (Amersham Biosciences).

RNA extraction and real-time polymerase chain
reaction (PCR)
Cells transduced with AdNell-1 or AdLacZ were
trypsinized at day 0, 3, 7, 14 and 21 after gene transfer and
RNA was harvested with a Rneasy Mini kit (Qiagen,
Germany). During this procedure, reverse transcription was
carried out in 20 µl volume containing 1 µg of template
RNA. Samples were incubated at 37°C for 2 hours.

Real-time PCR analysis of bone marker genes performed
with ABI Prism 7300 real-time PCR system (Applied
Biosystems, USA) for rat bMSCs transduced with
AdNell-1 and AdLacZ. The primers for rat genes are
presented in Table.

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Chin Med J 2009;122(8):972-979
974
Table. PCR primer pairs
Sequences
Forward 5′- CACCAGCACCAACTCCAC-3′
Reverse 5′- CTCGTAGCCTTCATAGCC-3′
Forward 5′- TAAGGTGGTGAATAGACTCCG-3′
Reverse 5′- GTGCCGTCCATACTTTCG-3′
Forward 5′- TGAAGCCTGACCCATCTC-3′
Reverse 5′- GGTCTTCCCGTTGCTGTC-3′
Forward 5′-GGATTTGGTCGTATTGGG-3′
Reverse 5′-GGAGATGGTGATGGGATT-3′
Genes
BSP

OC

OP

GAPDH


The house-keeping gene, glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) was used as an endogenous
control.22 mRNA expression of genes of interest was
firstly normalized to GAPDH and given as normalized
ratio, then the ratio of Nell-1 group was compared with
that of LacZ group.23 The relative expressions of every
gene of interest at day 0, 3, 7, 14 and 21 were calculated.
Each sample was assessed in triplicate (n=3).

ALP activity and von Kossa staining
ALP activity was assessed at day 3, 6, 9 after gene
transfer. As modified from Roostaeian et al,24 cells were
washed in PBS and suspended in lysis buffer with 0.2 %
NP-40. Absorbance at 405 nm was determined after the
cell lysate supernatant was mixed with freshly made 20
mg/ml p-nitrophenyl phosphate (Sigma-Aldrich, USA).
Each assay condition was done in triplicate. Enzyme
activity was expressed as nanomoles of p-nitrophenol
produced per hour per milligram of total cellular proteins.

With von Kossa staining, cultures were fixed in 70%
ethanol at day 21 and 28 after gene transfer and stained
with 5% silver nitrate under ultraviolet rays for 10
minutes. After treated with 5% NaS2O3 for 2 minutes and
washed with ethanol, samples were subjected for
observation. Calcium nodules with a diameter more than
0.5 mm at six-well plates were counted and compared for
all these 3 groups (n=6 wells per group).

Fabricating tissue-engineered bone construct with
β-TCP
The β-TCP scaffolds were purchased from Bio-Lu
company (Shanghai, China). The average diameter of the
pores was 450 µm and the average void volume was 85%.
In this study, β-TCP disks of 5 mm in diameter with
thickness of 2 mm were used. The method of cell seeding
was essentially the same as reported by Maniatopoulos et
al.20 Briefly, 72 hours after gene transfer, bMSCs were
released from the culture substratum. Then bMSCs were
seeded onto β-TCP scaffolds at a concentration of 2×107
cells/ml. The surgical procedures were performed
immediately after the seeding saturation was reached.

Scanning electron microscopy (SEM)
The degree of cell adhesion and growth was visually
assessed 4 days after seeding in extra samples treated
similarly as described above through SEM (Philips
Quanta-200, Netherlands). The samples were rinsed in
PBS, fixed in 4% paraformaldehyde, and then prepared
for SEM. The procedure included rinsing samples with

distilled water, incubating samples with 1% osmium
tetroxide in 0.1 mol/L sodium cacodylate for 30 minutes,
then rinsing samples with distilled water and dehydrated
using a gradation series of ethanol/distilled water
solutions. Afterward, critical point drying was achieved
using hexamethyldisilazane overnight. After critical point
drying, samples were placed onto SEM stubs and coated
using gold and palladium sputter coating for 90 seconds
and then were imaged.

