|Year : 2014 | Volume
| Issue : 4 | Page : 347-353
|Bone formation in rabbit's leg muscle after autologous transplantation of bone marrow-derived mesenchymal stem cells expressing human bone morphogenic protein-2
Licheng Wei1, Guang-Hua Lei2, Han-Wen Yi1, Pu-yi Sheng3
1 Department of Orthopaedics, The 8th Hospital, Changsha, Hunan 410008, China
2 Department of Orthopaedics, XiangYa Hospital, Central South University, 87 XiangYa Road, Changsha, Hunan 410008, China
3 The First Affilliated Hospital, Sun YAT-SEN University, 58 The Second ZhongShan Road, GuangZhou, GuangDong 510080, China
Click here for correspondence address and email
|Date of Web Publication||8-Jul-2014|
| Abstract|| |
Background: To test whether autologous transplantation of bone marrow-derived mesenchymal stem cells (BM-MSCs) expressing human bone morphogenic protein-2 (hBMP-2) can produce bone in rabbit leg muscles.
Materials and Methods: MSCs were isolated from BM of the iliac crest of rabbits and then infected with lentiviral vectors (LVs) bearing hBMP-2 and green fluorescent protein under the control of the cytomegalovirus (immediate early promoter). Differentiation of transduced MSCs to osteoblasts in vitro was evaluated with an alkaline phosphatase activity assay and immuohistochemistry against osteoblast specific markers. MSCs expressing hBMP-2 were placed in an absorbable gelatin sponge, which was then transplanted into the gastrocnemius of rabbits from which MSCs were isolated. Bone formation was examined by X-ray and histological analysis.
Results: LVs efficiently mediated hBMP-2 gene expression in rabbit BM-MSCs. Ectopic expression of hBMP in these MSCs induced osteoblastic differentiation in vitro. Bone was formed after the MSCs expressing hBMP-2 were transplanted into rabbit muscles.
Conclusion: Ectopic expression of hBMP-2 in rabbit MSCs induces them to differentiate into osteoblasts in vitro and to form a bone in vivo.
Keywords: Autologous transplantation, bone morphogenic protein-2, lentiviral vector, mesenchymal stem cells
MeSH terms: Autologus, transplantation, stem cells, mesenchymal, bone marrow
|How to cite this article:|
Wei L, Lei GH, Yi HW, Sheng Py. Bone formation in rabbit's leg muscle after autologous transplantation of bone marrow-derived mesenchymal stem cells expressing human bone morphogenic protein-2. Indian J Orthop 2014;48:347-53
|How to cite this URL:|
Wei L, Lei GH, Yi HW, Sheng Py. Bone formation in rabbit's leg muscle after autologous transplantation of bone marrow-derived mesenchymal stem cells expressing human bone morphogenic protein-2. Indian J Orthop [serial online] 2014 [cited 2019 Aug 20];48:347-53. Available from: http://www.ijoonline.com/text.asp?2014/48/4/347/136208
| Introduction|| |
Bone defects from congenital anomalies, trauma, infection, or tumors are common clinical problems. Autologous or allogeneic transplantation of osteoblasts are popular strategies to treat bone defects. However, osteoblast transplantation has been limited due to the lack of available transplantable cells. In recent times, tissue engineered transplantable bones have been developed to meet the clinical needs to treat bone defects. ,, This strategy requires the use of various growth and differentiation factors, which unfortunately have short half-lives in vivo. Thus, cells over-expressing the genes encoding these factors have been implemented. For example, cells transfected with the bone morphogenic protein-2 (BMP-2) gene stably express BMP-2 in artificial tissues.  A successful tissue engineering technique requires transplantable cells, signaling molecules for bone formation and a scaffold to support cell proliferation.
Mesenchymal stem cells (MSCs) can be isolated from various adult tissues, such as bone marrow (BM), fat, bone, placenta, and skeletal muscle. ,, Under appropriate conditions, MSCs can differentiate into osteogenic, chondrogenic, and adipogenic cells. ,,, For example, the over-expression of osteogenic factors, including BMP-2, induces MSCs to differentiate into osteoblasts.  Thus, MSCs have been thought as a useful cell source for bone tissue engineering.
