Abstract
BACKGROUND: Mesenchymal stem cells (MSCs) can differentiate into osteoblasts in the action of dexamethasone; Meanwhile, bone morphogenetic protein-2 (BMP-2) can promote the cell proliferation and differentiation, and matrix secretion in the process of bone repair. BMP-2 plays an important role in treating fracture and bone defects by inducing bone formation both in vivo and in vitro.
OBJECTIVE: To analyze whether dexamethasone induction in vitro could enhance the ability of BMP-2 modified MSCs in osteogenic conversion.
DESIGN: A randomized paired design.
SETTING: Xijing Hospital of the Fourth Military Medical University of Chinese PLA.
MATERIALS: The experiments were carried out in the Orthopaedic Oncology Institute of Chinese PLA, the Fourth Military Medical University of Chinese PLA from February to August in 2004. Twenty 20-month-old New Zealand rabbits were provided by the experimental animal center of the Fourth Military Medical University of Chinese PLA [Certificate number: 2005C00117]. The rabbits were raised normally at room temperature with normal humidity. The samples were collected bilaterally, the cells from the left limb were taken as the dexamethasone-induced group, and those from the right limb as the control group.
METHODS: MSCs treated and untreated with dexamethasone were transfected with BMP-2 gene, then the BMP-2 expression was determined by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemical staining. The growths of the MSCs were observed, and the activity of alkaline phosphatase (ALP) and osteocalcin of MSCs were determined with ALP kit and osteocalcin radioimmunoassay kit after Ad-BMP-2 transfection.
MAIN OUTCOME MEASURES: ¢Ù BMP-2 expression in MSCs; ¢Ú Morphological changes of the MSCs; ¢Û ALP activity and osteocalcin content in the MSCs.
RESULTS: ¢Ù Expressions of BMP-2 gene could be observed in MSCs both treated and untreated with dexamethasone after transfection. ¢Ú The forms were irregular in most of the MSCs treated with dexamethasone, appeared as triangular or polygonal changes.They grew more slowly than those cultured with basic medium. There were no obvious changes of cellular forms after gene transfection. ¢Û Five days after transgene, the ALP activity in the MSCs supernatant in the dexamethasone-induced group was higher than that in the control group [(134.36¡À8.84), (104.02¡À7.83) nkat/L, t =3.350 6, P < 0.01, n =20]. The amount of osteocalcin secretion in MSCs in the dexamethasone-induced group was higher than that in the control group [(14.68¡À0.73), (6.52¡À1.21) ¦Ìg/L, t =3.568 2, P < 0.01, n =20].
CONCLUSION: Dexamethasone induction before transgene can promote the proliferation of BMP-2 modified MSCs and the conversion into osteoblasts.

INTRODUCTION

In recent years, bone marrow mesenchymal stem cells (MSCs) have been extensively applied in bone tissue engineering as seed cells[1]. It has been demonstrated that MSCs can differentiate into osteoblasts and mediate osteogenesis in vivo in the action of dexamethasone or other bone inducers. Plenty of experiments have indicated that bone morphogenetic protein-2 (BMP-2) can promote the cell proliferation and differentiation, and matrix secretion in the process of bone repair[2], induces bone formation both in vivo and in vitro, and plays an important role in treating fracture and bone defects[3]. The above results suggest that the combined application of them may have certain synergistic action. In order to confirm whether dexamethasone induction in vitro could enhance the ability of BMP-2 modified MSCs in osteoblastic conversion, the main indexes for osteoblastic conversion of the treated and untreated MSCs before transfection were observed after BMP-2 transfection, so as to provide experimental basis for the ideal construction of tissue-engineered bone.
ÿÿ
MATERIALS AND METHODS

Materials
The experiments were carried out in the Orthopaedic Oncology Institute of Chinese PLA, the Fourth Military Medical University of Chinese PLA from February to August in 2004. Twenty 20-month-old New Zealand rabbits of 1.0-1.5 kg, either male or female, were provided by the experimental animal center of the Fourth Military Medical University of Chinese PLA [Certificate number: 2005C00117]. The rabbits were raised normally at room temperature with normal humidity. The samples were collected bilaterally, the cells from the left limb were taken as the dexamethasone-induced group, and those from the right limb as the control group.
Main reagents: Ad-BMP-2 (Academy of Military Medical Sciences of Chinese PLA); Lymphocyte separating medium and dexamethasone (Jiangsu Xinghua Pharmaceutical Factory, batch number: 970273); Vitamin C, beta (¦Â)- glycerol phosphate (Sigma); Dulbecco's modified Eagle medium (DMEM), trypsin (Gibco); Fetal bovine serum
(Sijiqing, Hangzhou); Trizol total RNA extract kit, SuperscriptTM¢ò reverse transcription No.1 chain synthesis kit (1nVitrogen); Kit for detecting alkaline phosphatase (ALP) (Shanghai Kehua-Dongling, Co.,Ltd.); Kit for detecting osteocalcin (Beijing Dongya Immunological Technology Institute).

