The repair of the long bone defects by combined grafting of homogenous deealcified bene matrix(DBM ) with centrally enveloped vascularized periosteum Was reported as a new techniqe. Theroentgenograms,bone mineral count and histologic examination were done. The results showed thatthis method was beneficial and had better effect on prornoting healing of the long bene defeets fromone stage operation The oporative proeedure was described on deatil It was considered that the homogenous DBM ...
Objective To evaluate the tensile mechanical characteristics of decalcified cortical bone matrix with different thicknesses so as to provide an experimental basis for the scaffold of tissue engineering. Methods Decalcified cortical bone matrix was prepared from fresh bovine tibia with rapid decalcification techniques. Its physical characteristics including colour, texture, and so on, were observed. Then the decalcified rate was calculated. Decalcified cortical bone matrices were radially cut into sl ices with different thicknesses along longitudinal axis and divided into 4 groups: group A (100- 300 μm), group B (300-500 μm), group C (500-700 μm), and group D (700-1 000 μm). Then the sl ice specimens of each group were characterized with tensile test and histological examination. Results General observation showed that decalcified cortical bone matrix with hydrogen peroxide treatment was ivory white with good elasticity and flexibil ity. The decalcified rate was 97.6%. The tensile strength and elastic modulus of groups B, C, and D were significantly higher than those of roup A (P lt; 0.05); there was no significant difference among groups B, C, and D (P gt; 0.05). The stiffness in 4 groups increased gradually with the increasing thickness, it was significantly lower in group A than those in groups B, C, and D (P lt; 0.05), and in groups B and C than that in group D (P lt; 0.05). While there was no significant difference in ultimate strain within 4 groups (P gt; 0.05). Histologically, intact osteon was observed in every group, with an average maximum diameter of 182 μm (range, 102- 325 μm). Conclusion The mechanical properties of decalcified cortical bone matrix might depend on the integrity of the osteons. Sl ices with thickness of 300 μm or more could maintain similar mechanical properties when decalcified cortical bone matrix is used as a scaffold for tissue engineering.
Objective To fabricate a novel composite scaffold with acellular demineralized bone matrix/acellular nucleus pulposus matrix and to verify the feasibility of using it as a scaffold for intervertebral disc tissue engineering through detecting physical and chemical properties. Methods Pig proximal femoral cancellous bone rings (10 mm in external diameter, 5 mm in internal diameter, and 3 mm in thickness) were fabricated, and were dealed with degreasing, decalcification, and decellularization to prepare the annulus fibrosus phase of scaffold. Nucleus pulposus was taken from pig tails, decellularized with Triton X-100 and deoxycholic acid, crushed and centrifugalized to prepare nucleus pulposus extracellular mtrtix which was injected into the center of annulus fibrosus phase. Then the composite scaffold was freeze-dryed, cross-linked with ultraviolet radiation/carbodiimide and disinfected for use. The scaffold was investigated by general observation, HE staining, and scanning electron microscopy, as well as porosity measurement, water absorption rate, and compressive elastic modulus. Adipose-derived stem cells (ADSCs) were cultured with different concentrations of scaffold extract (25%, 50%, and 100%) to assess cytotoxicity of the scaffold. The cell viability of ADSCs seeded on the scaffold was detected by Live/Dead staining. Results The scaffold was white by general observation. The HE staining revealed that there was no cell fragments on the scaffold, and the dye homogeneously distributed. The scanning electron microscopy showed that the pore of the annulus fibrosus phase interconnected and the pore size was uniform; acellular nucleus pulposus matrix microfilament interconnected forming a uniform network structure, and the junction of the scaffold was closely connected. The novel porous scaffold had a good pore interconnectivity with (343.00 ± 88.25) μm pore diameter of the annulus fibrosus phase, 82.98% ± 7.02% porosity and 621.53% ± 53.31% water absorption rate. The biomechanical test showed that the compressive modulus of elasticity was (89.07 ± 8.73) kPa. The MTT test indicated that scaffold extract had no influence on cell proliferation. Live/Dead staining showed that ADSCs had a good proliferation on the scaffold and there was no dead cell. Conclusion Novel composite scaffold made of acellular demineralized bone matrix/acellular nucleus pulposus matrix has good pore diameter and porosity, biomechanical properties close to natural intervertebral disc, non-toxicity, and good biocompatibility, so it is a suitable scaffold for intervertebral disc tissue engineering.