BMP-2 is a member of the transforming growth factor-β superfamily. It stimulates heterotopic bone growth, enhances the healing of bone defects, and strongly induces de novo bone formation at orthotopic and heterotopic sites. ,,,, Expression of BMP-2 in MSCs has been shown to induce MSC differentiation into osteoblasts in vitro and bone formation in vivo in mice, rats, rabbits.  Cheng et al. used a recombinant adenoviral vector to deliver the human BMP-2 (hBMP-2) gene into rabbit BM-derived MSCs. These BM-MSCs expressed BMP-2, differentiated into osteoblasts expressing osteogenic marker genes, such as alkaline phosphatase (ALP), osteocalcin and collagen I, in vitro, as well as formed new bones when transplanted in vivo. Sugiyama et al. used a lentiviral vector (LV) to deliver hBMP-2 into rat BM-MSCs and achieved a long term BMP-2 expression, which resulted in bone formation in immune deficient mice. LVs can efficiently infect relatively quiescent stem cells and incorporate into the host genome, achieving long term transgene expression. Furthermore, LVs have been shown to be less immunogenic. ,,, Artificially synthesized biodegradable poly (lactic acid) has been widely used as the scaffold for bone construction in vitro and in vivo because it can be hydrolytically degraded by de-esterification. ,,,, In contrast, absorbable gelatin sponge is composed of purified porcine derived gelatin.  This material is cheap, easy to obtain and has been extensively used in the clinics for years.
In this study, we used rabbits as an animal model to develop a simple and efficient MSC autologous transplantation approach to generate new bone. This approach should be suitable for future clinical application.
| Materials and Methods|| |
New Zealand white rabbits (8-10-month-old) were used for this study. Rabbits were housed in a clean environment according to the criteria set by the Ministry of Science and Technology of the People's Republic of China.  The experimental procedure was approved by the animal ethics committee at the Central South University of China (approval ID: SCXK2009-0012).
Isolation and culture of MSCs
MSCs were isolated from the BM of rabbits as described previously.  Briefly, a rabbit was anesthetized by 2% pentobarbital sodium. BM was aspirated from its iliac crests using 18G needle. After lysing the red blood cells, we cultured cells in DMEM/F12 supplemented with 10% fetal bovine serum (Gibco BRL) at a density of 1 × 10  cells/cm  . Cells were incubated at 37°C with 5% CO 2 . Culture medium was changed every 3 days to remove nonadherent cells. After 7-10 days, adherent cells were trypsinized and replated in plates according to the experimental design.
After passaging the third time, MSCs were harvested for flow cytometry analysis to determine whether they expressed MSC surface markers. Cells were stained with primary mouse anti rabbit monoclonal antibodies (anti-CD29, anti-CD44, anti-CD105, anti-CD34, anti-CD45, anti-CD146, and anti-HLA-DR) and then stained with fluorescein isothiocyanate-conjugated goat anti-mouse IgG. An identical antibody isotype was used as a negative control. All antibodies were purchased from Roche Biotechnology Inc., (Roche, Switzerland). Stained cells were then subjected to flow cytometry (EPICS® ALTRA™ , USA). All analyses were performed in triplicate.
Viral production and tittering
A LV bearing hBMP-2-green fluorescent protein (GFP) under the control of the cytomegalovirus (CMV) immediate early promoter was kindly provided by Dr. Pu Qin (Department of Biochemistry, The Fourth Military Medical University, PR of China). Viral particles were produced and tittered as described previously.  Briefly, LV-hBMP-2-GFP viral vectors were produced in 293 cells by co-transfecting cells with the LV-hBMP-GFP transfer plasmid and three packaging plasmids. Viral vectors were purified by centrifugation, and viral tittering was assayed by measuring the ability to infect 293 cells.
MSCs (5 × 10  cells/well) were seeded into a 6-well plate and cultured for 24 h before lentiviral infection. Viral infection was carried out in 1 ml of serum-free growth medium in the presence of 6 g of polybrene (Sigma, USA) at multiplicities of infection (MOI) of 1, 10 and 100. After the cells were incubated with medium mixed with viral vectors at 37°C and 5% CO 2 for 1 h, viral medium was replaced with 3 ml of fresh growth medium. The GFP expression in infected cells was monitored with a fluorescence microscope to estimate transduction efficiency. An optimal MOI had a high transduction efficiency and low rate of cell death.
ALP activity assay
The ALP activity of the MSCs was measured using an ALP detection kit (Sigma, St. Louis, MO) according to manufacturer's instruction. MSCs were trypsinized and lysed with TritonX-100 lysis buffer. The ALP activity was expressed as the optical density times 100.
