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 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 evaluate the adhesion, prol iferation and osteogenic differentiation of rabbit BMSCs after cultured on freeze-dried demineral ized bone matrix (FDBM) modified with type II cadherin ectodomain (Cad- II). Methods BMSCs isolated from 10 Japanese white rabbits (male and female, 4-week-old, 0.61-0.88 kg) were cultured. The second generation of BMSCs (cell density 1 × 106 /mL) were seeded onto the Cad-II modified allogenic FDBM (experimental group) and only FDBM (control group) respectively, and then cocultured in vitro. The densities of seeded cells, the adhesion rate and their ALP activity were measured. The complex was observed through inverted phase contrast microscope and scanning electron microscope to evaluate the interaction between cells and FDBM. Another group of second generation of BMSCs (cell density 5 × 105 /mL) were seeded onto the Cad-II modified FDBM (experimental group) and only FDBM (control group) respectively, and then cocultured in vitro too. The ALP activity and osteocalcin immunohistochemical was measured. Results There was no significant difference in cell prol iferation between experimental group and control group. The adhesion rate of cells in the experimental group was 87.41% ± 5.19%, higher than that in the the control group 35.56% ± 1.75% (P lt; 0.01); the densities of seeded cells reached 5.0 × 105, showing significant difference compared with the control group (2.6 × 104, P lt; 0.05). Inverted phase contrast microscope showed that in the experimental group, more cultured BMSCs pasted in the hole and edge of the scaffold than that in the control group. HE staining showed the densities of seeded cells in the experimental group was higher than that in the control group. Scanning electron microscope showed that in the experimental group, a lot of cultured BMSCs adhered, spreaded in the scaffold, in the control group only a few BMSCs unevenly distributed in the scaffold. After 7 days of culture, the cultured BMSCs on modified FDBM expressed higher ALP activity; after 14 days of culture, the ALP activity (29.33 ± 1.53) was higher than that cultured on unmodified FDBM (18.31 ± 1.32), the positive rates of osteocucl in were 83% ± 7% in the experimental group and 56% ± 7% in the control group, showing significant difference (P lt; 0.01). Conclusion Cad-II enhanced cell adhesion to FDBM and promoted BMSCs differentiate to osteoblast, but no obvious effects were observed in cell prol iferation.
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 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.
Objective Tissue engineered bone (TEB) lacks of an effective and feasible method of storage and transportation. To evaluate the activity of osteogenesis and capabil ity of ectopic osteogenesis for TEB after freeze-dried treatment in vitro and in vivo and to explore a new method of preserving and transporting TEB. Methods Human bone marrow mesenchymal stem cells (hBMSCs) and decalcified bone matrix (DBM) were harvested from bone marrow and bone tissue of the healthy donators. TEB was fabricated with the 3rd passage hBMSCs and DBM, and they were frozen and dried at extremely low temperatures after 3, 5, 7, 9, 12, and 15 days of culture in vitro to obtain freeze-dried tissue engineered bone (FTEB). TEB and FTEB were observed by gross view and scanning electron microscope (SEM). Western blot was used to detect the changes of relative osteogenic cytokines, including bone morphogenetic protein 2 (BMP-2), transforming growth factor β1 (TGF-β1), and insul in-l ike growth factor 1 (IGF-1) between TEB and FTEB. The ectopic osteogenesis was evaluated by the methods of X-ray, CT score, and HE staining after TEB and FTEB were transplanted into hypodermatic space in athymic mouse. Results SEM showed that the cells had normal shape in TEB, and secretion of extracellular matrix increased with culture time; in FTEB, seeding cells were killed by the freeze-dried process, and considerable extracellular matrix were formed in the pore of DBM scaffold. The osteogenic cytokines (BMP-2, TGF-β1, and IGF-1) in TEB were not decreased after freeze-dried procedure, showing no significant difference between TEB and FTEB (P gt; 0.05) except TGF-β1 15 days after culture (P lt; 0.05). The ectopic osteogenesis was observed in TEB and FTEB groups 8 and 12 weeks after transplantation, there was no significant difference in the calcified level of grafts between TEB and FTEB groups by the analysis of X-ray and CT score. On the contrary, there was no ectopic osteogenesis in group DBM 12 weeks after operation. HE staining showed that DBM scaffold degraded and disappeared 12 weeks after operation. Conclusion The osteogenic activity of TEB and FTEB is similar, which provides a new strategy to preserve and transport TEB.
Objective To evaluate the biocompatibil ity of manufactured heterogeneous demineral ized bone matrix(DBM) particles and to provide basis for further experimental study and cl inical application. Methods Heterogeneous DBMparticles A (degreased and demineralized) and B (degreased, demineralized and acellular), particle size from 250 to 810 μm, and leaching l iquor were made with a series of physical and chemical methods from pig l imbs cortical bone. The residual calcium and phosphorus contents of bone particles were measured after degreased and demineral ized. The acute toxicity test, skin stimulating test, pyrogeneous test, hemolysis test, cellular toxicity test and muscular embedded test were carried out according standard toxicological method. Results The contents of calcium and phosphorus in cortical bone were (189.09 ± 3.12) mg/g and (124.73 ± 2.87) mg/g, and in demineral ized bone matrix particles were (3.48 ± 0.09) mg/g and (3.46 ± 0.07) mg/ g. The residual calcium content was 1.87%, of phosphorus was 2.69%. The activity of mice was normal in the acute toxicity test. No animal died and no toxicity symptom or adverse effects were shown within 7 days. The mean weight daily increased showed no statistically significant difference (P gt; 0.05) between two groups after 7 days. Skin stimulating reactions were not found in the two experimental groups and negative control group by intradermal stimulation test. The maximal increase of body temperature in two experimental groups were 0.4℃ , which meet the national standard (lt; 0.6 ). The rate of haemolysis to the leaching liquor was 1.14% (A) and 0.93% (B), which was lower than the national standard (lt; 5%). The cell prol iferation rates of two experimental groups when compared with control group showed no statistically significant difference (P gt; 0.05). The toxicity of DBM particlesleaching liquor was graded from 0 to 1, which means the material has no cytotoxicity. All the animals survived well. There was no tissue necrosis, effusion or inflammation at all implantation sites. For the index of HE and Masson staining, there were no effusion around the material and inflammatory cell infiltrate obviously in two experimental groups. Inflammatory cell infiltrate is sl ight in control group 2 weeks postoperatively. The inflammatory cell infiltration was mitigate gradually over time in two experimental groups after 4, 8 and 12 weeks. New bone and collagen fibers formation were observed when the material was degraded and absorpted. Score evaluation of local cellular immune response at different time after operation of two experimental groups showed no statistically significant difference (P gt; 0.05). Conclusion Heterogeneous DBM has no obvious toxicity, skin irritation, pyrogenicity, and no cytotoxicity with a rate of haemolysis lt; 5%, so it has good biocompatibility and partial osteoinductive.
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.
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.
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.