Surgical procedure and harvesting
Eighteen constructs were assigned to 3 groups:
untransduced group (bMSCs/TCP composites, n=6),
LacZ group (LacZ/bMSCs/TCP composites, n=6) and
Nell-1 group (Nell-1/bMSCs/TCP composites, n=6).
Scaffolds alone previously proved not being able to
induce any ectopic bone formation were not included in
our design. Six nude mice were used in the experiment,
constructs from each of the 3 above groups were
implanted into the back of each animal subcutaneously
with a distance more than 5 mm between each implant.
The procedure of implantation was performed according
to the protocol adopted by Tsuda et al.25 Animals were
anesthetized by intramuscular injection of pentobarbital
(Nembutal 3.5 mg/100 g) after light ether inhalation.
Through a midlongitudinal skin incision in the back of
each mouse, the subcutaneous pockets were created by
blunt dissection. Four weeks after implantation, the
implants were harvested for the histological analyses.
Every implant was fixed and decalcified in 10% EDTA
for 2 weeks, then embedded in paraffin wax. Serial cross
sections parallel to the round underside were made, and
three randomly selected cross sections from each implant
were stained with haematoxylin and eosin. Then the
sections were analyzed histomorphometrically using
Image Pro 5.0 system (Media Cybernetics, USA). In this
study, the percentage of new bone area (including both
mature bone and premature osteochondral-like tissue)
among the total implanted area observed was calculated
by determining the mean of the 3 horizontal sections of a
given specimen.

Statistical analysis
All the results comparing among the 3 groups were
analyzed by one-way analysis of variance (ANOVA) with
Student-Neuman-Keuls (SNK) procedure. A SAS 6.04
statistical software package was used with the level of
significance set at P <0.05.

RESULTS

Gene transfer
The expression of LacZ was climaxed 72 hours after
AdLacZ gene transduction. At the current working titre,
the efficiency of (57.9±6.8)% was achieved (Figure 1).
Untransduced cells showed negative β-galactosidase
activity. At the same time point, the Nell-1 protein was
detected in Nell-1 transduced bMSCs and were not
detected in LacZ transduced bMSCs or untransduced
bMSCs (Figure 1B).

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Figure 1. A: A transfer efficiency of up to 60% was achieved
under optimal transfer conditions as determined by LacZ
expression using phase contrast
magnification ×100). B: Western blotting analysis of Nell-1
protein in bMSCs. Nell-1 protein was detected in AdNell-1
transduced bMSCs but not detected in untransduced bMSCs or
AdLacZ transduced bMSCs.
microscopy (Original

Nell-1 stimulates osteogenic differentiation of bMSCs
in vitro
The transcriptions of a few osteogenic markers were
analyzed after bMSCs transduced with AdNell-1 in this
study. The expressions of osteopontin (OP) and bone
sialoprotein (BSP) were significantly increased in the
intermediate stage of osteogenic differentiation (OP: 2.1-,
3.4-, 2.3- and 1.8-fold; BSP: 4.5-, 6.7-, 26.2- and
16.1-fold, at day 3, 7, 14 and 21 respectively). The
expression of osteocalcin (OC), a late marker of
osteogenic differentiation, was slightly decreased at day 3
(0.63-fold) but increased at day 7, 14 (1.29-, 1.46-fold),
and was significantly up-regulated at day 21 (55.5-fold)
(Figure 2).

ALP activity and von Kossa staining
To assess the phenotype of the genetically modified
bMSCs, ALP expression was quantified. At day 3, 6, 9
after gene transfer, ALP expression normalized by
cellular protein was significantly enhanced in the Nell-1
group than those of the control groups at the same time.
As an earlier osteogenic marker, it is not surprising that
ALP showed highest activity at day 3 than at later stages
for each group of cells (Figure 3). Calcium nodules were
counted with a diameter more than 0.5 mm at day 21 and
day 28 after gene transfer. The number of nodules formed
in Nell-1 group was larger than those in the untransduced


Figure 2. Real-time PCR analysis of OP (A) , BSP (B) and OC
(C) transcription in rat bMSCs at day 0, 3, 7, 14 and 21. Values
are expressed as fold changes relative to cells transduced with
AdLacZ.



