Objective To investigate the effects of flow shear stress and mass transport on the construction of largescale tissue engineered bone using a perfusion bioreactor. Methods Bone marrow (20 mL) was harvested from the il iac crestof the healthy volunteer, and then hBMSCs were isolated, cultured and identified. The hBMSCs at passage 3 were seeded on the critical-size β-TCP scaffold and cultured in a perfusion bioreactor for 28 days. Different flow shear stress (1 ×, 2 × and 3 ×) and different mass transport (3, 6 and 9 mL/min) were exerted on the cells seeded on the scaffold by changing the viscosity of media or perfusion flow rate. The cell prol iferation and ALP activity of cells seeded on the scaffold were detected, and histology observation and morphology measurement of cell/scaffold complex were conducted. Results When the perfusion flow rabe was 3 mL/min, the cell viabil ity of 2 × group was higher than that of other groups (P lt; 0.05). When the flow shear stress was 3 ×, no significant differences were found among 3, 6 and 9 mL/min in cell viabil ity (P gt; 0.05). When the perfusion flow rate was 3 mL/min, the activity of ALP of 2 × and 3 × groups was higher than that of 1 × group (P lt; 0.05). When the flow shear stress was 3 ×, the activity of ALP of 6 mL/min group was the highest (P lt; 0.05). After 28 days of perfusion culture, the ECM of all the groups distributed throughout the scaffold, and the formation and mineral ization of ECM was improved with the increase of flow shear stress when the perfusion flow rate was 3 mL/min. However, the increase of perfusion flow rate decreased the mineral ization of ECM when the flow shear stress was 3 ×. Conclusion As two important fluid dynamics parameters affecting the construction of large-scale tissue engineered bone, the flow shear stress and the mass transport should be measured duringthe process of constructing large-scale tissue engineered bone so as to maximize their roles.
Objective To evaluate the feasibility of poly-L-lactide(PLLA)/porcinederived xenogeneic bone(PDXB) composite as a scaffold for the bone tissue engineering. Methods The film and the scaffold of the PLLA-PDXB composite were respectively prepared by a solution casting method and a solution casting-particle leaching method. The composite film and scaffold were further treated by the surface alkaline hydrolysis. The surface morphology of the composite was observed by the scanning electron microscopy, and hydrophilicity degree of the composite was measured. The OCT-1 osteoblastlike cells were cultured and amplified in vitro as the seeding cells, which werethen implanted on the film and scaffold. The adherence rate, adherence shape,proliferating activity, and growing morphology of the OCT-1 osteoblastlikecells were observed on the film. Results The PDXB particle 50 μm in diameter on average had a similar phase structure to that of hydroxyapatite. But its Ca/P ratio was lower than that of hydroxyapatite. After the surface alkaline hydrolysis, the PDXB particle could be exposed on the surface of the PLLA-PDXB composite. The surface roughness and hydrophilicity of the PLLAPDXB composite were obviously enhanced. The cell adherence rate and the cell proliferation activity of the PLLAPDXB composite were higher than those of the pure PLLA material. The cells tended to grow on the exposed surface of the PDXB particles. The cells seeded on the composite scaffold could migrate to the inside of the composite scaffold and grew well. Conclusion The PLLA-PDXB composite has a good cell affinity, and this kind of composite can hopefullybecome a new scaffold material to be used in the bone tissue engineering.
Objective To construct the recombinant adeno-associated virus vector with human bone morphogenetic protein 4 gene（AAV-hBMP4）. Methods The hBMP-4 gene primer was designed basing on the corresponding gene sequence in GenBank. EcoR I site was introduced into the upstream of the primer and Sal Ⅰ site into downstream. The hBMP-4 gene was amplifiedwith the template of EX-A0242-M01-hBMP-4, then was cloned into pUC18 vectorto construct recombinant plasmid pUC18-hBMP-4. The plasmids pUC18-hBMP-4 and plasmid pSNAV cut by EcoR Ⅰ and Sal Ⅰenzyme, the fragments were collected and linked with T4 DNA ligase at 16℃ over night, recombinant plasmid pSNAVhBMP-4 was obtained. The recombinant plasmid was then transfected into BHK21 cells using Lipofectamine TM2000. The G418 resistant cells were obtained consequently. Thesecells were infected with HSV1-rc/△UL2 which has the function of packaging andcopying the recombinant AAV. After purification, the construction of recombinant AAV-hBMP-4 was completed. Results The construction of the recombinant pSNAV-hBMP-4 was confirmed by PCR electrophoresis and digestion with restriction enzyme. The gene sequence in the recombinant pSNAV-hBMP-4 wascorrect. The virus titer was about 1.5×1012 μg/ml.The purity of the virus was more than 95% using the SDSPAGE method. Conclusion With this method, high virus titers and purity of AAV-hBMP-4 can be acquired successfully and it is useful to bone tissue engineering.
