Objective To investigate the possibility of differentiation of theisolated and cultured adipose-derived adult stem cells into chondrocytes, which is induced by the recombinant human bone morphogenetic protein 2 (rhBMP-2). Methods The rabbit adipose tissue was minced and digested by collagenase Type Ⅰ. The adposederived adult stem cells were obtained and then they were cultured inthe micropellet condition respectively in the rhBMP-2 group, the rhTGF-β1 group, the combination group, and the control group for 14 days. The differentiation of the adiposederived stem cells into chondrocytes was identifiedby the histological methods including HE, Alcian blue, Von kossa, and immunohistochemical stainings. Results After the continuous induction by rhBMP-2 and continuous culture for 14 days, the HE staining revealed a formation of the cartilage lacuna; Alcian blue indicated that proteoglycan existed in the extracellular matrix; the immunohistochemical staining indicated that collagen Ⅱ was in the cellular matrix; and Von kossa indicated that the adipose-derived stem cells couldnot differentiate into the osteoblasts by an induction of rhBMP-2. Conclusion In the micropellet condition, the adipose-derived adult stemcells can differentiate into the chondrocytes, which is initially induced by rhBMP-2. This differentiation can provide a foundation for the repair of the cartilage injury.
Objective To evaluate the bone regenerative potential of reconbinant human bone morphogenetic protein 2(rhBMP-2) / collagen on adult rat calvarial bone. Methods A tight subperiosteal pocket was produced under both sides ofthe temporal muscle in rats. rhBMP-2 / collagen was implanted in one side and collagen alone was implanted in the other side as control. The rats were sacrificed 2, 4 and 8 weeks after operation. The specimen was harvested and examined histologically. For morphometric analysis, the thickness of the temporal bone of both sides was measured and compared. Results The rhBMP-2 / collagen onlay implant resulted in active bone formation and the augmented bone was connected directly with the original bone, whereas the collagen alone resulted in neither bone nor cartilage production. The ossification process in the rhBMP-2 / collagen occurred directly through bone formation, similar to intramembranous ossification. Conclusion rhBMP-2 / collagen is an effective material as a biological onlay implant.
Objective To investigate the effects of the recombinanthuman bone morphogenetic protein 2 (rhBMP-2) and/or the osteogenic agents on proliferation and expression of the osteoblast phenotype differentiation of the SD rat mesenchymal stem cells（MSCs）. Methods The rat MSCs were cultured in vitro and were randomly divided into the experimental groups(Groups A-I) and the control group. In the experimental group, MSCs were induced by rhBMP2 in different doses (10, 50, 100 and 200 μg/L) in Groups BE, the osteogenic agent alone (Group A) and by the combined use of rhBMP-2 ［in different doses (10,50, 100 and 200 μg/L)］ and the osteogenic agent in Groups F-I. The MTT colorimetric assay was used to evaluate the proliferation, and the activities of alkaline phosphatase (ALP) and osteocalcin (OC) were observed at 3, 6, 9, 12 days, respectively. Results The inverted phase contrast microscopy showed that MSCs by primary culture for 12 hours were adhibited, with a fusiform shape at 48 hours. At 4 days they were polygonal or atractoid, and were spread gyrately or radiately at 6 days. At 10 days, they were spread at the bottom of the bottle.The statistical analysis showed that the expression of the osteoblast phenotype differentiation of MSCs could be induced in the experimental groups. The proliferation of MSCs could be enhanced in a dosedependent manner in GroupsB-E. The expression of the osteoblast phenotype differentiation, which was tested by the activities of ALP and OC, was significantly higher in Groups F-I than in Groups A-E. Conclusion The combined use of rhBMP-2 and the osteogenic agents can enhance the MSC proliferation and induce an expressionand maintenance of the osteoblast phenotype differentiation of the rat MSCs.
