Objective To observe effects of the direct impaction onthe cell survival and the bone formation of the tissue engineered bone modified by the adenovirus mediated human bone morphogenetic protein 2 (Adv-hBMP2) gene and to verify the feasibility of the impacted grafting with it. Methods The marrow stromal cells (MSCs) were separated from the canine bone marrow and were cultured. MSCs were transfected with the Adv-hBMP2 gene and combined with the freeze-dried cancellous bone (FDB) to form the tissue engineered bone. Four days after the combination, the tissue engineered bone was impacted in a simulated impactor in vitro and implanted in the mouse. The cell survivals were evaluated with SEM 1 and 4 days after the combination, immediately after the impaction, and 1 and 4 days after the impaction, respectively. The bone formation and the allograft absorption were histologically evaluated respectively. Results There were multiple layers of the cells and much collagen on FDB before the impaction. Immediately after the impaction, most of the cells on the direct contact area disappearedand there was much debris on the section. Some of the cells died and separatedfrom the surface of FDB at 1 day, the number of the cells decreased but the collagen increased on the surface at 4 days. Histologically, only the fibrous tissue was found in FDB without the cells, the bone formation on FDB was even in distribution and mass in appearance before the impaction, but declined and was mainly on the periphery after the impaction in the AdvhBMP2 modified tissue-engineered bone. Conclusion The simulated impaction can decrease the cells survival and the bone formation of the AdvhBMP-2 modified tissue-engineered bone. The survival cells still function well.It is feasible to use the tissue engineered bone in the impaction graft.
Objective To explore the osteogenesis and angiogenesis effect of bone marrow mesenchymal stem cells (BMSCs) derived osteoblasts and endothelial cells compound with chitosan/hydroxyapatite (CS/HA) scaffold in repairing radialdefect in rats. Methods The BMSCs were isolated from Sprague Dawley rats and the 3rd generation of BMSCs were induced into osteoblasts and endothelial cells. The endothelial cells, osteoblasts, and mixed osteoblasts and endothelial cells (1 ∶ 1) were compound with CS/HA scaffold in groups A, B, and C respectively to prepare the cell-scaffold composites. The cell proliferation was detected by MTT. The rat radial segmental defect model was made and the 3 cell-scaffolds were implanted, respectively. At 4, 8, and 12 weeks after transplantation, the graft was harvested to perform HE staining and CD34 immunohistochemistry staining. The mRNA expressions of osteopontin (OPN) and osteoprotegerin (OPG) were detected by RT-PCR. Results Alkal ine phosphatase staining of osteoblasts showed that there were blue grains in cytoplasm at 7 days after osteogenic induction and the nuclei were stained red. CD34 immunocytochemical staining of the endothelial cells showed that there were brown grains in the cytoplasm at 14 days after angiogenesis induction. MTT test showed that the proliferation level of the cells in 3 groups increased with the time. HE staining showed that no obvious osteoid formation, denser microvessel, and more fibrous tissue were seen at 12 weeks in group A; homogeneous osteoid which distributed with cord or island, and many osteoblast-l ike cells were seen in groups B and C. The microvessel density was significantly higher in groups A and C than group B at 3 time points (P lt; 0.05), and in group A than in group C at 12 weeks (P lt; 0.05). The OPN and OPG mRNA expressions of group A were significantly lower than those of groups B and C at 3 time points (P lt; 0.05). In groups B and C, the OPN mRNA expressions reached peak t8 and 12 weeks, respectively, and OPG mRNA expressions reached peak at 4 weeks. Conclusion BMSCs derived steoblasts and endothelial cells (1 ∶ 1) compound with CS/HA porous scaffold can promote bone formation and vascularization in bone defect and accelerate the healing of bone defect.
