ObjectiveTo investigate the feasibil ity of the domestic porous tantalum as scaffold material of bone tissue engineering by observing the expressions of osteogenesis related factors of MG63 cells co-cultured with domestic porous tantalum. MethodsMG63 cells were cultured with porous tantalum scaffolds (group A), with porous tantalum leaching solution (group B), and with MEM as control group (group C). The cell adhesion of group A was observed on the scaffolds at 3, 5, and 7 days after culture by scanning electron microscopy (SEM); immunohistochemistry and Western blot methods were used to detect the expressions of Runt-related transcri ption factor 2 (Runx-2), osteocalcin (OC), and fibronectin (FN). ResultsAt 3 days after culture, the cells of group A adhered the surface and pore of the porous tantalum scaffolds, with sparse cell arrangement and less protuberances; at 5 days after culture, adjacent cells connected to be a flat each other, which covered the surface and pore of the scaffold; at 7 days after culture, cells secreted plenty of extracellular matrix, covering most of the material surface. The expressions of Runx-2, OC, and FN were positive in 3 groups; darker staining of the cytoplasm was observed in group A, the expressions were significantly higher in group A than in other 2 groups. The results of immunohistochemistry and Western blot showed that the expressions of Runx-2 and OC were significantly increased in group A when compared with those in groups B and C (P < 0.05), but no significant difference was found between groups B and C (P > 0.05). The expression of FN had no significant difference among 3 groups (P > 0.05). ConclusionDomestic porous tantalum could promote MG63 cells adhesion and growth, and may promote the expressions of Runx-2 and OC, so it can be used as a scaffold material of bone tissue engineering.
Objective To review the research progress on bone repair biomaterials with the function of recruiting endogenous mesenchymal stem cells (MSCs). Methods An extensive review of the relevant literature on bone repair biomaterials, particularly those designed to recruit endogenous MSCs, was conducted, encompassing both domestic and international studies from recent years. The construction methods and optimization strategies for these biomaterials were summarized. Additionally, future research directions and focal points concerning this material were proposed. Results With the advancement of tissue engineering technology, bone repair biomaterials have increasingly emerged as an ideal solution for addressing bone defects. MSCs serve as the most critical “seed cells” in bone tissue engineering. Historically, both MSCs and their derived exosomes have been utilized in bone repair biomaterials; however, challenges such as limited sources of MSCs and exosomes, low survival rates, and various other issues have persisted. To address these challenges, researchers are combining growth factors, bioactive peptides, specific aptamers, and other substances with biomaterials to develop constructs that facilitate stem cell recruitment. By optimizing mechanical properties, promoting vascular regeneration, and regulating the microenvironment, it is possible to create effective bone repair biomaterials that enhance stem cell recruitment. Conclusion In comparison to cytokines, phages, and metal ions, bioactive peptides and aptamers obtained through screening exhibit more specific and targeted recruitment functions. Future development directions for bone repair biomaterials will involve the modification of peptides and aptamers with targeted recruitment capabilities in biological materials, as well as the optimization of the mechanical properties of these materials to enhance vascular regeneration and adjust the microenvironment.
