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 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.
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 explore the method of fabricating freeze-dried demineralized bone matrix with nanoscale topography (nFDBM) and to investigate the feasibility of reconstruction of tissueengineered bone with the novel scaffold. Methods Allogenic dogs’ phalangeal cortical bone was fabricatedinto freeze-dried demineralized bone (FDBM) with modified Urist’s method. FDBM was subjected toNd∶YAG laser irradiation under special conditions. The surface topography was identified by atomic force microscope(AFM) and scanning electron microscope (SEM). The osteoblasts were induced from autologous mesenchymal stem cells (MSCs) and mixed with nFDBM and FDBM in vitro.The effects of the different topography oncellbehavior was identified by SEM. The complex of nFDBM and osteoblasts wereimplanted into fascial bags on dogs’ back (experimental group A) and dogs’ phalangeal defects on right (experimental group C), while FDBMosteoblast complex (control group B) and unique FDBM (control group D) were implanted into the corresponding sites on left as control groups. The osteogenic status was assessed by X-ray, HE and SEM at 4, 8 and 12 weeks after surgery. Results The surface of FDBM subjected to Nd∶YAG laser irradiation resulted in well-defined three-dimensional nanoscale grooves (150 nm in depth and 600 to 800 nm in width). When the osteoblasts were implanted on the scaffold, the cells adhering to nFDBM were morethan those to FDBM and secreted more extracellular matrix. Either new bone-likethin layer on the nanoscale surface or a lot of new boneformation inner the experimental complex was observed by HE after 12 weeks of surgery and the experimental complexes were partially calcified at the same time, while the control groups almost had no osteogenic phenomena. Conclusion Nd∶YAG laser could produce nanoscale grooves on the FDBM surface. The nanoscale grooves are conductive to adherence, proliferation and matrix secretion of osteoblasts. Complexes by tissue engineering and nanoscale technology have some osteogenic abilities in vivoafter implanted the animal model.
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 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 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.
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.
ObjectiveBased on the cell-extracellular matrix adhesion theory in selective cell retention (SCR) technology, demineralized bone matrix (DBM) modified by simplified polypeptide surface was designed to promote both bone regeneration and angiogenesis.MethodsFunctional peptide of α4 chains of laminin protein (LNα4), cyclic RGDfK (cRGD), and collagen-binding domain (CBD) peptides were selected. CBD-LNα4-cRGD peptide was synthesized in solid phase and modified on DBM to construct DBM/CBD-LNα4-cRGD scaffold (DBM/LN). Firstly, scanning electron microscope and laser scanning confocal microscope were used to examine the characteristics and stability of the modified scaffold. Then, the adhesion, proliferation, and tube formation properties of CBD-LNα4-cRGD peptide on endothelial progenitor cells (EPCs) were detected, respectively. Western blot method was used to verify the molecular mechanism affecting EPCs. Finally, 24 10-week-old male C57 mice were used to establish a 2-mm-length defect of femoral bone model. DBM/LN and DBM scaffolds after SCR treatment were used to repair bone defects in DBM/LN group (n=12) and DBM group (n=12), respectively. At 8 weeks after operation, the angiogenesis and bone regeneration ability of DBM/LN scaffolds were evaluated by X-ray film, Micro-CT, angiography, histology, and immunofluorescence staining [CD31, endomucin (Emcn), Ki67].ResultsMaterial related tests showed that the surface of DBM/LN scaffold was rougher than DBM scaffold, but the pore diameter did not change significantly (t=0.218, P=0.835). After SCR treatment, DBM/LN scaffold was still stable and effective. Compared with DBM scaffold, DBM/LN scaffold could adhere to more EPCs after the surface modification of CBD-LNα4-cRGD (P<0.05), and the proliferation rate and tube formation ability increased. Western blot analysis showed that the relative expressions of VEGF, phosphorylated FAK (p-FAK), and phosphorylated ERK1/2 (p-ERK1/2) proteins were higher in DBM/LN than in DBM (P<0.05). In the femoral bone defect model of mice, it was found that mice implanted with DBM/LN scaffold had stronger angiogenesis and bone regeneration capacity (P<0.05), and the number of CD31hiEmcnhi cells increased significantly (P<0.05).ConclusionDBM/LN scaffold can promote the adhesion of EPCs. Importantly, it can significantly promote the generation of H-type vessels and realize the effective coupling between angiogenesis and bone regeneration in bone defect repair.
OBJECTIVE To investigate the feasibility of freeze-dried demineralized bone matrix (FDBM) as scaffold material in bone tissue engineering. METHODS Osteoblasts which were isolated from cranial periosteum of New Zealand rabbits were cultured as the seeding cells, then the cells were cocultured with heterogenous FDBM in vitro. The cell-material complex was observed under phase microscope, light microscope and electronic scanning microscope in order to evaluate the interaction between cells and FDBM. RESULTS Eight hours after coculture, the osteoblasts adhered to FDBM scaffolds. Seven days later, the osteoblasts differentiated and proliferated in FDBM network. Extracellular matrix was secreted and calcium nodes were formed among osteoblasts. CONCLUSION FDBM is a good scaffold material for the bone tissue engineering.