Objective To review the research progress of osteoblastextracellular matrix(ECM) and its application in bone tissue engineering. Methods The recentrelated literatures were extensively reviewed. Results The ECM was complex in its components. The configuration of cell and cell’s adhesion, migration, proliferation, and differentiation were subject to the ECM. The bioactivity of the tissue engineering products was revealed by ECM, which predicted the product’s efficiency in clinic application. Conclusion ECM has the potential to become the effective index in evaluating tissue engineered products.
Objective To study the feasibil ity of preparation of the poly-D, L-lactide acid (PDLLA) scaffolds treated by ammonia plasma and subsequent conjugation of Gly-Arg-Gly-Asp-Ser (GRGDS) peptides via amide l inkage formation. Methods PDLLA scaffolds (8 mm diameter, 1 mm thickness) were prepared by solvent casting/particulate leaching procedure and then treated by ammonia plasma. The consequent scaffolds were labeled as aminated PDLLA (A/ PDLLA). The pore size, porosity, and surface water contact angle of groups 0 (un-treated control), 5, 10, and 20 minutes A/ PDLLA were measured. A/PDLLA scaffolds in groups above were immersed into the FITC labelled GRGDS aqueous solutionwhich contain 1-[3-(dimethylamino) propyl]-3-ethylcarbodiimide hydrochloride (EDC.HCl) and N-hydroxysuccinimide(NHS), the molar ratio of peptides/EDC.HCL /NHS was 1.5 ∶ 1.5 ∶ 1.0, then brachytely sloshed for 24 hours in roomtemperature. The consequent scaffolds were labelled as peptides conjugated A/PDLLA (PA/PDLLA). The scaffolds in groups 0, 5, 10, and 20 minutes A/PDLLA and groups correspondingly conjugation of peptides were detected using X-ray photoelectron spectroscopy (XPS). The scaffolds in groups of conjugation of peptides were measured by confocal laser scanning microscope and high performance l iquid chromatography (HPLC), un-treated and un-conjugated scaffolds employed as control. Bone marrow mesenchymal stem cells (BMSCs) from SD rats were isolated and cultured by whole bone marrow adherent culture method. BMSCs at the 3rd–6th passages were seeded to the scaffolds as follows: 20 minutes ammonia plasma treatment (group A/PDLLA), 20 minutes ammonia plasma treatment and conjugation of GRGDS (group PA/PDLLA), and untreated PDLLA control (group PDLLA). After 16 hours of culture, the adhesive cells on scaffolds and the adhesive rate were calculated. After 4 and 8 days of culture, the BMSCs/scaffold composites was observed by scanning electron micorscope (SEM). Results No significant difference in pore size and porosity of PDLLA were observed between before and after ammonia plasma treatments (P gt; 0.05). With increased time of ammonia plasma treatment, the water contact angle of A/PDLLA scaffolds surface was decreased, and the hydrophil icity in the treated scaffolds was improved gradually, showing significant differences when these groups were compared with each other (P lt; 0.001). XPS results indicated that element nitrogen appeared on the surface of PDLLA treated by ammonia plasma. With time passing, the peak N1s became more visible, and the ratio of N/C increased more obviously. AfterPDLLA scaffolds treated for 0, 5, 10, and 20 minutes with ammonia plasma and subsequent conjugation of peptides, the ratio of N/C increased and the peak of S2p appeared on the surface. The confocal laser scanning microscope observation showed that the fluorescence intensity of PA/PDLLA scaffolds increased obviously with treatment time. The amount of peptides conjugated for 10 minutes and 20 minutes PA/PDLLA was detected by HPLC successfully, showing significant differences between 10 minutes and 20 minutes groups (P lt; 0.001). However, the amount of peptides conjugated in un-treated control and 0, 5 minutes PA/PDLLA scaffolds was too small to detect. After 16 hours co-culture of BMSCs/scaffolds, the adhesive cells and the adhesive rates of A/PDLLA and PA/PDLLA scaffolds were higher than those of PDLLA scaffolds, showing significant difference between every 2 groups (P lt; 0.01). Also, SEM observation confirmed that BMSCs proliferation in A/PDLLA and PA/PDLLA groups was more detectable than that in PDLLA group, especially in PA/PDLLA group. Conclusion Ammonia plasma treatment will significantly increase the amount of FITC-GRGDS peptides conjugated to surface of PDLLA via amide l inkage formation. This new type of biomimetic bone has stablized bioactivities and has proved to promote the adhesion and proliferation of BMSCs in PDLLA.
