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 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 evaluate the biomechanicalproperties and structuralcharacteristics of various composites of partially decalcified allogenic bone matrix gelatin and bone cement at different ratios. Methods According to Urist method, partially decalcified allogenic bone matrix gelatin was prepared and mixedwith bone cement at different ratios of 0, 400, 500, and 600mg/g. Then the comparisons of these composites were performed in microstructure, ultimate compression strength and ultimate bending strength properties. Results The electronic microscope showed that the bone particles and bone cement were distributed evenly in the composite, irregularly connecting by multiple points; with the increase ofbone particles and decrease of bone cement in the composite, there were more and more natural crevices, varying from 100 μm to 400 μm in width, in the biomaterials. Of all the composites with the ratios of 0, 400,500, and 600 mg/g, the measurements of ultimate compression strength were (71.7±2.0) MPa, (46.9±3.3) MPa, (39.8±4.1) MPa, and (32.2±3.4) MPa, respectively; and the measurements ofultimate bending strength were (65.0±3.4) MPa, (38.2±4.0) MPa, (33.1±4.3) MPa and (25.3±4.6) MPa, respectively. Conclusion The compositeof partially decalcified allogenic bone matrix gelatin and bone cement has a good biomechanical property and could be easily fabricated and re-shaped, which make it available to be used clinically as an idea bone graft biomaterial.
Because of its high biological compatibility, titanium has been a good biomaterial. The implanted artificial bone made from titanium can contact with the vital and mature osseous tissue directly within 3-6 months, the so-called osteointergration. In order to promote the process of osteointergration, FDBM of rabbit was prepared and was combined with pure titanium so as to speed up osteointergration. The study focused on bone density, bone intergration rate, new bone growth rate around the pure titanium, and the Ca2+ and PO(4)3- density of titanium-bone interface. A control group of pure titanium inplant without FDBM was set up. The results showed FDBM had no antigenicity. It could induce and speed up the new bone formation at titanium-bone interface. The titanium-bone intergration time was within 2 months. It was suggested that there were more bone morphogenesis protein (BMP) or other bone induction and bone formation factors in brephobone than that in child and adult bone. As a kind of bone induction material, FDBM was easy prepared, cheap in price, easy to storage, no antigenicity and obvious bone-inductive function.
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 investigate whether combining use of platelet-rich plasma (PRP) and decalcified bone matrix (DBM) has synergistic action on promoting bone consol idation and heal ing. Methods Forty male New Zealand rabbits (weighing 2.2-2.8 kg) were randomly divided into 4 groups (n=10). The whole blood was extracted from the central aural artery and PRP was prepared with the Landesberg’s method. An 1 cm-defect was made below the tibiofibular joint of the lefttibia through osteotomy. In group A, defect was repaired by distraction osteogenesis (1 cm); in group B, defect was repaired with 0.5 cm DBM and then by distraction osteogenesis (0.5 cm); in group C, defect was repaired by distraction osteogenesis (1 cm) and local injection of 1 mL PRP; in group D, defect was repaired by 0.5 cm DBM combined with 1 mL PRP and then by distraction osteogenesis (0.5 cm). Then lengthening started at 7 days after operation, at a rate of 1 mm/day and 0.5 mm every time for 10 days (groups A and C) or for 5 days (groups B and D). After the lengthening, the consolidation was performed. The X-ray films were taken at 0, 12, 17, 27, and 37 days after operation. At 37 days after operation, the tibial specimens were harvested for Micro-CT scanning, three-dimensional reconstruction and biomechanical test. Results The X-ray films showed that new bone formation in groups B and C was obviously better than that in groups A and D at 37 days. The bone mineral density (BMD), bone mineral content (BMC), and bone volume fraction (BVF) of groups B and C were significantly higher than those of groups A and D (P lt; 0.05); the BMD and BMC of group C were significantly higher than those of group B (P lt; 0.05); the BVF had no significant difference between groups B and C (P gt; 0.05). There was no significant difference in BMD, BMC, and BVF between groups A and D (P gt; 0.05). The trabecula number (Tb.N) of group C was significantly more than that of other groups (P lt; 0.05), and the trabecula spacing (Tb.Sp) of group C was significantly smaller than that of other groups (P lt; 0.05), but no significant differencewas found among other groups (P gt; 0.05). There was no significant difference in the trabecula thickness among 4 groups (P gt; 0.05). The ultimate angular displacement had no significant difference among 4 groups (P gt; 0.05). The maximum torque of groups B and C was significantly higher than that of groups A and D (P lt; 0.05); the maximum torque of group C was significantly higher than that of group B (P lt; 0.05); no significant difference was found between groups A and D (P gt; 0.05). Conclusion In the rabbit bone defect/lengthening model, local injection of PRP can enhance bone consol idation effectively during consol idation phase. In normal distraction rate, DBM can promote bone consol idation during distraction osteogenesis. In the early stage of distraction osteogenesis, combining use of DBM and PRP can not further promote bone consolidation and healing.