OBJECTIVE: To explore a new method of preparing the composite of DL-polylactic acid (PDLLA), hydroxyapatite(HA), decalcium bone matrix (DBM), and to observe the degradation characteristics of PDLLA/HA/DBM in vitro. METHODS: An emulsion blend method was developed to prepare the composite of PDLLA/HA/DBM based on the weight rate of PDLLA:HA:DBM = 1.5-2:1-1.5:1. The characteristics of the particles was observed by scanning electron microscope. In vitro, PDLLA/HA/DBM and PDLLA were put into PBS(pH7.4) respectively; the pH value, weight and biomechanics of them were determined during the degradation. RESULTS: Without heating, the emulsion blend method could be developed to prepare PDLLA/HA/DBM. Scanning electron microscope showed that the gap diameter in the compound material was 100 to 400 microns, and the porosity was 71.3%; During degradation, the pH value of PDLLA decreased little within 2 weeks, then decreased obviously and decreased to 4.0 at the end of the 4th week; while the pH value of PDLLA/HA/DBM kept quite steady and was 6.4 at the end of the 12th week. The weight of PDLLA decreased little within 4 weeks, then decreased obviously and remained 50% of its prime weight at the end of the 12th week; while the weight of PDLLA/HA/DBM decreased little within 5 weeks, then decreased obviously and remained 60% of the prime at the end of the 12th week. The prime biomechanical strength was 1.33 MPa in PDLLA and 1.71 MPa in PDLLA/HA/DBM. There was significant difference between them (P lt; 0.05). The strength of PDLLA decreased little within 3 weeks, then decrease obviously and was 0.11 MPa at the end of the 12th week; the strength of PDLLA/HA/DBM decreased little within 4 weeks, then decrease obviously and was 0.21 MPa at the end of the 12th week. CONCLUSION: The emulsion blend method is a new method to prepare bone repair materials. As a new bone repair material, PDLLA/HA/DBM is suitable for bone tissue engineering for its good characteristics of porosity and degeneration.
ObjectiveTo investigate the bone regeneration potential of cell-tissue engineered bone constructed by human bone marrow mesenchymal stem cells (hBMSCs) expressing the transduced human bone morphogenetic protein 2 (hBMP-2) gene stably.
MethodsThe full-length hBMP-2 gene was cloned from human muscle tissues by RT-PCR and connected into a vector to consturct a eukaryotic expression system. And then the gene expression system was transduced to hBMSCs with lipidosome. hBMSCs were transfected by hBMP-2 gene (experimental group) and by empty plasmid (negative control group), untransfected hBMP-2 served as blank control group. RT-PCR, dot-ELISA, immunohistochemical analysis and ALP activity were performed to compare and evaluate the situation of hBMP-2 expression and secretion after transfection. hBMSCs transfected by hBMP-2 gene were seeded on hydroxyapatite (HA) and incubated for 4 days to construct the hBMP-2 gene modified tissue engineered bone, and then the tissue engineered bone was observed by the inverted phase contrast microscope and scanning electron microscope. Then the hBMP-2 gene modified tissue engineered bone (group A, n=3), empty plasmid transfected hBMSCs seeded on HA (group B, n=3), hBMSCs suspension transfected by hBMP-2 gene (group C, n=3), and hBMP-2 plasmids and lipidosome (group D, n=3) were implanted into bilateral back muscles of nude mice. The osteogenic activity was detected by HE staining and alcian blue staining after 4 weeks.
ResultsAt 48 hours and 3 weeks after transfection, RT-PCR and dot-ELISA results indicated that the transfected hBMSCs could express and secrete active and exogenous hBMP-2 stably. The immunohistochemical staining was positive, and the ALP activity in the transfected hBMSCs was significantly higher than that in two control groups (P < 0.05). The transfected hBMSCs had a good attaching and growing on the three-demension suface of HA under inverted phase contrast microscope and scanning electron microscope. In vivo study indicated that a lot of new bone formation was obviously found at 4 out of 6 sides of back muscles in group A. Some new bone formation at both sides of back muscles was observed in 1 of 3 mice in group B. No new bone formation was found in group C. A few new bone formation was observed at one side of back muscles in group D.
ConclusionThe tissue engineered bone constructed by hBMP-2 gene modified hBMSCs and HA is able to express and secrete active hBMP2 stably and can promote new bone formation effectively in muscles of nude mice.
Objective To investigate the feasibility of repairing goat tibia defect with marrow stromal cells (MSCs).Methods MSCs were cocultured with the bio-derived bone in vitro, and the 20 mm tibia defectswere made and fixed with plate in 35 goats, and they were divided into the experimental group, control group and blank group. The defects on the right side were filled with tissue engineering bone as the experimental group, the defects onthe left side with bio-derived bone as the control group in 33 goats, and the defect on the both sides were not filled with any materials as the blank group in 2 goats. Threpair capability was assessed physically, histopathologically and biomechanically at 2, 4, 6, 8, 12,16 and 24 weeks after operation in 3 groups.Results By physical, histopathological and biomechanical examinations, the bio-derived bone was partially absorbed in the experimental group and was rarely absorbed in the control group in the 4th week; the defects were partially repaired in the experimental group, and in the control group, few new bones were observed in the two ends of the implants, in which there was fibrous tissue. The effects of biomechanics had no statistically significant difference between the experimental group and the control group(P＞0.05) in the 8th week; the defects were perfectly repaired in the experimental group and the effects of biomechanics had statistically significant difference between two groups (P＜0.05) in the 12th weeks. The defects were not repaired in the 24th week in the blank group.Conclusion The tissue engineering bone can efficiently repair bone defect, and itsrepair capability is better than that of bio-derived bone alone both in quantity and in quality of bone formation.