Figure 3. Quantitative ALP activity was assessed in triplicate at
day 3, 6, and 9 after gene transfer. Enzyme activity was
expressed as nanomoles of p-nitrophenol produced per hour per
milligram of total cellular proteins. ALP in the Nell-1 group
showed significantly higher expression than those in other
groups 3 days after gene transfer (ANOVA; n=6, *P <0.05).

and LacZ groups at each time point (Figure 4).

These results of ALP activity and von Kossa staining
suggested that Nell-1 gene transfer led to the
enhancement of bMSCs differentiation into osteoblastic
cells in vitro.

SEM
Four days after the bMSCs were combined with the

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Chin Med J 2009;122(8):972-979
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Figure 4. At day 21 and 28 after gene transfer, calcium nodules
with a diameter greater than 0.5 mm in Nell-1 group were
significantly more than those in untransduced and LacZ groups
(ANOVA; n=6, *P <0.05).

implant, cells were fully spread and growing (Figure 5).
Nominal differences in cellular adhesion and proliferation
were observed among bMSCs transduced with AdNell-1,
AdLacZ and left untransduced.



Figure 5. BMSCs spreading and proliferation along the surface
was observed through SEM 4 days after gene transfer (Original
magnification ×500).

Histological examination
Four weeks after surgery, the implants were harvested,
sectioned and evaluated histomorphologically. No
inflammation or giant cell-type reaction was observed in
any of the groups. Bone formation occurred only inside
the pores, and was not found outside of β-TCP for all the
groups. The percentage of new bone area in Nell-1 group
was (18.1±5.0)%, while was (11.3±3.2)% in untransduced
group and (12.3±3.1)% in LacZ group respectively. The
percentage of new bone area in Nell-1 group was
significantly higher than those of untransduced group and
LacZ group (Figure 6). The results mentioned above
indicate that AdNell-1 improved the bone formation of the
tissue engineered composites in vivo.

DISCUSSION

A protocol of obtaining bMSCs from bone marrow of
young adult rats was adopted as described by
Maniatopoulos et al.20 Hollinger et al26 believed that such
a rat mandibular model of bone defects, was constant
regardless of age.

Figure 6. Four weeks after surgery, new bone formation was
evaluated by HE staining (A, original magnification ×100). The
percentage of new bone formed out of total area observed were
(11.3±3.2)% in untransduced group, (12.3±3.1)% in LacZ group
and (18.1±5.0)% in Nell-1 group. Histomorphological analysis
confirmed that the percentage of new bone formed in Nell-1
group was highest among the three groups 4 weeks
postoperatively (B) (ANOVA; n=6, *P <0.05).

The approaches of tissue engineering are generally
centered on the delivery of osteoinductive growth factors,
using direct protein delivery or gene therapy approaches,
implantation of osteogenic cells and combining these
approaches with osteoconductive scaffolds to promote
bone regeneration.27 In the current study, the osteogenic
differentiation and new bone formation ability of novel
gene Nell-1 modified bMSCs were evaluated with an ex
vivo gene therapy method. Adenoviral vector was used to
deliver Nell-1 gene since it can deliver foreign genes to a
variety of cell types with a comparatively higher transfer
efficiency.28 As a transient gene expression system, it is
preferable in certain situations to avoid side effects which
longer term over-expression probably would bring with.
For the above reasons, adenovirus has been extensively
investigated to promote bone regenerations.15,29-31 Besides,
using an ex vivo gene deliver method, which allows the
release of gene products to be localized and
target-oriented, may minimize systemic side effects and
maximize local therapeutic effects.

At the current working titre, data showed the transfer
efficiency reached (57.9±6.8)% and no obvious cell death
was observed. The Western blotting analysis confirmed
the expression of Nell-1 protein in AdNell-1 transduced
bMSCs, whereas Nell-1 remained undetectable in
AdLacZ transduced cells or untransduced cells. The
results revealed at this working titre we took, gene
transfer was effective and safe.