Objective To prepare a new plastic bone filler material with adhesive carrier and matrix particles derived from human bone, and evaluate its safety and osteoinductive ability through animal tests. MethodsThe human long bones donated voluntarily were prepared into decalcified bone matrix (DBM) by crushing, cleaning, and demineralization, and then the DBM was prepared into bone matrix gelatin (BMG) by warm bath method, and the BMG and DBM were mixed to prepare the experimental group’s plastic bone filler material; DBM was used as control group. Fifteen healthy male thymus-free nude mice aged 6-9 weeks were used to prepare intermuscular space between gluteus medius and gluteus maximus muscles, and all of them were implanted with experimental group materials. The animals were sacrificed at 1, 4, and 6 weeks after operation, and the ectopic osteogenic effect was evaluated by HE staining. Eight 9-month-old Japanese large-ear rabbits were selected to prepare 6-mm-diameter defects at the condyles of both hind legs, and the left and right sides were filled with the materials of the experimental group and the control group respectively. The animals were sacrificed at 12 and 26 weeks after operation, and the effect of bone defect repair were evaluated by Micro-CT and HE staining. Results In ectopic osteogenesis experiment, HE staining showed that a large number of chondrocytes could be observed at 1 week after operation, and obvious newly formed cartilage tissue could be observed at 4 and 6 weeks after operation. For the rabbit condyle bone filling experiment, HE staining showed that at 12 weeks after operation, part of the materials were absorbed, and new cartilage could be observed in both experimental and control groups; at 26 weeks after operation, the most of the materials were absorbed, and large amount of new bone could be observed in the 2 groups, while new bone unit structure could be observed in the experimental group. Micro-CT observation showed that the bone formation rate and area of the experimental group were better than those of the control group. The measurement of bone morphometric parameters showed that the parameters at 26 weeks after operation in both groups were significantly higher than those at 12 weeks after operation (P<0.05). At 12 weeks after operation, the bone mineral density and bone volume fraction in the experimental group were significantly higher than those in the control group (P<0.05), and there was no significant difference between the two groups in trabecular thickness (P>0.05). At 26 weeks after operation, the bone mineral density of the experimental group was significantly higher than that of the control group (P<0.05). There was no significant difference in bone volume fraction and trabecular thickness between the two groups (P>0.05). Conclusion The new plastic bone filler material is an excellent bone filler material with good biosafety and osteoinductive activity.
ObjectiveTo evaluate the physical and chemical properties, immunogenicity, and osteogenesis of two antigen-extracted xenogeneic bone scaffolds—decalcified bone matrix (DBM) and calcined bone.MethodsBy removing the inorganic and organic components of adult pig femus, xenogeneic DBM and calcined bone were prepared respectively. The density and pH value of the two materials were measured and calculated, the material morphology and pore diameter were observed by scanning electron microscope, and the surface contact angle was measured by automatic contact angle measuring instrument. The safety, osteogenic activity, and immunogenicity of the two materials were evaluated by cytotoxicity test, osteoblast proliferation test, DNA residue test, and human peripheral blood lymphocyte proliferation test. The two materials were implanted into the 5 mm full-thickness skull defect of 6-week-old male Sprague Dawley rats (the blank control group was not implanted with materials). The materials were taken at 4 and 8 weeks after operation, the repair effect of the materials on the rat skull was observed and evaluated by gross observation, Micro-CT scanning, and HE staining observation.ResultsCompared with calcined bone, DBM has lower density and poor hydrophilicity; the pH value of the two materials was 5.5-6.1, and the pore diameter was 160-800 μm. The two materials were non-cytotoxic and could promote the proliferation of osteoblasts. The absorbance (A) values of osteoblast proliferation at 1, 4, and 7 days in the DBM group were significantly higher than those in the calcined bone group (P<0.05). The DNA residues of the two materials were much lower than 50 ng/mg dry weight, and neither of them could stimulate the proliferation and differentiation of human peripheral blood lymphocytes. The results of animal experiments in vivo showed that the bone volume/total volume (BV/TV) in DBM group and calcined bone group were significantly higher than that in blank control group at 4 weeks after operation (P<0.05), and that in calcined bone group was significantly higher than that in DBM group (P<0.05); at 8 weeks after operation, there was no significant difference in BV/TV between groups (P>0.05). HE staining showed that at 4 and 8 weeks after operation, the defect in the blank control group was filled with fibrous connective tissue, the defect was obvious, and no bone growth was found; the defect in DBM group and calcined bone group had been repaired to varying degrees, and a large number of new bone formation could be seen. The material degradability of DBM group was better than that of calcined bone group.ConclusionThe physical and chemical properties and degradability of the two kinds of xenogeneic bone scaffolds were slightly different, both of them have no immunogenicity and can promote the repair and reconstruction of skull defects in rats.
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.