Objective To construct polyhydroxyalkanoate (PHA) microspheres loaded with bone morphogenetic protein 2 (BMP-2) and human β-defensin 3 (HBD3), and evaluate the antibacterial activity of microspheres and the effect of promoting osteogenic differentiation, aiming to provide a new option of material for bone tissue engineering. Methods The soybean lecithin (SL)-BMP-2 and SL-HBD3 were prepared by SL-mediated introduction of growth factors into polyesters technology, and the functional microsphere (f-PMS) containing BMP-2 and HBD3 were prepared by microfluidic technology, while pure microsphere (p-PMS) was prepared by the same method as the control. The morphology of microspheres was observed by scanning electron microscopy and the water absorption was detected; the release curves of BMP-2 and HBD3 in f-PMS were detected by ELISA kit. The antibacterial effect of microspheres in Staphylococcus aureus and Escherichia coli was tested with the LIVE/DEADTM BacLightTM bacterial staining kit; the biocompatibility of microspheres was tested using Transwell and cell counting kit 8 (CCK-8). The effect of microspheres on osteogenic differentiation was determined by collagen type Ⅰ (COL-1) immunofluorescence staining and alkaline phosphatase (ALP) concentration. Results In this experiment, the f-PMS and p-PMS were successfully constructed. Morphological characteristics showed that p-PMS surface was rough and distributed with micropores of 1-3 μm, while f-PMS surface was smooth and existed white granular material. There was no significant difference in water absorption between the two groups (P>0.05). The release curves of BMP-2 and HBD3 in the f-PMS and p-PMS were basically the same, showing both early sudden release and late slow release. The antibacterial activity of f-PMS was significantly higher than that of p-PMS in the test that against Staphylococcus aureus and Escherichia coli (P<0.05), but there was no significant difference in biocompatibility between the two groups (P>0.05). The results of osteogenic differentiation of human BMSCs showed that the fluorescence intensity of osteogenic specific protein COL-1 of f-PMS was significantly higher than that in p-PMS, and the activity of ALP in f-PMS was also significantly higher than that in p-PMS (P<0.05). Conclusion The p-PHA have good antibacterial activity and biocompatibility, and can effectively promote the osteogenic differentiation of human BMSCs, which is expected to be applied to bone tissue engineering in the future.
ObjectiveTo evaluate the effect of tissue engineered periosteum on the repair of large diaphysis defect in rabbit radius, and the effect of deproteinized bone (DPB) as supporting scaffolds of tissue engineering periosteum. MethodsBone marrow mesenchymal stem cells (BMSCs) were cultured from 1-month-old New Zealand Rabbit and osteogenetically induced into osteoblasts. Porcine small intestinal submucosa (SIS) scaffold was produced by decellular and a series mechanical and physiochemical procedures. Then tissue engineered periosteum was constructed by combining osteogenic BMSCs and SIS, and then the adhesion of cells to scaffolds was observed by scanning electron microscope (SEM). Fresh allogeneic bone was drilled and deproteinized as DPB scaffold. Tissue engineered periosteum/DPB complex was constructed by tissue engineered periosteum and DPB. Tissue engineered periosteum was "coat-like" package the DPB, and bundled with absorbable sutures. Forty-eight New Zealand white rabbits (4-month-old) were randomly divided into 4 groups (groups A, B, C, and D, n=12). The bone defect model of 3.5 cm in length in the left radius was created. Defect was repaired with tissue engineered periosteum in group A, with DPB in group B, with tissue engineered periosteum/DPB in group C; defect was untreated in group D. At 4, 8, and 12 weeks after operation, 4 rabbits in each group were observed by X-ray. At 8 weeks after operation, 4 rabbits of each group were randomly sacrificed for histological examination. ResultsSEM observation showed that abundant seeding cells adhered to tissue engineered periosteum. At 4, 8, and 12 weeks after operation, X-ray films showed the newly formed bone was much more in groups A and C than groups B and D. The X-ray film score were significantly higher in groups A and C than in groups B and D, in group A than in group C, and in group B than in group D (P<0.05). Histological staining indicated that there was a lot of newly formed bone in the defect space in group A, with abundant newly formed vessels and medullary cavity. While in group B, the defect space filled with the DPB, the degradation of DPB was not obvious. In group C, there was a lot of newly formed bone in the defect space, island-like DPB and obvious DPB degradation were seen in newly formed bone. In group D, the defect space only replaced by some connective tissue. ConclusionTissue engineered periosteum constructed by SIS and BMSCs has the feasibility to repair the large diaphysis defect in rabbit. DPB isn't an ideal support scaffold of tissue engineering periosteum, the supporting scaffolds of tissue engineered periosteum need further exploration.