Objective To construct inducible lentiviral vector containing human bone morphogenetic protein 2 (hBMP-2) gene and to study its expression in human umbil ical cord blood mesenchymal stem cells (HUMSCs). Methods hBMP-2 gene was ampl ified by PCR from a plasmid and was cloned into pDown by BP reaction. pLV/EXPN2-Neo-TRE-hBMP-2 and pLV/EXPN2-Puro-EF1A-reverse transactivator (rtTA) were obtained with GATEWAY technology, and then were sequenced and analyzed by PCR. The recombinant vectors were transfected into 293FT cells respectively through l ipofectamine, and the lentiviral viruses were harvested from 293FT cells, then the titer was determined. Viruses were used to infect HUMSCs in tandem. In order to research the influence of induction time and concentration, one group of HUMSCs was induced by different doxycl ine concentrations (0, 10, 100 ng/mL, and 1, 10, 100 μg/mL) in the same induction time (48 hours), and the other by the same concentration (10 μg/mL) in different time points (12, 24, 48, and 72 hours). The expression of target gene hBMP-2 was indentified by ELISA method. After 2-week osteogenic induction of transfected HUMSCs, the mineral ization nodes were detected with Al izarin bordeaux staining method. Results Therecombinant inducible lentiviral vectors (pLV/EXPN2-Neo-TRE-hBMP-2 and pLV/EXPN2-Puro-EF1A-rtTA) were successfully constructed. The lentiviruses were also obtained and mediated by 293FT cells, and the virus titers were 3.5 × 108 TU/mL and 9.5 × 107 TU/mL respectively. HUMSCs could expression hBMP-2 by induction of doxycycl ine. The expression of hBMP-2 reached the peak at 10 μg/mL doxycl ine at 48 hours of induction. After 2-week osteogenic induction, a lot of mineral ization nodes were observed. Conclusion The recombinant inducible lentiviral vectors containing hBMP-2 gene can be successfully constructed, which provide an effective and simple method for the further study of stem cells and animal experiment in vivo.
Objective To investigate if the course of intervertebral disc degeneration (IDD) is delayed by injecting lentivirus (Lv) vector carrying bone morphogenetic protein 2 (BMP-2) and inhibitor of differentiation 1 (Id1) genes directly into the nucleus pulposus. Methods Thirty-two New Zealand white rabbits, 2.0-2.5 kg in weight and 4 months in age, were used to establish the IDD models at L3, 4, L4, 5, and L5, 6 discs with annular puncture via transabdominal approach. Thirty rabbits with successful modeling were randomly divided into 5 groups, 6 rabbits every group. At 4 weeks after modeling, rabbits were injected with Lv-BMP-2 (group A), with Lv-BMP-2 and Lv-Id1 (group B), with Lv-Id1 (group C), with Lv-green fluorescent protein (group D), and with PBS (group E). At 2, 4, and 8 weeks after injection, T2-mapping MRI was performed on 2 rabbits each group to obtain the T2 values, and then subsequently the lumbar disc tissues were harvested to test the mRNA expressions and contents of collagen type II and proteoglycan by real-time fluorescent quantitative PCR and ELISA methods. Results T2-mapping MRI demonstrated that there was no significant difference in the T2 value between different groups at immediate and 2 weeks after injection (P>0.05). The T2 value of groups A and B was significantly higher than that of groups C, D, and E at 4 weeks after injection (P<0.05), but no significant difference was observed between group A and group B (P>0.05). The T2 value of group B was significantly higher than that of the other groups at 8 weeks after injection (P<0.05). The real-time fluorescent quantitative PCR and ELISA showed that the expressions and contents of collagen type II and proteoglycan in group B were significantly higher than those in the other groups at 2, 4, and 8 weeks after injection (P<0.05). Conclusion Combined application of Lv-BMP-2 and Lv-Id1 can delay IDD changes in rabbit IDD models.
