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 study in vitro sustained release behaviour of the recombinant human bone morphogenetic protein 2(rhBMP-2) from the sample which porous calcium phosphate cement (PCPC) was combined with rhBMP-2, and to evaluate the effect of PCPC/rhBMP-2 composite on repairing bone defect in the animalstudy.Methods rhBMP-2 was absorbed into PCPC by vacuum-adsorption and freeze-dried at -40℃, the PCPC/rhBMP-2 enwrapped with chitosan as the experimental group, the pure PCPC/rhBMP-2 as the control group, then the sustained release ofrhBMP-2 from PCPC was determined in simulated body fluid (SBF) by UV-VIS spectrophotometer. At same time, the PCPC/rhBMP-2 composites with chitosan were implanted into the (4.2 mm×5.0 mm femora defects of rabbits, which were considered as the experimental group, whereas in the control group only PCPC was implanted. The effect of repairingbone defect was evaluated in the 4th and 8th week postoperatively by radiograph and histomorphology.Results The PCPC have a high absorption efficiency to rhBMP-2, and the release of rhBMP-2 was sustained release system. The release of rhBMP-2 from PCPC in the experimental group (99% after 350 hours) was slowerthan that in the control group (100% after 150 hours). In the experimental group, the radiological and histomorphological evaluations showed that theinterfaces between the materials and host bones became blurred both at 4th and 8th week. The implanted materials were partially absorbed, and the implanted areas exhibited the formation of new bone. In the control group, a little amount of new bones was observed. Conclusion The PCPC shows great clinical potential as a carrier for rhBMP-2. The PCPC/rhBMP-2 composite possesses much potentialities of osteoinductivity and the ability of repairing bone defect, so it can be used as a novel bone substitute clinically.
ObjectiveTo investigate the correlation between the content of bone morphogenetic protein 2 (BMP-2) in demineralized bone matrix (DBM) and its osteogenic activity in vitro and in vivo, in order to choose a simple and convenient method to evaluate the osteogenic activity of DBM.MethodsThe left mid-femoral tissues of 9 donors were taken, and DBMs (S1-S9) were prepared by dynamic decalcification process, and inactivated DBM (control group) was prepared at the same time. Protease inhibitor method, collagenase method, guanidine hydrochloride/ethylene diamine tetraacetic acid (EDTA) method, and RIPA lysate method were used to extract BMP-2 in S1-S9 and inactivated DBMs. The BMP-2 content was measured and the differences between DBMs were compared. Then the S1-S9 and inactivated DBMs were co-cultured with mouse embryonic osteoblasts MC3T3-E1, respectively. The cell proliferation was detected by MTT method and fluorescence staining, and alkaline phosphatase (ALP) activity was detected at the same time. Thirty BALB/c male nude mice were divided into 10 groups, namely S1-S9 DBM groups (S1-S9 groups) and inactivated DBM group (control group), with 3 mice in each group. Muscle pockets of the middle thighs were prepared on both hindlimbs of mice in each group, and implanted corresponding DBM materials. At 4 weeks after operation, the samples were taken for HE staining observation and semi-quantitative evaluation, and the new bone formation score was calculated.ResultsThe BMP-2 content of DBM derived from different donor bones was distinct. The BMP-2 content obtained by different extraction methods for DBM prepared from the same donor bone was also different, and the extraction efficiency of the guanidine hydrochloride/EDTA method was the highest. In vitro cell experiments, MTT test displayed that cell proliferations and ALP activity were significantly higher in S4 and S6 groups than in other groups at each time point after co-cultivation (P<0.05). Moreover, the cell proliferation of S4 group was the most significant at 7 days (P<0.05); fluorescence staining demonstrated that the osteoblasts of each group was in good condition, but the osteoblasts of S1, S2, S3, S4, and S6 groups were significantly more than other groups. In vivo ectopic osteogenesis experiments, the cartilage and new bone formation could be seen in the bone graft area of S1-S6 groups at 4 weeks after operation, and with the increase of BMP-2 content, the more new bone formation induced by the material, the higher the score of new bone formation of the material (P<0.05). Among them, S4 and S6 groups contained a large number of chondrocytes and osteoblasts in the osteogenesis area.ConclusionThe osteogenic activity of DBM can be evaluated through BMP-2 quantitative detection combined with in vitro osteoblast proliferation and differentiation experiments.
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.
ObjectiveTo study the ectopic osteogenesis and biocompatibility of bone morphogenetic protein 2 (BMP-2)-derived peptide P24 loaded chitosan-4-thio-butylamidine (CS-TBA) hydrogel.MethodsFirst, the CS-TBA/hydroxyapatite (HA) solution was prepared by using chitosan, 2-iminothiolane hydrochloride, and HA. Then, the different amount of P24 peptides were added to the CS-TBA/HA to prepare the CS-TBA/5%P24/HA and CS-TBA/10%P24/HA solutions. Finally, β-glycerophosphate disodium (β-GP) was added to the CS-TBA/HA, CS-TBA/5%P24/HA, and CS-TBA/10%P24/HA to prepare the CS-TBA/HA/β-GP, CS-TBA/5%P24/HA/β-GP, and CS-TBA/10%P24/HA/β-GP hydrogels, respectively. Eighteen Sprague Dawley female rats were randomly divided into 3 groups (n=6), which were injected into the back muscle pouches with equal volume CS-TBA/HA/β-GP hydrogel (group A), CS-TBA/5%P24/HA/β-GP hydrogel (group B), and CS-TBA/10%P24/HA/β-GP hydrogel (group C). The animals were sacrificed at 4 and 8 weeks and conducted micro-CT. The ability of biodegradation and osteogenesis of hydrogl was detected by trabecular thickness (Tb.Th), trabecular number (Tb.N), bone mineral density (BMD), and histological staining (HE and Masson).ResultsAll the rats survived to the time point of the harvest. Micro-CT results showed that the new bones gradually increased in each group after operation. At the same time, the new bone formation was more obvious in groups B and C than in group A, and with the increase of P24 concentration, new bone formation in group C was much more than that in group B. The Tb.Th, Tb.N, and BMD increased gradually in 3 groups, and the differences between 4 and 8 weeks were significant (P<0.05) except the Tb.Th in group A. At different time points, the Tb.Th, Tb.N, and BMD were significantly higher in groups B and C than in group A (P<0.05), and in group C was higher than in group B (P<0.05), showing significant differences between groups. Histological staining showed that the materials of groups B and C were biodegradable, and the osteogenic effect was increased with the increase of P24 concentration.ConclusionP24 peptide can improve the ectopic osteogenesis of CS-TBA hydrogel, and the 10% concentration is more effective.