Immunohistochemical staining was performed as previously described.  Briefly, cultured cells were rinsed with phosphate-buffered saline, and then fixed in cold acetone for 5-10 min. After blocking with normal horse serum (1:20) for 10 min, the cells were incubated with primary antibodies at 37°C for 60 min followed by incubation with biotinylated anti-goat IgG (1:1000) at room temperature for 30 min. To detect hBMP-2, a goat anti-human BMP-2 antibody (1:800) was used, and a goat anti-rabbit osteocalcin antibody (1:400) was used to detect osteocalcin. Labeled cells were then stained with 3, 3-diaminobenzidine solution at room temperature for 3-10 min. Samples were also counterstained with hematoxylin, and then dehydrated and mounted. Stained cells were imaged with an Olympus microscope (Olympus, Japan) connected to an Olympus camera.
In situ hybridization
To determine type I collagen expression in MSCs, we performed in situ hybridization as described previously.  The type I collagen specific probe used in the in situ hybridization was 5'- GATTGTGGGATGTCTTCGTCTT-3'.
Cell transplantation and examination of bone formation
At 3 weeks after viral infection, MSCs transduced with LV-hBMP-2-GFP or mock from each rabbit were harvested and transplanted, respectively, into each gastrocnemius of the same rabbit. For transplantation, rabbits were anesthetized with Phenobarbital (150 ~ 200 mg/kg), and a 4-cm incision was created in each hind limb. Ten million cells were loaded into an absorbable gelatin sponge, which was then implanted into a gastrocnemius muscle. Rabbits were administered Gentamicin antibiotics for the following 3 days.
New bone formation in the hind limb of each rabbit was examined using X-ray every 2 weeks for up to 12 weeks post cell transplantation. Rabbits were then sacrificed using air embolism, and ossified tissues were harvested for histological analyses. To prepare tissue sections, the harvested tissues were cut into cubes of 1.0 cm × 1.0 cm × 0.3 cm in size and fixed in 10% neutral formalin. After decalcifying in 10% nitric acid and 2% sodium hydroxide, the tissues cubes were dehydrated in a gradient series of alcohol solutions, embedded in paraffin, and cut into 5 μm sections. The sections were stained with H and E dyes before being imaged [Figure 1].
Statistical analyses were performed using Statistical Product and Service Solutions 17.0. Paired-Student's t-test was used. P < 0.05 was considered as statistically significant.
| Results|| |
Characterization of MSCs
MSCs were isolated from six rabbits (three males and three females). They were long and spindle-like. After three passages, MSCs from each rabbit were harvested and analyzed with flow cytometry for the expression of MSC surface markers. Most cells (70%) expressed high levels of CD29, CD44 and CD105 and low levels of CD34, CD45 and HLA-DR (data not shown), indicating that they were MSCs.
LVs efficiently transduce MSCs
To determine whether LV-hBMP-2-GFP could transduce MSCs efficiently, MSCs (5 × 10  ) were infected with LV-hBMP-2-GFP at MOI of 1, 10 and 100 in 6-well plates. At day 3, percentages of cells expressing GFP were 30%, 50%, and 60% at a MOI of 1, 10, and 100, respectively, when analyzed by flow cytometry. A representative of fields of MSCs expressing GFP which infected with LVs at MOI of 100 [Figure 2]a.
|Figure 2: Lentiviral vector (LV)-human bone morphogenic protein-2 (hBMP-2)-green fluorescent protein (GFP) efficiently transduced mesenchymal stem cells, which were infected with LV-hBMP-2-GFP at multiplicities of infection of 100 (a) Expressions of transgenes were observed by fluorescence microscopy and immunohistochemical analysis against hBMP-2 (b and c)|
Click here to view
hBMP-2 expression in MSCs transduced with LV-hBMP-2-GFP
Next, we examined the expression of hBMP-2 in MSCs infected with LV-hBMP-2-GFP at MOI of 100 by immunohistochemical analysis. Nearly 85% cells expressed hBMP-2 [Figure 2]b and c, suggesting that the LVs efficiently induced ectopic expression of hBMP-2 in MSCs.