Methods
Isolation and primary culture of MSCs in rabbits
According to literature [4], rabbit medullary cavity was punctured via femur to draw bone marrow (3-4 mL), mononuclear cell layers were obtained with lymphocyte separating medium after density gradient centrifugation, washed with phosphate buffer solution (PBS) for three times, then added by basic medium containing fetal bovine serum (0.15 in volume fraction), penicillin (100 U/mL) and streptomycin (100 U/mL), then inoculated into 50 mL culture bottle at the density of 1¡Á104 cells/cm2, and cultured in the incubator containing CO2 (0.05 in volume fraction) and 95% relative humidity at 37 ¡æ. The medium was changed firstly after 4 days, then the unattached cells were all removed, and the medium was changed every two or three days afterwards.

MSCs osteoblast induction culture and gene transfection
After covered the complete monolayer, the primary cells were digested with 2.5 g/L pancreatin, and washed with PBS. The cells in the dexamethasone-induced group were resuspended with induction medium, which contained dexamethasone (5¡Á10-8 mol/L), vitamin C (160 ¦Ìmol/L) and ¦Â- glycerol phosphate (10 mmol/L), whereas those in the control group were resuspended with basic medium, then they were inoculated into 100 mL culture bottle at 1¡Á104 cells/cm2, and cultured routinely for 7 days. When 60%-70% of the cells became confluent, medium containing serum of 0.02 in volume fraction was changed, and transfected with Ad-BMP-2 according to multiplicity of infection (MOI) of 100, then normal medium containing serum of 0.1 in volume fraction was changed for culture continuously.

Morphological observation
The growth and morphological characteristics of primary cells, passaged cells, and those after induction and transfection were observed daily with inverted phase contrast microscope.

BMP-2 expression detected with reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemical staining
After transfection for 48 hours, the total RNA of cells were extracted using Trizol one-step method for RT-PCR, and glyceraldehydes phosphate dehydrogenase (GAPDH) was taken as internal control. The instructions of the kits were followed. Meanwhile, the cells covering the glasses were immunocytochemically stained with SABC method, the primary antibody was goat-anti-human polyclonal antibody, and the second antibody was biotinylated rabbit-anti-goat IgG, and stained with diaminobenzidine (DAB).

Determination of ALP activity
MSCs after Ad-BMP-2 transfection for 5 days in the dexamethasone-induced group and those in the control group were collected, and the ALP activity was quantitatively determined with ALP detection kit, then the effect of pre-induction on the ALP activity of Ad-BMP-2 transfected MSCs was analyzed.

Detection of osteocalcin in supernatant
The content of osteocalcin in supernatant of cells transfected with Ad-BMP-2 for 5 days was detected with osteocalcin radioimmunoassay kit. According to the principle of radio-immune competitive combination, 125I-labeled osteocalcin and that in samples competitively combined with osteocalcin antibody, the binding antibody was separated using separating agent, centrifugated at 4 ¡æ, the depositor counting was determined with gamma counter (r/min), and the content of osteocalcin in the samples was obtained according to the standard curve.

Statistical analysis
The SPSS 11.5 software was used by the first author for paired t test, and the data were expressed as Mean¡ÀSD.

RESULTS

Results of morphological observation of MSCs after induction and transfection
All 230 patients participated in the final analysis.

Biocompatibility of allogeneic bones
It was observed under the inverted phase contrast microscope that the MSCs began to attach after inoculation for 12-16 hours. There were plenty of suspending cells, which were mainly hematopoietic cells, in the culture bottle at early period. The hematopoietic cells were gradually removed as the culture and changing solution. The attached cells were in attachment completely at 5-7 days, and appeared as fibroblast-like colony growth. They became confluent into monolayer. The forms were irregular in most of the cells treated by dexamethasone, appeared as triangular or polygonal changes. They grew more slowly than those cultured in basic medium. More round pycnotic cellular nodes appeared after 7 days, and the node center became dark gradually with bad light permeability (Figure 1). There were no obvious changes of cellular forms after gene transfection.

BMP-2 expression in MSCsenchymaltF
The RT-PCR results at 48 hours after Ad-BMP-2 transfection showed that there was specific band at the site of 1 200 bp after transfection, whereas the untreated blank control cells were negative (Figure 2). The results of immunohistochemical staining displayed that there was buffy strong positive reaction in the transgenic cytoplasm (Figure 3), whereas the untreated cells were negative.

Five days after transgene, the ALP activity in the MSCs supernatant was higher in the dexamethasone-induced group than in the control group [(134.36¡À8.84), (104.02¡À7.83) nkat/L, t =3.350 6, P < 0.01, n =20]. The amount of osteocalcin secretion in MSCs was higher in the dexamethasone-induced group than in the control group [(14.68¡À0.73), (6.52¡À1.21) ¦Ìg/L, t =3.568 2, P < 0.01, n =20]. The results indicated that dexamethasone induction before transfection could obviously promote the mature differentiation of MSCs into osteoblasts.