Objective To study the potential of a bioderived material combined with Pluronic F-127 in vitro as a delivery vehicle for WO-1 in the bone repair therapy. Methods Bio-derived materials were fabricated and loaded with WO-1 by Pluronic F-127. Micromorphology and porosity were detected by the scanning electron microscope and the digital image analysis system respectively. The WO-1 release from the system in vitro was studied by the high performance liquid chromatography. Results Bio-derived material-WO-1 drug delivery systems were created with the interconnected pore network. Theporosity and pore size of the system were 55% and 522.43±16.75 μm respectively, compared with those of bio-derived materials, which were 75% and 623.67±12.31 μm respectively. And the main composition of the system was HA. The in vitrorelease kinetics of WO-1 revealedthat an effective therapeutic concentration(0.2-0.8 μg/ml) of WO-1 was maintained for 6 days after a high initial burst release. Conclusion The bio-derived material-WO-1 drug delivery system can be used in the bone repair therapy. However, the in vivostudy on it is still needed.
Objective To provide the chosen scaffold materials for experiment and application of tissue engineering and to detect the properties of the collagenbio-derived bone scaffold material loading WO-1. Methods The purebio-derived bone scaffold material, bio-derived bone scaffold material loading collagen, collagen bio-derived bone scaffold material loading WO-1 were made by use of allograftbone, and typeI collagen, and WO-1. The morphological features, constitute components and mechanical properties were examined by scanning electron microscopy,X- rays diffraction and mechanical assay. Results The bio-derived bone scaffold material maintained natural network pore system; the bio-derived bone scaffold material loading collagen maintained natural network pore system, the surface of network pore system was coated by collagen membrane; the collagen bio-derived bone scaffold material loading WO-1 maintained natural network pore system, thesurface of network pore system was coated by collagen membrane. The pore sizes of the 3materials were 90-700 μm, 75-600 μm and 80-600 μm, respectively, and the porosities were 87.96%, 80.47%, 84.2%. There was no significant difference between them(P＞0.05).The collagen bio-derived bone scaffold material loading WO-1 consisted of ［HA,Ca10(OH)2(PO4)6］. There was no significant difference in the mechanical strength of the three scaffold materials. Conclusion The bio-derived bone scaffold material loading WO-1 is as good as bio-derived bone scaffold material and collagen bio-derived bone scaffold material, and it is an effective scaffold material for tissue engineering bone.
Objective To study the vascularization of the compositeof bio-derived bone and marrow stromal stem cells(MSCs) in repairing goat tibial shaft defect.Methods Bio-derived bone was processed as scaffold material. MSCs were harvested and cultured in vitro. The multiplied and induced cells were seeded onto the scaffold to construct tissue engineered bone. A 20 mm segmental bone defect inlength was made in the middle of the tibia shaft in 20 mature goats and fixed with plate. The right tibia defect was repaired by tissue engineered bone (experimental side), and the left one was repaired by scaffold material (control side).The vascularization and osteogenesis of the implants were evaluated by transparent thick slide, image analysis of the vessels, and histology with Chinese ink perfusion 2, 4, 6, and 8 weeks after operation.Results More new vessels were found in control side than in experimental side 2 and 4 weeks after implantation (Plt;0.05). After 8 weeks, there was no significant difference in number of vessels between two sides(Pgt;0.05), and the implants were vascularized completely. New bone tissue was formed gradually as the time and the scaffold material degraded quickly after 6 and 8 weeks in the experimental side. However, no new bone tissue was formed andthe scaffold degraded slowly in control side 8 weeks after operation.Conclusion Bio-derived bone has good quality of vascularization. The ability of tissue-engineered bone to repair bone defect is better than that of bio-derived bone alone.