Objective To introduce an injectable andin situ gelling gelatin hydrogel, and to explore the possibility as a carrier for demineralized bone matrix (DBM) powder delivery. Methods First, thiolated gelatin was prepared and the thiol content was determined by Ellman method, and then the injectable andin situ gelling gelatin hydrogel (Gel) was formed by crosslinking of the thiolated gelatin and poly (ethylene oxide) diacrylate and the gelation time was determined by inverted method. Finally, the DBM-Gel composite was prepared by mixing Gel and DBM powder. The cytotoxicity was tested by live/dead staining and Alamar blue assay of the encapsulated cells in the DBM-Gel. Forin vitro cell induction, C2C12 cells were firstly incubated onto the surface of the DBM and then the composite was prepared. The experiment included two groups: DBM-Gel and DBM. The alkaline phosphatase (ALP) activity was determined at 1, 3, 5,and 7 days after culture.In vivo osteoinductivity was evaluated using ectopic bone formation model of nude rats. Histological observation and the ALP activity was measured in DBM-Gel and DBM groups at 4 weeks after implantation. Results The thiol content in the thiolated gelatin was (0.51±0.03) mmol/g determined by Ellman method. The gelation time of the hydrogel was (6±1) minutes. DBM powder can be mixed with the hydrogel and injected into the implantation site within the gelation time. The cells in the DBM-Gel exhibited spreading morphology and connected each other in part with increasing culture time. The viability of the cells was 95.4%±1.9%, 97.3%±1.3%, and 96.1%±1.6% at 1, 3, and 7 days after culture, respectively. The relative proliferation was 1.0±0.0, 1.1±0.1, 1.5±0.1, and 1.6±0.1 at 1, 3, 5, and 7 days after culture respectively.In vitro induction showed that the ALP activity of the DBM-Gel group was similar to that of the DBM group, showing no significant difference (P>0.05). With increasing culture time, the ALP activities in both groups increased gradually and the activity at 5 and 7 days was significantly higher than that at 1 and 3 days (P<0.05), while there was no significant difference between at 1 and 3 days, and between 5 and 7 days (P>0.05). At 4 weeks after implantationin vivo, new bone and cartilage were observed, but no bone marrow formation in DBM-Gel group; in DBM group, new bone, new cartilage, and bone marrow formation were observed. The histological osteoinduction scores of DBM-Gel and DBM groups were 4.0 and 4.5, respectively. The ALP activities of DBM-Gel and DBM groups were respectively (119.4±22.7) and (146.7±13.0) μmol/mg protein/min, showing no significant difference (t=–2.085,P=0.082). Conclusion The injectable andin situ gelling gelatin hydrogel for delivery of DBM is feasible.
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 explore the possibility of repair long segmental bone defects and preventing infection with cefazolin loaded bone matrix gelatin (C-BMG). METHODS: C-BMG was made from putting cefazolin into BMG by vacuum adsorption and freeze-drying techniques. The sustaining period of effective drug concentration in vitro and in vivo was detected by inhabition bacteria, and the drug concentration in local tissues (bone and muscle) and plasma after implantation of C-BMG was examined by high performance liquid chromatography(HPLC). RESULTS: The effective inhibition time to staphylococcus aureus of C-BMG was 22 days in vitro, while 14 days in vivo. The drug concentration in local tissues(bone and muscle) were higher than that of plasma, and the drug concentration in local tissues was higher in early stage, later it kept stable low drug release. It suggested that C-BMG had excellent ability to repair segmental long bone defects. CONCLUSION: C-BMG can gradually release cefazolin with effective drug concentration and has excellent ability to repair segmental long bone defects. It may be a novel method to repair segmental long bone defects and prevent infection after the operation.
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.
Objective To investigate the effect of a porous calcium phosphate/bone matrix gelatin (BMG) composite cement (hereinafter referred to as the " porous composite cement”) for repairing lumbar vertebral bone defect in a rabbit model. Methods BMG was extracted from adult New Zealand rabbits according to the Urist’s method. Poly (lactic-co-glycolic) acid (PLGA) microsphere was prepared by W/O/W double emulsion method. The porous composite cement was developed by using calcium phosphate cement (CPC) composited with BMG and PLGA microsphere. The physicochemical characterizations of the porous composite cement were assessed by anti-washout property, porosity, and biomechanical experiment, also compared with the CPC. Thirty 2-month-old New Zealand rabbits were used to construct vertebral bone defect at L3 in size of 4 mm×3 mm×3 mm. Then, the bone defect was repaired with porous composite cement (experimental group, n=15) or CPC (control group, n=15). At 4, 8, and 12 weeks after implantation, each bone specimen was assessed by X-ray films for bone fusion, micro-CT for bone mineral density (BMD), bone volume fraction (BVF), trabecular thickness (Tb. Th.), trabecular number (Tb.N.), and trabecular spacing (Tb. Sp.), and histological section with toluidine blue staining for new-born bone formation. Results The study demonstrated well anti-washout property in 2 groups. The porous composite cement has 55.06%±1.18% of porosity and (51.63±6.73) MPa of compressive strength. The CPC has 49.38%±1.75% of porosity and (63.34±3.27) MPa of compressive strength. There were significant differences in porosity and compressive strength between different cements (t=4.254, P=0.006; t=2.476, P=0.034). X-ray films revealed that the zone between the cement and host bone gradually blurred with the time extending. At 12 weeks after implantation, the zone was disappeared in the experimental group, but clear in the control group. There were significant differences in BMD, BVF, Tb. Th., Tb. N., and Tb. Sp. between 2 groups at each time point (P<0.05). Histological observation revealed that there was new-born bone in the cement with the time extending in 2 groups. Among them, bony connection was observed between the new-born bone and the host in the experimental group, which was prior to the control group. Conclusion The porous composite cement has dual bioactivity of osteoinductivity and osteoconductivity, which are effective to promote bone defect healing and reconstruction.