Objective To investigate the feasibility of a new kind of porous β tricalcium phosphate (β-TCP) as a scaffold for the bone tissue engineering Methods The inverted phase contrast microscope was used to observe the growth of the marrow mesenchymal stem cells (MSCs) in the experimentalgroup and the control group at 10 days.In the experimental group, the MSCs were cultured with β-TCP(3 mm×3 mm×3 mm) in the 24-hole cultivation board, and in the control to control group, only MSCs were cultivated. The scanning electron microscope was used to observe growth of MSCs at 6 days. Cultivated with β-TCP at 3, 6, 9, 12 days, the MTT assay was used to judge the biocompatibility. The cytotoxicity was analyzed with the method that used the different density(100%， 50%， 10%， 1%，0%) leaching liquor gained from β-TCP to raise MSCs. MSCs were induced into the osteoblasts and were mixed with β-TCP, and the composite was used to repair a large radius bone defect in the rabbit. The specimens were made at 2,6,12 weeks. The histology imageology, and the radionuclide bone scan were used to analyze the bone formation. Results Some MSCs had a good adherence 4 hours after MSCs were inoculated and had a complete adherence at 12 hours. The cells were shaped like polyangle, spindle or converge monolayer after 8-10 days. The cells in the two groups had no difference. The cell adhesion was good, when observed by the inverted phase contrast microscope and the scanning electron microscope at 6 days. MTT showed that the absorbance (A)was not statistically different between the experimental group and the control group (P＞0.05); the different density leaching liquor had no cytotoxicity at the different time points. Histology, X-ray, and CT tomograph showed that itcould repair the large radius bone defect in the rabbit and its in vivo degradationrate was the same as the bone formation rate. Conclusion The new porous β-TCP has a unique three dimensional (3D) stereochemical structure and superordinary physicochemical property, and so it is a good scaffold for the bone tissue engineering.
ObjectiveTo investigate the formation of nanostructure on cuttlefish bone transformed hydroxyapatite (CB-HA) porous ceramics and the effects of different nanostructures on the osteoblasts adhesion, proliferation, and alkaline phosphatase (ALP) expression.MethodsThe cuttlefish bone was shaped as plate with diameter of 10 mm and thickness of 2 mm, filled with water, and divided into 4 groups. The CB-HA in groups 1-4 were mixed with different phosphorous solutions and then placed in an oven at 120℃ for 24 hours. In addition, the samples in group 4 were further sintered at 1 200℃ for 3 hours to remove nanostructure as controls. The chemical composition of CB-HA were analyzed by X-ray diffraction spectroscopy, Fourier transform infrared spectrum, and inductively coupled plasma (ICP). The physical structure was analyzed using scanning electron microscopy, specific surface tester, and porosity tester. The MC3T3-E1 cells of 4th generation were co-cultured with 4 groups of CB-HA. After 1 day, the morphology of the cells was observed under scanning electron microscopy. After 1, 3, and 7 days, the cell proliferation was analyzed by MTT assay. After 7 and 14 days, the ALP expression was measured by pNPP method.ResultsX-ray diffraction spectrum showed that the four nanostructures of CB-HA were made of hydroxyapatite. The infrared absorption spectrum showed that the infrared absorption peak of CB-HA was consistent with hydroxyapatite. ICP showed that the ratio of calcium to phosphorus of all CB-HA was 1.68-1.76, which was consistent with hydroxyapatite. Scanning electron microscopy observation showed that the nanostructure on the surface of CB-HA in groups 1-3 were large, medium, and small cluster-like structures, respectively, and CB-HA in group 4 had no obvious nanostructure. There were significant differences in the specific surface areas between groups (P<0.05). There was no significant difference in the porosity between groups (P>0.05). Compared with group 4, groups 1-3 have more pores with pore size less than 50 nm. After co-cultured with osteoblasts, scanning electron microscopy observation and MTT assay showed that the cells in groups 2 and 3 adhered and proliferated better and had more ALP expression than that in groups 1 and 4 (P<0.05).ConclusionThe size of cluster-like nanostructure on the surface of CB-HA can be controlled by adjusting the concentration of ammonium ions in the phosphorous solution, and the introduction of small-sized cluster-like nanostructure on the surface of CB-HA can significantly improve the cell adhesion, proliferation, and ALP expression of the material which might be resulted from the enlarged surface area.