Osteogenic activity in MSCs transduced with LV-hBMP-2-GFP
To determine whether the ectopic expression of hBMP-2 in MSCs induced osteogenic differentiation, we first measured the ALP activities in LV-hBMP-2-GFP or mock transduced cells at various time points post viral infection. As shown in [Figure 3]a, the ALP activity increased in LV-hBMP-2-GFP transduced cells gradually with time and reached a maximum at day 18 post viral infection.
|Figure 3: Osteogenic activity of lentiviral vector (LV)-human bone morphogenic protein-2 (hBMP-2) transduced mesenchymal stem cells in vitro. Mesenchymal stem cells were infected with LV-hBMP-2-green fluorescent protein at multiplicities of infection of 100. (a) Bar diagram showing ALP activity at various time points were measured and reported as optical density (OD) times 100. The values are shown as the average of the OD plus standard derivation from six samples. (b) Immunohistochemical analysis was used to detect osteocalcin and in situ hybridization was used to detect type I collagen|
Click here to view
We then examined whether osteoblast-specific markers, osteocalcin and type I collagen, were expressed in LV-hBMP-2-GFP transduced cells using immunohistochemical staining and in situ hybridization, respectively. At 4 weeks post viral infection, 75% LV-hBMP-2-GFP transduced cells expressed osteocalcin and type I collage. Taken together, these data suggest that ectopic expression of hBMP-2 induced osteogenic differentiation of MSCs [Figure 3]b.
MSCs expressing hBMP-2 form bones in vivo
We then investigated whether MSCs expressing hBMP-2 could form bones in vivo. For this purpose, MSCs transduced with LV-hBMP-2-GFP were loaded in an absorbable gelatin sponge, and then the sponge was transplanted into gastrocnemius muscles of the rabbit from which the MSCs were isolated. The vital signs of six rabbits receiving transplantation were monitored every day. No apparent immune reactions, such as redness of transplantation site, fever, wound infection and local inflammatory secretions, were observed. X-ray examination revealed new bone formation in the left gastrocnemius muscles (receiving LV-hBMP-2-GFP transduced MSCs), but not in the right gastrocnemius muscles (receiving mock transduced MSCs) of six rabbits [Figure 4]a. At 12 weeks, the newly formed bones were confirmed by histological analysis [Figure 4]b and c.
|Figure 4: Lentiviral vector (LV)-human bone morphogenic protein (hBMP) transduced mesenchymal stem cells (MSCs) form bones in vivo. Ten million mesenchymal stem cells (MSCs) transduced with LV-hBMP-2-green fluorescent protein (left muscle) or mock (right muscle) were loaded into an absorbable gelatin sponge, which was then transplanted into a gastrocneminus muscle of the same rabbit from which the MSCs were isolated. (a) Bone formation shown by X-ray rabbits hind foot. The red arrow points to newly formed bone. (b) Newly formed bone stained with H and E. (c) Normal muscle tissue stained with H and E|
Click here to view
| Discussion|| |
Musgrave et al.  have compared five different cell types include a BM stromal cell line, primary muscle derived cells, primary BM stromal cells, primary articular chondrocytes, and primary fibroblasts in ex vivo gene therapy to produce bone, and demonstrated that BM stromal cells showed more responsiveness to recombinant human bone morphogenetic protein-2. In this study, we demonstrated that ectopic expression of hBMP-2 in rabbit BM-derived MSCs could induce the MSCs toward osteogenic differentiation in vitro and in vivo. Furthermore, we successfully used a simple and economic scaffold to induce new bone formation in the muscles of rabbits after transplanting MSCs expressing hBMP-2. Isolated MSCs were long and spindle-like and expressed MSC-specific surface markers, including CD29, CD44, CD105, CD34, CD45, CD146 and HLA-DR (data not shown), which is consistent with other groups.  These MSCs were transduced efficiently with LVs bearing two transgenes, hBMP-2 and GFP, and they expressed both transgenes [Figure 1]. Transduced cells altered their morphology from long spindle-like to triangle-like. Moreover, these cells had increased ALP activity and expressed osteocalcin and type I collagen, which are specific markers for osteoblasts  [Figure 2]. After the cells were transplanted into the same rabbits from which the MSCs were isolated, new bone formation was observed [Figure 3]. Our data were consistent with another report, concluding that hBMP-2 is an osteoinductive factor. 
Ectopic expression of hBMP-2 in MSCs from various tissues of mouse, rat and rabbit has been mediated with adenoviral, AAV, and LVs. In this study, we successfully used LVs to mediate both hBMP-2 and GFP simultaneously. The CMV promoter once was used as the standard promoter in most BMP transgene expression systems.  However, Ferreira et al.  have proved a fact that elongation factor-1α, (EF-1α), β-actin and GAPDH promoters are more efficient than the viral promoters such as CMV in driving gene expression, and provided the first in vitro evidence for a safe alternative to viral methods that permit efficient BMP-2 gene delivery and expression in MSCs. We have no experiences about above cellular promoters. Based on our laboratory experience and convenience, we always used to apply CMV as a promoter since 2000. Moreover in practice, we also acquired satisfactory results.