Objective To observe the tissue engineered bonefabricated with the cultured mesenchymal stem cells (MSCs) by the green fluorescent protein (GFP) gene transfer. Methods The recombinant Adeno-XTM-GFP expression vector was purified after being packed and proliferated by the HEK293 cells, and then it was used to infect the rabbit’s MSCs directly afer the virus titer was assayed. The cell morphological changes were observed under the inverted phase contrast microscope, and the expression of GFP was observed under the fluorescence microscope to confirm success of the labeling of MSCs.The GPFlabeled MSCs and the pure MSCs were cultured together in the conventional osteogenic supplements for 3 weeks, and then they were seeded onto the compound scaffold of the calcium phosphate cement (CPC) and the fibrin glue (FG) to form a new kind of the tissue engineered bone. It was implanted into the donator rabbit subcutaneously to be used as the experimental group; in contrast, the pure compound scaffold of the CPC-FG without any MSCs was implanted in the same rabbit as a control. The alkline phosphatase (ALP) activity assay was performed respectively at 1, 2 and 3 weeks after operation. GFP was observed under the laserconfocal microscope at 4 weeks after operation, and the formed new bone was harvested at 4 weeks and evaluated by the Masson staining, the immunohistochemistry staining of osteocalcin (OC) and collagen typeⅠ.Results The virus titer was 3×108pfu/ml after proliferation and purification. Expresstionof GFP was confirmed 96 h after MSCs were infected by the Adeno-XTM-GFP expression vector and the infection rate was proximally 50%-70%. In contrast to MSCs, division and proliferation of the GPF-labeled MSCs were not significantly different. The ALP activity in the experimental group (12.546±1.091, 16.567±0.659, 20.443±0.706) was significantly higher than that in the control group (0.453±0.113, 0.243±0.018, 0.308±0.056), respectively at 1, 2 and 3 weeks after operation (Plt;0.05). The tissue engineered bone formed at 4 weeks. There were newly-formed trabeculae around the pore of the compound scaffold, and theimmunohistochemistry staining of OC and collagen typeⅠ were positive. The laser confocal microscope revealed that the GFP-labeled cells existed in many newlyformed tissues，and the compound scaffold of CPC-FG was partly degraded. Conclusion The engineered bone is similar to the spongy bone and the composed cells originate from the cultured MSCs, all of which can be confirmed by the GFP gene transfer technique.
Objective To investigate the effect of repairing bone defect with tissue engineered bone seeded with the autologous red bone marrow (ARBM) and wrapped by the pedicled fascial flap and provide experimental foundation for cl inicalappl ication. Methods Thirty-two New Zealand white rabbits (male and/or female) aged 4-5 months old and weighing2.0-2.5 kg were used to make the experimental model of bilateral 2 cm defect of the long bone and the periosteum in the radius. The tissue engineered bone was prepared by seeding the ARBM obtained from the rabbits on the osteoinductive absorbing material containing BMP. The left side of the experimental model underwent the implantation of autologous tissue engineered bone serving as the control group (group A). While the right side was designed as the experimental group (group B), one 5 cm × 3 cm fascial flap pedicled on the nameless blood vessel along with its capillary network adjacent to the bone defect was prepared using microsurgical technology, and the autologous tissue engineered bone wrapped by the fascial flap was used to fill the bone defect. At 4, 8, 12, and 16 weeks after operation, X-ray exam, absorbance (A) value test, gross morphology and histology observation, morphology quantitative analysis of bone in the reparative area, vascular image analysis on the boundary area were conducted. Results X-ray films, gross morphology observation, and histology observation: group B was superior to group A in terms of the growth of blood vessel into the implant, the quantity and the speed of the bone trabecula and the cartilage tissue formation, the development of mature bone structure, the remolding of shaft structure, the reopen of marrow cavity, and the absorbance and degradation of the implant. A value: there was significant difference between two groups 8, 12, and 16 weeks after operation (P lt; 0.05), and there were significant differences among those three time points in groups A and B (P lt; 0.05). For the ratio of neonatal trabecula area to the total reparative area, there were significant differences between two groups 4, 8, 12, and 16 weeks after operation (P lt; 0.05), and there were significant differences among those four time points in group B (P lt; 0.05).For the vascular regenerative area in per unit area of the junctional zone, group B was superior to group A 4, 8, 12, and 16 weeks after operation (P lt; 0.05). Conclusion Tissue engineered bone, seeded with the ARBM and wrapped by the pedicled fascial flap, has a sound reparative effect on bone defect due to its dual role of constructing vascularization and inducing membrane guided tissue regeneration.