DISCUSSION

In recent years, the combined application of bone tissue engineering, which is the combination of cell transplantation and biomaterials, and the gene engineering, which takes recombinant DNA as the core technique will become the ideal model for repairing bone defects[5]. The selection of seed cells is the premise and key point for tissue engineered bone[6, 7]. MSCs have attracted more and more attentions and have become the seed cells firstly selected in bone tissue engineering[7, 8] due to its convenient to obtain, small injury, high reproductive activity and definite osteoblastic ability[9]. As a kind of multipotent stem cells, MSCs have multiple differentiative potencies, but its differentiation is not a complete spontaneous process, some cytokines and internal environment can promote the differentiation[10, 11]. It has been demonstrated that the ossific process was slower when MSCs as seed cells were used alone. When the formation of extracellular matrix was insufficient early after cell transplantation in vivo, the implanted MSCs would lose support and dense intercellular adhesion, and the formation of the local osteoblastic microenvironment will be hindered, and dominant osteoblast group cannot be defined, which will block the osteogenesis[12]. Some scholars compared the bioactivities of osteoblasts from different sources, and believed that MSCs had powerful proliferative ability, weak osteogenic activity, and they could become ideal seed cells after modification. Therefore, it will be the key link for optimizing seed cells in bone tissue engineering how to improve the osteogenic potency of MSCs so that they could stably transform into osteoblasts abundantly[13].
The osteogenic potency of MSCs is affected by various factors. It has been demonstrated that dexamethasone treated MSCs led to early cell proliferation, increase of ALP expression and deposition of calcium salts, also mediate osteogenesis in vitro[14]. When MSCs were cultured in vitro, adding suitable ¦Â-glycerol phosphate, vitamin C and dexamethasone could promote the osteogenesis, thus MSCs were called as ossific adjunct[15-17]. Peter et al[18] showed in an experiment in vitro that dexamethasone could induce the osteogenesis of human MSCs, the osteogenetic ability of the treated MSCs was obviously stronger than that of the untreated ones. Our results also confirmed the pro-osteogenesis of ossific adjunct, whereas the mechanism was still unclear, and it was suspected that glycerol phosphate was a kind of substrate of ALP, and it might provide phosphate, which was good for mineralization. Vitamin C could stimulate the synthesis of type ¢ñ collagen. However, further explorations are needed to investigate what substances can induce the osteogenesis of MSCs, whether there is specificity and species differences, which phase of differentiation do the inducers act, and whether the roles are the same in vivo and in vitro.
Plenty of domestic and foreign experiments have confirmed that BMP-2 can promote bone formation directly or through the local application of vectors[19]. The application of BMP-2 is restricted due to multiple factors, such as natural BMP is hard to extract, recombinant BMP cannot endure the internal environment of 37 ¡æ, short half life is difficult to maintain the effective concentration, cytokines are easy to lose in local application, etc. However, the transgenic technique introduces the objective gene of codocyte protein into proper target cells, so that they can continuously release the objective gene expressed codocyte protein, and act on the target cells by means of autocrine or paracrine, which can greatly make up the defect of local application of exogenous cytokines.
The characteristic phenotypes of osteoblasts are the high expression of ALP, synthesis of type I collagen and secretion of osteocalcin, which have become the standard acknowledged by domestic and foreign scholars. In this study, the ALP activities were compared after transfection between the treated and untreated BMP-2, and found that it was obviously higher in the treated cells than in the untreated ones. The results indicated that dexamethasone induction before transfection could obviously promote the MSCs to convert into osteoblasts. The enzymological action of ALP and the accumulation of extracellular matrixes provide suitable calcium and phosphorus concentrations and pH values, it is good for inducing the formation of osteogenic microenvironment, so that the osteogenic potency of MSCs can be significantly improved. ALP is one of the marker enzymes of mature osteoblasts. The main mechanism is that ALP can hydrolyze organic phosphatase, increase the local concentration of PO4+, destroy the calcification inhibitor, and then activate the calcification. Our results of ALP activities indicated that induction beforehand could obviously promote the conversion of MSCs into osteoblasts after BMP-2 transfection. In this study, the significant differences of osteocalcin, which was the characteristic secretory product of osteoblasts, further confirmed that dexamethasone induction before transfection could greatly improve the osteogenic ability of MSCs.
Our results suggested that dexamethasone could obviously improve the osteogenic ability of BMP-2 modified MSCs. However, the product of BMP-2 expression was not quantitatively analyzed due to the restriction of the experimental conditions. Therefore, it should be further quantitatively studied whether the promotion was caused by dexamethasone alone, or the increase of BMP-2 expressed product after dexamethasone induction further promoted the conversion into osteoblasts.

REFERENCES

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