At a MOI of 100, 65% MSCs expressed both transgenes [Figure 1], suggesting that LVs can efficiently transduce BM-derived MSCs in vitro. At day 28 post viral infection, GFP expression was still apparent, suggesting that long term transgene expression was obtained. Long term hBMP-2 expression is one of the advantages of using LVs, but may cause a safety concern due to its integration into transcriptional active regions in chromosomes.
In this study, we applied lentiviruses as transgene vehicle. Many authors have explicitly addressed the advantages and disadvantages about nonviral and viral gene transfer. , Nonviral vector has advantages such as low immunogenicity, relative safety. However, the efficiency of transfection is relative lower and short transgene expression. Transgene electrotransfer is first highly efficient nonviral gene transfer method for most primary cells and for hard-to-transfect cell lines; it looks like a promising method. Ferreira et al.  have compared a BMP-2 secretion rate of the control under CMV, eIF4A1, EF-1α, β-actin, GAPDH, fibronectin or osteocalcin promoters using electrotransfer into rat MSCs and showed that BMP-2 production correlated with sustained (up to 21 d) hBMP-2 mRNA expression. But some authors also pointed out that the major demerits of which are the cost and the induced cell mortality, and improvements must be made to increase the expression efficiency of the transfected cells stable expression of transgene.  Furthermore, in the past recombinant adeno-associated virus and lipidosome transfection reagent Fugene6 were used in our laboratory, we found the transfection efficiency of lipidosome transfection reagent Fugene6 was relatively lower. In this study, we did not observe oncogenicity in rabbits until 12 weeks after autologous cell transplantation. Another safety concern of using LVs is the potential to generate replication-competent lentiviruses. We carefully recommended that LV is a high efficiency and relative safety transgene vehicle. Meanwhile, further investigations to evaluate the bio-safety of using LVs are needed in large-scale animal experiments before they can be implemented in the clinics.
Previous studies often used complicated and expensive scaffolds to load cells for bone formation.  And Lό et al.  have described the osteogenic differentiation after β-tricalcium phosphate calcium phosphate cement seeded with pBMP-2 modified canine MSCs through polyethylenimine transgene. Fujita et al.  also have found no significant difference between the gelatin sponge incorporating BMP-2 and the gelatin-β TCP sponge incorporating BMP-2 groups and although gelatin has been applied in a number of tissue-engineering studies and clinical practices. To our limited knowledge, sole application of absorbable gelatin sponge as a scaffold in gene-mediated bone tissue engineering is rare. Mehanna et al.  have reported a clinical case about used recombinant human morphogenetic protein-2 on absorbable gelatin sponge for augmentation of severe lateral ridge defects. In this study, an absorbable gelatin sponge was used as a scaffold to load MSCs expressing hBMP-2 for bone formation in vivo. Although, this scaffold was simple and economic, it was efficient in our procedure. In our study, all six rabbits formed bones of 10-15 mm  after transplantation with 10  LV-hBMP-2-GFP transduced MSCs, suggesting that the gelatin sponge functioned as an efficient scaffold for bone formation. Absorbable gelatin sponge used as Scaffolds is degradable and has good biocompatibility. However, we also acknowledge the main limitation of it is less osteoconductivity compared with calcium phosphate cement.
In our study, efficient bone formation may also be ascribed to the autologous cell transplantation used in our experiments. Autologous cell transplantation can prevent the immune response, which commonly occurs during allogenic cell transplantation.
Using rabbit as an animal model, we demonstrated that ectopic expression of hBMP-2 in rabbit BM-derived MSCs induced MSCs toward osteogenic differentiation and formed a bone in vivo. In addition, we developed a simple and economic absorbable gelatin sponge scaffold to induce new bone formation in vivo. The procedure established in the rabbit could be translated for human clinical applications for bone defects.
| Acknowledgments|| |
We thank Dr. Qin Pu for providing the LV-hBMP-2-GFP lentiviral vector and Dr. Zhang Zheng for his technical assistance.