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 study the effect of Schwann cells (SCs) promoting the function of nitric oxide (NO) secretion of bone marrow mesenchymal stem cells (BMSCs) derived endothelial cells so as to lay the experimental foundation for research of the effect of nerves on vessels during the process of tissue engineering bone formation. MethodsSCs were collected from 1-day-old Sprague Dawley (SD) rats,and identified through S100 immunohistochemistry (IHC).BMSCs were collected from 2-week-old SD rats and induced into endothelial cells (IECs),which were identified through von Willebrand factor (vWF) and CD31 immunofluorescence (IF).Transwell system was used for co-culture of SCs and IECs without contact as the experimental group,and simple culture of IECs served as the control group.The NO concentration in the medium was measured at 1,3,5,and 7 days after culture; the mRNA expressions of nitric oxide synthetase 2 (NOS2) and NOS3 were detected by real-time fluorescence quantitative PCR (RT-qPCR) at 1,3,7,and 10 days. ResultsSCs and IECs were identified through morphology and immunology indexes of S100 IHC,vWF and CD31 IF.Significant differences were found in the NO concentration among different time points in 2 groups (P<0.05); the NO concentration of the experimental group was significantly higher than that of the control group at the other time points (P<0.05) except at 3 days.NOS2 mRNA expression of the experimental group was significantly higher than that of the control group (P<0.05); difference was significant in the NOS2 mRNA expression among different time points in 2 groups (P<0.05).NOS3 mRNA expression of the experimental group was significantly higher than that of the control group at the other time points (P<0.05) except at 10 days.No significant difference was found in NOS3 mRNA expression among different time points in the experimental group (F=6.673,P=0.062),but it showed significant differences in the control group (F=36.581,P=0.000). ConclusionSCs can promote NO secretion of BMSCs derived endothelial cells,which is due to promoting the activity of NOS.
Objective Tissue engineered bone implanted with sensory nerve can effectively promote angiogenesis and repair of bone defects. To investigate the effects of calcitonin gene-related peptide (CGRP) on proliferation and migration of human umbilical vein endothelial cells (HUVECs) for further revealing the mechanism of tissue engineered bone implanted with sensory nerve promoting angiogenesis. Methods HUVECs were collected from human umbilical core, and identified through von Willebrand factor (vWF) and CD31 immunofluorescence. The HUVECs were treated with CGRP and were ivided into 6 groups according to CGRP concentration: group A (0 mol/L), group B (1 × 10—12 mol/L), group C (1 × 10—11 mol/L), group D (1 × 10—10 mol/L), group E (1 × 10—9 mol/L), and group F (1 × 10—8 mol/L). The expression of the CGRP1 receptor (CGRP1R) was observed in HUVECs by cell immunofluorescence. The growth rate of HUVECs was detected through AlarmarBlue at 1, 2, 3, 4, and 5 days. Transwell chamber was used to detect the abil ity of cell migration. ELISA assay was used to detect the vascular endothel ial growth factor (VEGF) secretion and the protein expression of focal adhesion kinase (FAK) was examined using Western blot. Results HUVECs were identified through morphology, vWF and CD31 immunofluorescence. HUVECs expressed CGRP1R. CGRP could stimulate HUVECs prol iferation in a time- and concentration-dependent manners; the cell growth rates of groups B-F were significantly higher than that of group A at all time (P lt; 0.05); group F had highest cell growth rate. The number of cell migration of group B-F was significantly higher than that of group A (P lt; 0.05), which increased more than 3 times. Groups B-F had higher amount of VEGF than group A (P lt; 0.05), and groups C and D had highest amount of VEGF. FAK expression of groups B-F was significantly increased at 3, 7, and 10 days after CGRP treatment when compared with group A (P lt; 0.05). Conclusion CGRP may enhance the proliferation and migration of HUVECs by increasing the secretion of VEGF and expression of FAK.
