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

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

Editor(s): JI, Yuan-yuan

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doi: 10.3760/cma.j.issn.0366-6999.2009.08.018
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The 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 appropriately 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{L-Start} 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 osteoinductivity 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 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 than in those with 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 report on fabricating 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 transduced bMSCs and β-tricalcium phosphate (β-TCP) scaffolds. The composites were implanted in nude mice subcutaneously. We intended to assess whether more bone tissues could be obtained after Nell-1 gene modification.


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 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 dexamethasone after 2 passages. Cells at passage 3 were used for the following gene transfer studies.

bMSCs were transduced with an adenovirus overexpressing 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.

PCR primer pairs

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.


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).

Figure 1. A:
Figure 1. A::
A transfer efficiency of up to 60% was achieved under optimal transfer conditions as determined by LacZ expression using phase contrast microscopy (Original 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.

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).

Figure 2.
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.

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

Figure 3.
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).
Figure 4.
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).

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.


Four days after the bMSCs were combined with the 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.
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.

Figure 6.
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).


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.

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.

For the role of Nell-1 in promoting osteoblastic differentiation, Zhang et al14 have demonstrated that AdNell-1 overexpression increased the expression of a few bone formation-related genes and increased mineralization in fetal rat calvarial cells. To address more on how Nell-1 regulate the osteogenic differentiation on rat bMSCs, we investigated Nell-1's regulation of osteogenic gene transcription by means of quantitative real-time PCR as well as the quantitative tests of ALP and calcium nodules.

The time-dependent expressions of bone marker genes may elucidate osteoblastic maturation and differentiation. OP, a potent inhibitor of nucleation, is largely considered as an intermediate or relatively earlier marker of osteogenic differentiation.14BSP was currently considered to be an intermediate up-regulated maker to monitor the extent of bMSCs osteogenic differentiation in vitro. The up-regulated expression of the BSP gene induced in vitro indicates the promotion of bone formation.11,32 OC is considered to be a later marker of osteogenic differentiation corresponding with matrix deposition and mineralization.33

Our data demonstrated that the expression of OP was more significantly increased in intermediate stage. The transcription of BSP, similar to that of OP, was also increased in intermediate stage. It is generally believed that in the process of ossification, the upregulation of BSP was a little bit later than OP. This was also the case in the current study. In the later stage, the transcription of OC was increased. In the present study, the transcription of OP, BSP and OC reflected the time-dependent transcriptional change of a few marker of osteogenic differentiation, which verified that after Nell-1 gene transfer, osteogenic differentiation of bMSCs was induced.

Bone marrow stromal cells were also referred as mesenchymal stem cells capable of differentiation down multiple mesenchymal tissue lineages (e.g. bone, cartilage, muscle and tendon) when appropriately stimulated. In the present study, the osteoblast phenotype was induced by Nell-1 gene transfer. This may be one of the explanation of the fact we found in the study: although the trend of OC and OP expressions were similar, the turning points in rat bMSCs occurred later than those reported in MC3T3-E1 osteoblasts, a more differentiated cells than bMSCs.23

An increase in ALP staining as well as an increase in mineralized nodules was also found in Nell-1 group. These findings indicated that the generated Nell-1 was biologically active and Nell-1 overexpression increased osteoblastic differentiation. Given that these results suggested that adenovirus gene transfer with bMSCs as target cells could be effective for Nell-1 gene therapy, we further explored its osteoinductive properties in tissue engineering application.

With respect to therapeutic applications, BMP-2 is among the most potent osteoinductive factors but usually requires a large dose, which may lead to certain side effects including cyst formation.34 Compared with BMP-2, Nell-1 was believed to be more specific in promoting new bone formation. Recent findings by Truong et al35 supported the hypothesis since it is a direct down stream mediator of Cbfa1 containing three Cbfa1/Runx2 binding sites (osteoblast specific cis-acting element 2 (OSE2)) within its promoter. Together with other experiments,17,36 researchers have confirmed Nell-1 to be a novel osteoinductive factor with a more restrictive and specific targeting of cells of the osteoblast-lineage.15

A simple model of direct intramuscular injection of adenoviral transduced goat bMSCs into nude mice without the use of a carrier or scaffold had been chosen for an initial study, which demonstrated that AdNell-1-transduced bMSCs gained the ability to promote new bone in vivo.15 To pursue further tissue engineering applications of Nell-1, we chose β-TCP as the scaffold.

The in vivo new bone formation was observed in untransduced group and the LacZ group, demonstrated the osteoconductive ability of β-TCP scaffold for bMSCs, which has been reported previously.37 The results of histomophometric analyses in our study showed much higher ((18.1±5.0)%) new bone formation achieved 4 weeks postoperatively in Nell-1 group. These represented a statistically significant increase over the other two groups assessed. It can be inferred from our study that the generated Nell-1 was biologically active and might function as an osteogenetic growth factor for the use of tissue engineering.

In conclusion, the current data demonstrate that Nell-1 gene is a promoter of osteoblast differentiation and of bone formation in vivo. The results suggest that Nell-1 may be a potential osteogenic gene to be used in bone tissue engineering and regeneration. As far as we know, this is the first report to evaluate Nell-1's osteoinductive properties in tissue engineering application using an ex vivo technique.


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

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