| References|| |
|1.||Quinonez R, Sutton RE. Lentiviral vectors for gene delivery into cells. DNA Cell Biol 2002;21:937-51. |
|2.||Gelse K, Schneider H. Ex vivo gene therapy approaches to cartilage repair. Adv Drug Deliv Rev 2006;58:259-84. |
|3.||Løken S, Jakobsen RB, Arøen A, Heir S, Shahdadfar A, Brinchmann JE, et al. Bone marrow mesenchymal stem cells in a hyaluronan scaffold for treatment of an osteochondral defect in a rabbit model. Knee Surg Sports Traumatol Arthrosc 2008;16:896-903. |
|4.||Yanoso-Scholl L, Jacobson JA, Bradica G, Lerner AL, O′Keefe RJ, Schwarz EM, et al. Evaluation of dense polylactic acid/beta-tricalcium phosphate scaffolds for bone tissue engineering. J Biomed Mater Res A 2010;95:717-26. |
|5.||Zuk PA, Zhu M, Mizuno H, Huang J, Futrell JW, Katz AJ, et al. Multilineage cells from human adipose tissue: Implications for cell-based therapies. Tissue Eng 2001;7:211-28. |
|6.||Limbert C, Ebert R, Schilling T, Path G, Benisch P, Klein-Hitpass L, et al. Functional signature of human islet-derived precursor cells compared to bone marrow-derived mesenchymal stem cells. Stem Cells Dev 2010;19:679-91. |
|7.||In ′t Anker PS, Scherjon SA, Kleijburg-van der Keur C, de Groot-Swings GM, Claas FH, Fibbe WE, et al. Isolation of mesenchymal stem cells of fetal or maternal origin from human placenta. Stem Cells 2004;22:1338-45. |
|8.||Caplan AI. Adult mesenchymal stem cells for tissue engineering versus regenerative medicine. J Cell Physiol 2007;213:341-7. |
|9.||Li F, Wang X, Niyibizi C. Bone marrow stromal cells contribute to bone formation following infusion into femoral cavities of a mouse model of osteogenesis imperfecta. Bone 2010;47:546-55. |
|10.||Izuta Y, Ochi M, Adachi N, Deie M, Yamasaki T, Shinomiya R. Meniscal repair using bone marrow-derived mesenchymal stem cells: Experimental study using green fluorescent protein transgenic rats. Knee 2005;12:217-23. |
|11.||Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999;284:143-7. |
|12.||Shafiee A, Seyedjafari E, Soleimani M, Ahmadbeigi N, Dinarvand P, Ghaemi N. A comparison between osteogenic differentiation of human unrestricted somatic stem cells and mesenchymal stem cells from bone marrow and adipose tissue. Biotechnol Lett 2011;33:1257-64. |
|13.||Kadiyala S, Young RG, Thiede MA, Bruder SP. Culture expanded canine mesenchymal stem cells possess osteochondrogenic potential in vivo and in vitro. Cell Transplant 1997;6:125-34. |
|14.||Kirker-Head CA. Potential applications and delivery strategies for bone morphogenetic proteins. Adv Drug Deliv Rev 2000;43:65-92. |
|15.||Kalwitz G, Endres M, Neumann K, Skriner K, Ringe J, Sezer O, et al. Gene expression profile of adult human bone marrow-derived mesenchymal stem cells stimulated by the chemokine CXCL7. Int J Biochem Cell Biol 2009;41:649-58. |
|16.||Valenti MT, Dalle Carbonare L, Donatelli L, Bertoldo F, Zanatta M, Lo Cascio V. Gene expression analysis in osteoblastic differentiation from peripheral blood mesenchymal stem cells. Bone 2008;43:1084-92. |
|17.||Bosnakovski D, Mizuno M, Kim G, Takagi S, Okumura M, Fujinaga T. Chondrogenic differentiation of bovine bone marrow mesenchymal stem cells (MSCs) in different hydrogels: Influence of collagen type II extracellular matrix on MSC chondrogenesis. Biotechnol Bioeng 2006;93:1152-63. |
|18.||Meinel L, Hofmann S, Betz O, Fajardo R, Merkle HP, Langer R, et al. Osteogenesis by human mesenchymal stem cells cultured on silk biomaterials: Comparison of adenovirus mediated gene transfer and protein delivery of BMP-2. Biomaterials 2006;27:4993-5002. |
|19.||Cheng SL, Lou J, Wright NM, Lai CF, Avioli LV, Riew KD. In vitro and in vivo induction of bone formation using a recombinant adenoviral vector carrying the human BMP-2 gene. Calcif Tissue Int 2001;68:87-94. |