Objective To investigate bone regeneration of the cell-biomaterial complex using strategies of tissue engineering based on cells.Methods Hydroxyapatite/collagen (HAC) sandwich composite was produced to mimic the natural extracellular matrix of bone, with type Ⅰ collagen servingas a template for apatite formation. A three-dimensional ploy-porous scaffoldwas developed by mixing HAC with poly(L-lactic acid) (PLA) using a thermally induced phase separation technique (TIPS). The rabbit periosteal cells were treated with 500 ng/ml of recombinant human bone morphogenetic protein 2(rhBMP-2), followed by seeded into pre-wet HAC-PLA scaffolds. Eighteen 3-month nude mice were implanted subcutaneously cell suspension (groupA, n=6), simple HAC-PLA scaffold (group B, n=6) and cell-biomaterial complex(group C, n=6) respectively.Results Using type Icollagen to template mineralization of calcium and phosphate in solution, we get HAC sandwich composite, mimicking the natural bone both in compositionand microstructure. The three dimensional HAC-PLA scaffold synthesized by TIPShad high porosity up to 90%, with pore size ranging from 50 μm to 300 μm. SEMexamination proved that the scaffold supported the adhesion and proliferation of the periosteal cells. Histology results showed new bone formation 8 weeks after implantation in group C. The surface of group A was smooth without neoplasma. Fibrous tissueinvasion occured in group B and no bone and cartilage formations were observed.Conclusion The constructed tissue engineering bone has emerged as another promising alternative for bone repair.
ObjectiveTo construct bone morphogenetic protein 2 (BMP-2) gelatin/chitosan hydrogel sustained-release system, co-implant with induced pluripotent stem cells (iPS) derived mesenchymal stem cells (MSCs) to hydroxyapatite (HA)/zirconium dioxide (ZrO2) bio porous ceramic foam, co-culture in vitro, and to explore the effect of sustained-release system on osteogenic differentiation of iPS-MSCs.MethodsBMP-2 gelatin/chitosan hydrogel microspheres were prepared by water-in-oil solution. Drug encapsulation efficiency, drug loading, and in vitro sustained release rate of the microspheres were tested. HA/ZrO2 bio porous ceramic foam composite iPS-MSCs and BMP-2 gelatin/chitosan hydrogel sustained release system co-culture system was established as experimental group, and cell scaffold complex without BMP-2 composite gelatin/chitosan hydrogel sustained release system as control group. After 3, 7, 10, and 14 days of co-culture in the two groups, ALP secretion of cells was detected; gene expression levels of core binding factor alpha 1 (Cbfa1), collagen type Ⅰ, and Osterix (OSX) were detected by RT-PCR; the expression of collagen type Ⅰ was observed by immunohistochemical staining at 14 days of culture; and cell creep and adhesion were observed by scanning electron microscopy.ResultsBMP-2 gelatin/chitosan hydrogel sustained-release system had better drug encapsulation efficiency and drug loading, and could prolong the activity time of BMP-2. The secretion of ALP and the relative expression of Cbfa1, collagen type Ⅰ, and OSX genes in the experimental group were significantly higher than those in the control group at different time points in the in vitro co-culture system (P<0.05). Immunohistochemical staining showed that the amount of fluorescence in the experimental group was significantly more than that in the control group, i.e. the expression level of collagen type Ⅰ was higher than that in the control group. The cells could be more evenly distributed on the materials, and the cell morphology was good. Scanning electron microscopy showed that the sustained-release system could adhere to cells well.ConclusioniPS-MSCs have the ability of osteogenic differentiation, which is significantly enhanced by BMP-2 gelatin/chitosan hydrogel sustained-release system. The combination of iPS-MSCs and sustained-release system can adhere to the materials well, and the cell activity is better.
OBJECTIVE To improve the osteoinduction of coral and provide a perfect bone graft substitute for clinical bone defects. METHODS By combining coral with collagen and recombinant human bone morphogenetic protein-2(rhBMP-2), coral/collagen/rhBMP-2 composite was obtained. The composite was implanted into the back muscle pouches of mice, and coral/collagen or coral/rhBMP-2 were implanted as control. The osteoinduction of the composite was assessed by histology and image analysis system. RESULTS The chondrocyte differentiation and matrix formation were observed in local sites after one week, lamellar bone with bone marrow were formed after 4 weeks, and coral were absorbed partially. The quantity of osteoinduction was time-related and rhBMP-2 dose-related(P lt; 0.01). Coral/collagen and coral/rhBMP-2 implants did not show any bone or cartilage formation. CONCLUSION The coral/collagen/rhBMP-2 composite possesses a superior osteoinduction and will be a new type of bone substitute to be used in orthopedic and maxillofacial surgery.