Objective To evaluate the tissue response induced by three kinds of bone transplantation materials implanted in rat so as to provide proper evidence for their cl inical appl ication. Methods Thirty-six healthy mature Sprague- Dawly mice, weighing from 229 g to 358 g, were randomly assigned to groups A and B (n=18). Three kinds of materials wereimplanted into muscles of rats. Calcium sulfate (CS) granular preparations and allogeneic demineral ized bone matrix (DBM) were transplanted into the left (group A1) and right (group A2) thigh muscle pouches of group A. Respectively, whereas xenogenic DBM were transplanted into the left (group B1) thigh muscle pouches of group B and the right (group B2) sites were taken as control without implant. The samples (n=6) were collected to make the observation of gross and histology and to analyze histological score after 2, 4, and 6 weeks. Results The gross observation: implanted materials were gradually absorbed at late stage in group A1. No obvious degradation and absorption, but fibrosis of tissues were observed in group A2 and B1. The inflammatory reactions were more severe in groups A2 and B1. In group B2, only the changes of scar were seen at operative site. The histological observation: no obvious inflammatory reactions were seen in group A1, CS were gradually absorbed and completely absorbed at 6 weeks, while fibrosis of tissues increased at late stage. Inflammatory reactions in group A2 and group B1 were alleviated gradually, no obvious absorption and degradation were observed. The different two DBM could induce granulation tissues and bone formation at different sites and secondary fibrosis with no obvious immune response was observed. In group B2, there was an increase in collagen fiber density and angiogenesis at late stage. The scores of inflammatory infiltration were significantly higher in groups A2, B1 than in groups A1, B2 (P lt; 0.05), and the scores of fibrosis was larger in groups A1, A2 and B1 than in group B2 (P lt; 0.05). Conclusion CS has rapid dissolution and good biocompatibil ity. It is a good replaceable packing materials of bone defects in some upper l imb’s or acute bone fracture. Both of two DBM have biocompatibil ity and osteoinductive potential, which dissolution are very slow. Due to these capacity, they can be served as an ideal materials in treatment of lower l imb’s bone defect and nonunion.
Objective To observe the histological structure and cytocompatibility of novel acellular bone matrix (ACBM) and to investigate the feasibility as a scaffold for bone tissue engineering. Methods Cancellous bone columns were harvested from the density region of 18-24 months old male canine femoral head, then were dealt with high-pressure water washing, degreasing, and decellularization with Trixon X-100 and sodium deoxycholate to prepare the ACBM scaffold. The scaffolds were observed by scanning electron microscope (SEM); HE staining, Hoechst 33258 staining, and sirius red staining were used for histological analysis. Bone marrow mesenchymal stem cells (BMSCs) from canine were isolated and cultured with density gradient centrifugation; the 3rd passage BMSCs were seeded onto the scaffold. MTT test was done to assess the cytotoxicity of the scaffolds. The proliferation and differentiation of the cells on the scaffold were observed by inverted microscope, SEM, and live/dead cell staining method. Results HE staining and Hoechst 33258 staining showed that there was no cell fragments in the scaffolds; sirius red staining showed that the ACBM scaffold was stained crimson or red and yellow alternating. SEM observation revealed a three dimensional interconnected porous structure, which was the microstructure of normal cancellous bone. Cytotoxicity testing with MTT revealed no significant difference in absorbance (A) values between different extracts (25%, 50%, and 100%) and H-DMEM culture media (P gt; 0.05), indicating no cytotoxic effect of the scaffold on BMSCs. Inverted microscope, SEM, and histological analysis showed that three dimensional interconnected porous structure of the scaffold supported the proliferation and attachment of BMSCs, which secreted abundant extracellular matrices. Live/dead cell staining results of cell-scaffold composites revealed that the cells displaying green fluorescence were observed. Conclusion Novel ACBM scaffold can be used as an alternative cell-carrier for bone tissue engineering because of thoroughly decellularization, good mircostructure, non-toxicity, and good cytocompatibility.