ObjectiveTo investigate the effect of graphene oxide (GO)-carboxymethyl chitosan (CMC) hydrogel loaded with interleukin 4 (IL-4) and bone morphogenetic protein 2 (BMP-2) on macrophages M2 type differentiation and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs).MethodsGO solution was mixed with CMC, then the phosphate buffered saline (PBS), IL-4, BMP-2, or IL-4+BMP-2 were added to prepare different GO-CMC hydrogel scaffolds with or without different cytokines under crosslinking agents. The characteristics of pure GO-CMC hydrogel were characterized by gross observation, scanning electron microscope (SEM), and Fourier transform infrared spectroscopy (FTIR), and the CMC hydrogel was used as control. The sustained release of GO-CMC hydrogels with different cytokines was also tested. Macrophages were isolated and cultured from female Sprague Dawley rats aged 4-5 weeks, and then cultured with GO-CMC hydrogels with and without different cytokines, respectively. CD206 immunofluorescence staining was used to detect the differentiation of macrophages after 24 hours. The 3rd generation of rats BMSCs were cultured with GO-CMC hydrogels with and without different cytokines respectively for osteogenic induction. The early osteogenesis was observed by alkaline phosphatase (ALP) staining after 10 days, and the late osteogenesis was observed by alizarin red staining after 21 days.ResultsGenerally, GO-CMC hydrogel was brown and translucent. SEM showed that the pore diameter and wall thickness of GO-CMC hydrogel were similar to that of CMC hydrogel, but the inner wall roughness increased. FTIR test showed that CMC polymerized to form hydrogel. In vitro, the sustained release experiments showed that the properties of GO-CMC hydrogels loaded with different cytokines were similar. CD206 immunofluorescence detection showed that GO-CMC hydrogels could induce macrophages differentiation into M2-type. ALP and alizarin red staining showed that GO-CMC hydrogels could induce BMSCs osteogenic differentiation, in which GO-CMC hydrogel loaded with IL-4+BMP-2 showed the most significant effect (P<0.05).ConclusionThe GO-CMC hydrogel loaded with IL-4 and BMP-2 can induce macrophages differentiation into M2-type and enhance the ability of BMSCs with osteogenic differentiation in vitro, which provide a new strategy for bone defect repair and immune regulation.
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 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.
Objective To measure the concentration of bone morphogenetic protein 2 (BMP-2) in demineralized bone matrix (DBM) prepared from different long bones and to evaluate the osteoinductivity of different DBM on MC3T3-E1 cells. Methods Different bones from the same cadaver donor were used as the initial materials for making DBM, which were divided into ulna group (uDBM), humerus group (hDBM), tibia group (tDBM), and femur group (fDBM) according to the origins, and boiled DBM (cDBM) was taken as the control group. The proteins of DBM were extracted by guanidine hydrochloride, and the concentrations of BMP-2 were determined by ELISA assay. Then the DBM were co-cultured with MC3T3-E1 cells, the proliferation of MC3T3-E1 cells was observed by cell counting kit 8 (CCK-8) assay. The osteogenic differentiation ability of MC3T3-E1 cells was qualitatively observed by alizarin red, alkaline phosphatase (ALP), and Van Gieson staining, and the osteogenic differentiation ability of MC3T3-E1 cells was quantitatively analyzed by ALP content. Linear regression was used to analyze the effect of BMP-2 concentration in DBM on ALP synthesis. ResultsThere were significant differences in the concentration of BMP-2 among the DBM groups (P<0.05). The concentrations of BMP-2 in the lower limb long bone were higher than those in the upper limb long bone, and the concentration of BMP-2 in the fDBM group was about 35.5 times that in the uDBM group. CCK-8 assay showed that the cells in each group continued to proliferate within 5 days of co-culture, and the absorbance (A) values at different time points were in the order of cDBM group<uDBM group<hDBM group<tDBM group<fDBM group. After co-culture for 14 days, the expressions of ALP, calcified nodules, and collagen fibers in each group were consistent with the distribution of BMP-2 concentration in DBM. The order of ALP content from low to high was cDBM group<uDBM group<hDBM group<tDBM group<fDBM group, and the differences between the groups were significant (P<0.05). Linear regression analysis showed that \begin{document}$\hat y $\end{document}=0.361+0.017x, the effect of BMP-2 concentration in DBM on cellular ALP content was significant (t=3.552, P=0.005); for every 1 ng/g increase in BMP-2 concentration, ALP content would increase by 0.017 [95%CI (0.006, 0.027)] U/100 mL. Conclusion The concentration of natural BMP-2 in different long bones varies greatly, and the lower limb long bone is higher than the upper limb long bone. The harvested location of bone material was an important factor affecting the osteoinductivity of DBM.
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.