Objective To explore the in vitro osteogenesis of the chitosan-gelatin scaffold compounded with recombinant human bone morphogenetic protein 2 (rhBMP-2). Methods Recombinant human BMP-2 was compounded with chitosan-gelatin scaffolds by freezedrying. 2T3 mouse osteoblasts and C2C12 mouse myoblasts were cultured and seeded onto the complexes at thedensity of 2×104/ml respectively. The complexes were divided into two groups. Group A: 2T3 osteoblasts seeded, consisted of 14 rhBMP-2 modified complexes. Each time three scaffolds were taken on the 3rd, 7th, 14th, and 21st day of the culturing, then the expression of osteocalcin gene (as the marker of bone formation) in adherent cells was detected by semiquantitative RT-PCR with housekeeping gene β-tubulin as internalstandard. The other 2 rhBMP-2 modified complexes were stopped being cultured on 14th day after cell seeding, and the calcification of the complexes was detected by Alizarian Red S staining. Five scaffolds without rhBMP-2 modification as the control group A, they were stopped being cultured on 14th day after cell seeding. Of the 5 scaffolds, 3 were subjected tothe detection of osteocalcin gene expression and 2 were subjected to the detection of calcification. Group B: C2C12 myoblasts seeded, had equal composition andwas treated with the same as group A. Besides these 2 groups, another 2 rhBMP2 modified complexes with 2T3 osteoblasts seeding were cultured for 3 days and then scanned by electron microscope (SEM) as to detect the compatibility of the cell to the complex. ResultsSEM showed that cells attached closely to the complex and grew well. In group A, the expression level(1.28±0.17)of osteocalcin gene in cells on rhBMP-2 modified complexes was higher than that (0.56±0.09) of the control group A, being statistically -significantly different(P＜0.05) control. C2C12 myoblasts which did not express osteocalcin normally could also express osteocalcin after being stimulated by rhBMP-2 for at least 7 days. Alizarian Red S staining showed that there was more calcification on rhBMP-2 modified complexes in both groups. There were more calcification in the group compounded with rhBMP-2, when the groups were seeded with the same cells. Conclusion The complexmade of rhBMP-2 and chitosan-gelatin scaffolds has b osteogenesis ability in vitro.
ObjectiveTo evaluate the combination of lipopolysaccharide-amine nanopolymersomes (LNPs), as a gene vector, with target gene and the transfection in bone marrow mesenchymal stem cells (BMSCs) so as to provide a preliminary experiment basis for combination treatment of bone defect with gene therapy mediated by LNPs and stem cells. MethodsPlasmid of bone morphogenetic protein 2 (pBMP-2)-loaded LNPs (pLNPs) were prepared. The binding ability of pLNPs to pBMP-2 was evaluated by a gel retardation experiment with different ratios of nitrogen to phosphorus elements (N/P). The morphology of pLNPs (N/P=60) was observed under transmission electron microscope (TEM) and atomic force microscope (AFM). The size and Zeta potential were measured by dynamic light scattering (DLS). The resistance of pLNPs against DNase I degradation over time was explored. The viability of BMSCs, transfection efficiency, and expression of target protein were investigated after transfection by pLNPs in vitro. ResultsAt N/P≥1.5, pLNPs could completely retard pBMP-2; at N/P of 60, pLNPs was uniform vesicular shape under AFM; TEM observation demonstrated that pLNPs were spherical nano-vesicles with the diameter of (72.07±11.03) nm, DLS observation showed that the size of pLNPs was (123±6) nm and Zeta potential was 20 mV; pLNPs could completely resist DNase I degradation within 4 hours, and such protection capacity to pBMP-2 decreased slightly at 6 hours. The cell survival rate first increased and then decreased with the increase of N/P, and reached the maximum value at N/P of 45; the cytotoxicity was in grade I at N/P≤90, which meant no toxicity for in vivo experiment. While the transfection efficiency of pLNPs increased with the increase of N/P, and reached the maximum value at N/P of 60. So it is comprehensively determined that the best N/P was 60. At 4 days, transfected BMSCs expressed BMP-2 continuously at a relatively high level at N/P of 60. ConclusionLNPs can compress pBMP-2 effectively to form the nanovesicles complex, which protects the target gene against enzymolysis. LNPs has higher transfection efficiency and produces more amount of protein than polyethylenimine 25k and Lipofectamine 2000.