ObjectiveTo investigate the influences of lactic acid (LA), the final degradation product of polylactic acid (PLA) on the prol iferation and osteoblastic phenotype of osteoblast-l ike cells so as to provide theoretical basis for bone tissue engineering. MethodsRos17/2.8 osteoblast-l ike cells were harvested and divided into 3 groups. In groups A and B, the cells were cultured with the medium containing 4, 8, 16, 22, and 27 mmol/L L-LA and D, L-LA, respectively. In group C, the cells were cultured with normal medium (pH7.4). The cell prol iferation was determined with MTT method after 1, 3, and 5 days. The relative growth ratio (RGR) was calculated, and the cytotoxicity was evaluated according to national standard of China. In addition, the alkal ine phosphatase (ALP) activity of cells cultured with medium containing 4 mmol/L L-LA (group A), 4 mmol/ L D, L-LA (group B), and normal medium (group C) after 1 and 5 days were detected with ALP kits, and the relative ALP ratio (RAR) was calculated; after 21 days, the calcium nodules were tested with von Kossa staining method, and were quantitatively analyzed. ResultsWhen LA concentration was 4 mmol/L, the mean RGR of both groups A and B were all above 80%, and the cytotoxic grades were grade 0 or 1, which meant non-cytotoxicity. When LA concentration was 8 mmol/L and 16 mmol/ L, groups A and B showed cytotoxicity after 5 days and 3 days, respectively. When LA concentration was above 22 mmol/L, cell prol iferations of groups A and B were inhibited evidently after 1-day culture. At each LA concentration, RGR of group A was significantly higher than that of group B at the same culture time (P<0.05) except those at 4 mmol/L after 1-day and 3-day culture. After 1 day, the RAR of group A was significantly higher than that of group B on 1 day (144.1%±3.2% vs. 115.2%±9.8%, P<0.05) and on 5 days (129.6%±9.8% vs. 78.2%±6.9%, P<0.05). The results of von Kossa staining showed that the black gobbets in group A were obviously more than those of groups B and C. The staining area of group A (91.2%±8.2%) was significantly higher than that of groups B (50.3%±7.9%) and C (54.2%±8.6%) (P<0.05). ConclusionThe concentration and composition of LA have significant effects on the cell proliferation and osteoblastic phenotype of osteoblast-l ike cells.
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.
Objective To find a new culture system to induce proliferation and osteodifferentiation of marrow stromal cells (MSCs) in vitro for bone tissueeng ineering． Methods There were four groups in this experiment to study effects of Passage 3 osteoblasts derived from the rat cranium and the osteogenic inductor (1 nmol/L dexamethasone，10 mmol/L beta-glycero-phosphate，50 μg/ml retin oic acid) on growth of MSCs isolated from the rat femur and the tibia． MSCs were cultured in the DMEM medium (the c ontrol group) and in the osteoinductive culture medium (the inductor group)；fur thermore, MSCs were co-cultured with the osteoblasts in the DMEM medium (the osteoblast group) and in the osteoinductive culture medium (the combined treatment group)．The cells in the four groups were counted every 2 days for 8 days and alkaline phosphatase (ALP) activity of MSCs at 10 days of cultivation was measured．The MRNA expression of osteocalcin (OC) of MSCs at 2 weeks was assayed with the reverse transcript polymase chain reaction (RT-PCR)． Results There were more cells in the osteoblast group than in the control group(31.73±3.31×104 V S. 24.33±3.04×104, Plt;0.05), but there were fewer cells in the inductor gro up(16.23±2.44×104, Plt;0.05). There was no significant difference in th e cell number between the combined treatment group (21.54±2.29×104) and th e control group（Pgt;0.05）．The ALP activity was higher in the combined trea tment group （2.01±0.56 U）than in the control group (1.27±0.43 U), in the inductor group（1.27±0.43 U）, and in the osteoblast group （0.77±0.19 U）.The osteocalcin mRNA was expressed in the three treat ment groups but was not expressed in the control group. The significantly higher leve l of the osteocalcin mRNA was expressed in the inductor group（0.783±0.094）and in the combined treatment group（0.814±0.071）than in the osteoblast group（0.302±0.026） (Plt;0.05). Conclusion The combined use of t he osteoblast and the inductor can induce marrow stromal cells. Their combined u se does not affect the normal proliferation but can obviously promote the osteodifferentiation of marrow stromal cells. This combined use can become a new culture system of the seed cells for bone tissue engineering．