|20.||Sugiyama O, An DS, Kung SP, Feeley BT, Gamradt S, Liu NQ, et al. Lentivirus-mediated gene transfer induces long term transgene expression of BMP-2 in vitro and new bone formation in vivo. Mol Ther 2005;11:390-8. |
|21.||Shi ZB, Wang KZ. Effects of recombinant adeno-associated viral vectors on angiopoiesis and osteogenesis in cultured rabbit bone marrow stem cells via co-expressing hVEGF and hBMP genes: A preliminary study in vitro. Tissue Cell 2010;42:314-21. |
|22.||Logan AC, Lutzko C, Kohn DB. Advances in lentiviral vector design for gene-modification of hematopoietic stem cells. Curr Opin Biotechnol 2002;13:429-36. |
|23.||Iwai R, Fujiwara M, Wakitani S, Takagi M. Ex vivo cartilage defect model for the evaluation of cartilage regeneration using mesenchymal stem cells. J Biosci Bioeng 2011;111:357-64. |
|24.||Azzouz M, Kingsman SM, Mazarakis ND. Lentiviral vectors for treating and modeling human CNS disorders. J Gene Med 2004;6:951-62. |
|25.||Huang J, Zhang L, Chu B, Peng X, Tang S. Repair of bone defect in caprine tibia using a laminated scaffold with bone marrow stromal cells loaded poly (L-lactic acid)/β-tricalcium phosphate. Artif Organs 2011;35:49-57. |
|26.||Terella A, Mariner P, Brown N, Anseth K, Streubel SO. Repair of a calvarial defect with biofactor and stem cell-embedded polyethylene glycol scaffold. Arch Facial Plast Surg 2010;12:166-71. |
|27.||Kim J, Kim IS, Cho TH, Lee KB, Hwang SJ, Tae G, et al. Bone regeneration using hyaluronic acid-based hydrogel with bone morphogenic protein-2 and human mesenchymal stem cells. Biomaterials 2007;28:1830-7. |
|28.||Grgurevic L, Macek B, Mercep M, Jelic M, Smoljanovic T, Erjavec I, et al. Bone morphogenetic protein (BMP) 1-3 enhances bone repair. Biochem Biophys Res Commun 2011;408:25-31. |
|29.||Inagaki N, Narushima K, Lim SK. Effects of aromatic groups in polymer chains on plasma surface modification. J Appl Polym Sci 2003;89:96-103. |
|30.||Tjia JS, Aneskievich BJ, Moghe PV. Substrate-adsorbed collagen and cell secreted fibronectin concertedly induce cell migration on poly (lactide-glycolide) substrates. Biomaterials 1999;20:2223-33. |
|31.||Guidance Suggestions for the Care and Use of Laboratory Animals The Ministry of Science and Technology of the People′s Republic of China. [Last accessed on 2006 Sep 30]. |
|32.||Chen F, Chen S, Tao K, Feng X, Liu Y, Lei D, et al. Marrow-derived osteoblasts seeded into porous natural coral to prefabricate a vascularised bone graft in the shape of a human mandibular ramus: Experimental study in rabbits. Br J Oral Maxillofac Surg 2004;42:532-7. |
|33.||Sena-Esteves M, Tebbets JC, Steffens S, Crombleholme T, Flake AW. Optimized large-scale production of high titer lentivirus vector pseudotypes. J Virol Methods 2004;122:131-9. |
|34.||Hirata K, Tsukazaki T, Kadowaki A, Furukawa K, Shibata Y, Moriishi T, et al. Transplantation of skin fibroblasts expressing BMP-2 promotes bone repair more effectively than those expressing Runx2. Bone 2003;32:502-12. |
|35.||de Girolamo L, Bertolini G, Cervellin M, Sozzi G, Volpi P. Treatment of chondral defects of the knee with one step matrix-assisted technique enhanced by autologous concentrated bone marrow: In vitro characterisation of mesenchymal stem cells from iliac crest and subchondral bone. Injury 2010;41:1172-7. |
|36.||Musgrave DS, Bosch P, Lee JY, Pelinkovic D, Ghivizzani SC, Whalen J, et al. Ex vivo gene therapy to produce bone using different cell types. Clin Orthop Relat Res 2000;378:290-305. |
|37.||Sacchetti B, Funari A, Michienzi S, Di Cesare S, Piersanti S, Saggio I, et al. Self-renewing osteoprogenitors in bone marrow sinusoids can organize a hematopoietic microenvironment. Cell 2007;131:324-36. |