ObjectiveTo solve the fixation problem between ligament grafts and host bones in ligament reconstruction surgery by using ligament-bone composite scaffolds to repair the ligaments, to explore the fabrication method for ligament-bone composite scaffolds based on three-dimensional (3-D) printing technique, and to investigate their mechanical and biological properties in animal experiments. MethodsThe model of bone scaffolds was designed using CAD software, and the corresponding negative mould was created by boolean operation. 3-D printing techinique was employed to fabricate resin mold. Ceramic bone scaffolds were obtained by casting the ceramic slurry in the resin mould and sintering the dried ceramics-resin composites. Ligament scaffolds were obtained by weaving degummed silk fibers, and then assembled with bone scaffolds and bone anchors. The resultant ligament-bone composite scaffolds were implanted into 10 porcine left anterior cruciate ligament rupture models at the age of 4 months. Mechanical testing and histological examination were performed at 3 months postoperatively, and natural anterior cruciate ligaments of the right sides served as control. ResultsBiomechanical testing showed that the natural anterior cruciate ligament of control group can withstand maximum tensile force of (1 384±181) N and dynamic creep of (0.74±0.21) mm, while the regenerated ligament-bone scaffolds of experimental group can withstand maximum tensile force of (370±103) N and dynamic creep of (1.48±0.49) mm, showing significant differences (t=11.617,P=0.000; t=-2.991,P=0.020). In experimental group, histological examination showed that new bone formed in bone scaffolds. A hierarchical transition structure regenerated between ligament-bone scaffolds and the host bones, which was similar to the structural organizations of natural ligament-bone interface. ConclusionLigament-bone composite scaffolds based on 3-D printing technique facilitates the regeneration of biomimetic ligament-bone interface. It is expected to achieve physical fixation between ligament grafts and host bone.
Objective To fabricate a nanohydroxyapatite-chitosan(nano-HA-CS) scaffold with high porosity by a simple and effective technique and to evaluate the physical and chemical properties and the cytocompatibility of the composite scaffold. Methods The threedimensional nano-HA-CS scaffolds with high porosity were prepared by the in situ hybridization-freeze-drying method. The microscopic morphology and components of the composite scaffolds were analyzed by the scanning electron microscopy (SEM), the transmission electron microscopy（TEM）, the X-ray diffraction（XRD）examination, and the Fourier transformed infrared spectroscopy（FTIR）. The calvarial osteoblasts were isolated from the neonatal Wistar rats. The serial subcultured cells (3rd passage) were respectively seeded onto the nanoHACS scaffold and the CS scaffold, and then were cocultured for 2, 4, 6 and 8 hours. At each time point,four specimens from each matrix were taken to determine the celladhesion rate. The cell morphology was observed by the histological staining and SEM. Results The macroporous nanoHACS scaffolds had a feature of high porosity with a pore diameter from 100 to 500 μm (mostly 400500 μm). The scaffolds had a high interval porosity; however, the interval porosity was obviously decreased and the scaffold density was increased with an increase in the contents of CS and HA. The SEM and TEM results showed that the nanosized HA was synthesized and was distributed on the pore walls homogeneously and continuously. The XRD and FTIR results showed that the HA crystals were carbonatesubstituded and not wellcrystallized. The cytocompatibility test showed that the seeded osteoblasts could adhere the scaffolds, proliferating and producing the extracellular matrix on the scaffolds. The adherence rate for the nanoHACS scaffolds was obviously higher than that for the pure CS scaffolds. Conclusion The nano-HA-CS scaffolds fabricated by the in situ hybridization-freeze-drying method have a good physical and chemical properties and a good cytocompatibility; therefore, this kind of scaffolds may be successfully used in the bone tissue engineering.
ObjectiveTo summarize the application status of hypoxia mimetic agents in bone tissue engineering.MethodsThe related literature about the hypoxia mimetic agents in bone tissue engineering was reviewed and analyzed. And the application status and progress of hypoxia mimetic agents in bone tissue engineering were retrospectively analyzed.ResultsHypoxia mimetic agents have the same effect as hypoxia in up-regulating the level of hypoxia inducible factor 1α (HIF-1α). The combination of hypoxia mimetic agents and scaffolds can up-regulate the level of HIF-1α in bone tissue engineering, thus promoting early vascularization and bone regeneration of the bone defect area, which provides a new idea for using bone tissue engineering to repair bone defect. At present, the commonly used hypoxia mimetic agents include iron chelating agents, oxoglutarate competitive analogues, proline hydroxylase inhibitors, etc.ConclusionHypoxia mimetic agents have a wide application prospect in bone tissue engineering, but they have been used in bone tissue engineering for a short time, more attention should be paid to their possible side effects. In the future research, the hypoxia mimetic agents should be developed in the direction of higher targeting specificity and safety, and the exact mechanism of hypoxia mimetic agents in promoting bone regeneration should be further explored.