|38.||Sutherland MS, Rao LG, Muzaffar SA, Wylie JN, Wong MM, McBroom RJ, et al. Age-dependent expression of osteoblastic phenotypic markers in normal human osteoblasts cultured long term in the presence of dexamethasone. Osteoporos Int 1995;5:335-43. |
|39.||Kawaguchi T, Yamaguchi A, Komaki M, Abe E, Takahashi N, Ikeda T, et al. bone morphogenetic protein-2 converts the differentiation pathway of C2C 12 myoblasts into the osteoblast lineage. J Cell Biol 1994;127:1755-66. |
|40.||Chuang CK, Sung LY, Hwang SM, Lo WH, Chen HC, Hu YC. Baculovirus as a new gene delivery vector for stem cell engineering and bone tissue engineering. Gene Ther 2007;14:1417-24. |
|41.||Ferreira E, Potier E, Vaudin P, Oudina K, Bensidhoum M, Logeart-Avramoglou D, et al. Sustained and promoter dependent bone morphogenetic protein expression by rat mesenchymal stem cells after BMP-2 transgene electrotransfer. Eur Cell Mater 2012;24:18-28. |
|42.||Wegman F, Bijenhof A, Schuijff L, Oner FC, Dhert WJ, Alblas J. Osteogenic differentiation as a result of BMP-2 plasmid DNA based gene therapy in vitro and in vivo. Eur Cell Mater 2011;21:230-42. |
|43.||Hamm A, Krott N, Breibach I, Blindt R, Bosserhoff AK. Efficient transfection method for primary cells. Tissue Eng 2002;8:235-45. |
|44.||Zaragosi LE, Billon N, Ailhaud G, Dani C. Nucleofection is a valuable transfection method for transient and stable transgene expression in adipose tissue-derived stem cells. Stem Cells 2007;25:790-7. |
|45.||Sun Y, Finne-Wistrand A, Albertsson AC, Xing Z, Mustafa K, Hendrikson WJ, et al. Degradable amorphous scaffolds with enhanced mechanical properties and homogeneous cell distribution produced by a three-dimensional fiber deposition method. J Biomed Mater Res A 2012;100:2739-49. |
|46.||Lü K, Zeng D, Zhang W, Xia L, Xu L, Jiang X, et al. Ectopic study of calcium phosphate cement seeded with pBMP-2 modified canine bMSCs mediated by a nonviral PEI derivative. Cell Biol Int 2012;36:119-28. |
|47.||Fujita N, Matsushita T, Ishida K, Sasaki K, Kubo S, Matsumoto T, et al. An analysis of bone regeneration at a segmental bone defect by controlled release of bone morphogenetic protein 2 from a biodegradable sponge composed of gelatin and β-tricalcium phosphate. J Tissue Eng Regen Med 2012;6:291-8. |
|48.||Mehanna R, Koo S, Kim DM. Recombinant human bone morphogenetic protein 2 in lateral ridge augmentation. Int J Periodontics Restorative Dent 2013;33:97-102. |
Department of Orthopaedics, The 8th Hospital, Changsha, No. 22, Xin Sha Road, Changsha, Hunan 410008
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
|This article has been cited by|
||Repair of bone defects using adipose-derived stem cells combined with alpha-tricalcium phosphate and gelatin sponge scaffolds in a rat model
| ||Adriana CORSETTI,Claudia BAHUSCHEWSKYJ,Deise PONZONI,Renan LANGIE,Luis Alberto dos SANTOS,Melissa CAMASSOLA,Nance Beyer NARDI,Edela PURICELLI |
| ||Journal of Applied Oral Science. 2017; 25(1): 10 |
|[Pubmed] | [DOI]|
||Possibility of Fixation of a Distal Radius Fracture With a Volar Locking Plate Through a 10 mm Approach
| ||Kiyohito Naito,Ahmed Zemirline,Yoichi Sugiyama,Hiroyuki Obata,Philippe Liverneaux,Kazuo Kaneko |
| ||Techniques in Hand & Upper Extremity Surgery. 2016; 20(2): 71 |
|[Pubmed] | [DOI]|
||Genetic Engineering of Mesenchymal Stem Cells for Regenerative Medicine
| ||Adam Nowakowski,Piotr Walczak,Miroslaw Janowski,Barbara Lukomska |
| ||Stem Cells and Development. 2015; 24(19): 2219 |
|[Pubmed] | [DOI]|
||Identification of optimal reference genes for quantitative PCR studies on human mesenchymal stem cells
| ||Xiuying Li,Qiwei Yang,Jinping Bai,Yanyan Yang,Lingzhi Zhong,Yimin Wang |
| ||Molecular Medicine Reports. 2014; |
|[Pubmed] | [DOI]|
| Article Access Statistics|
| Viewed||2140 |
| Printed||44 |
| Emailed||0 |
| PDF Downloaded||110 |
| Comments ||[Add] |
| Cited by others ||4 |