Osteogenesis Of Tissue-engineered Bone Research Articles

Schwann cells promote prevascularization and osteogenesis of tissue-engineered bone via bone marrow mesenchymal stem cell-derived endothelial cells

BackgroundTissue-engineered bone grafts (TEBGs) that undergo vascularization and neurotization evolve into functioning bone tissue. Previously, we verified that implanting sensory nerve tracts into TEBGs promoted osteogenesis. However, the precise mechanisms and interaction between seed cells were not explored. In this study, we hypothesized that neurotization may influence the osteogenesis of TEBGs through vascularization.MethodsWe cultured rat Schwann cells (SCs), aortic endothelial cells (AECs), and bone marrow-derived mesenchymal stem cells (BM-MSCs) and then obtained BM-MSC-derived induced endothelial cells (IECs) and induced osteoblasts (IOBs). IECs and AECs were cultured in an SC-conditioned medium (SC-CM) to assess proliferation, migration, capillary-like tube formation, and angiogenesis, and the vascular endothelial growth factor (VEGF) levels in the supernatants were detected. We established an indirect coculture model to detect the expression of nestin and VEGF receptors in IECs and tissue inhibitor of metalloproteinase (TIMP)-2 in SCs. Then, SCs, IECs, and IOBs were labeled and loaded into a β-tricalcium phosphate scaffold to induce prevascularization, and the scaffold was implanted into a 6-mm-long defect of rat femurs. Three groups were set up according to the loaded cells: I, SCs, and IECs (coculture for 3 days) plus IOBs; II, IECs (culture for 3 days) plus IOBs; III, IOBs. Nestin and TIMP-2 expression and osteogenesis of TEBGs were evaluated at 12 weeks post-implantation through histological and radiological assessments.ResultsWe found that SC-CM promoted IEC proliferation, migration, capillary-like tube formation, and angiogenesis, but no similar effects were observed for AECs. IECs expressed nestin extensively, while AECs barely expressed nestin, and SC-CM promoted the VEGF secretion of IECs. In the coculture model, SCs promoted nestin and VEGF receptor expression in IECs, and IECs inhibited TIMP-2 expression in SCs. The promotion of prevascularized TEBGs by SCs and IECs in group I augmented new bone formation at 6 and 12 weeks. Nestin expression was higher in group I than in the other groups, while TIMP-2 expression was lower at 12 weeks.ConclusionsThis study demonstrated that SCs can promote TEBG osteogenesis via IECs and further revealed the related specific characteristics of IECs, providing preliminary cytological evidence for neurotization of TEBGs.

Effects of Endothelial Progenitor Cells on Vascularization and Osteogenesis of Tissue-engineered Bones in Beagle Dogs

Objective The explore the effects of endothelial progenitor cells(EPCs)on vascularization and osteogenesis of tissue-engineered bones in Beagle dogs.Methods Bone marrow mesenchymal stem cells(BMSCs)and EPCs from bone marrow of Beagle dogs were isolated,cultured,and expanded and then seeded in β-tricalcium phosphate(TCP)scaffolds. The cell scaffold complexes(EPCs/ BMSCs/TCP group and BMSCs/TCP group)and the blank TCP scaffold were transplanted into the limb muscles of Beagle dogs,respectively. The relative CT values were calculated in the 3rd and 6th month through CT scan after operation. The vascularization and the osteogenesis were detected 6 months after operation by histological examination.Results Three months after the surgery,the relative CT value in the EPCs/BMSCs/TCP group was(366.67±19.51)HU,significantly higher than that[(163.00±30.81)HU] in the BMSCs/TCP group(t=2.10,P=0.0006);6 months after surgery,the relative CT value in the EPCs/BMSCs/TCP group was(553.34±26.86)HU,which was also significantly higher than that[(241.34±21.57)HU] in the BMSCs/TCP group(t=2.11,P=0.0006). The relative CT values at 6 months after operation were significantly higher than those at 3 months after operation in both BMSCs/TCP group(t=2.10,P=0.0255)and EPCs/BMSCs/TCP group(t=2.10,P=0.0006). Six months after the operation,HE staining showed no bone formation or calcium deposition in the blank TCP group,and osteoblasts,mature bone trabeculae,and calcium deposition were observed in EPCs/BMSCs/TCP group and BMSCs/TCP group. Immunohistochemical staining showed that the number of peripheral,central,and whole vessels in the EPCs/BMSCs/TCP group was 21.67±1.45,23.33±2.60,and 45.00±1.16,which were significantly higher than those in the BMSCs/TCP group[8.67±0.88(t=2.07,P=0.0016),9.33±0.67(t=2.07,P=0.0065),and 18.00±1.00(t=2.07,P=0.001),respectively]. The number of vessels was not significantly different between the BMSCs/TCP group and the blank TCP group(18.00±1.00 vs. 16.67±2.40;t=2.07,P=0.636). The osteogenic area of the EPCs/BMSCs/TCP group was(1 322 000±141 300)pixel,significantly higher than that of the BMSCs/TCP group[(874 900±49 430)pixel;t=2.10,P=0.04]. BMSCs/TCP group showed significantly higher bone area in the peripheral area than in the central region[(170 000±42 320)pixel vs.(613 900±90 290)pixel;t=2.10,P=0.02];in contrast,there was no statistically significant difference in bone area between the peripheral region and the central region in the EPCs/BMSCs/TCP group[(376 400±20 160)pixel vs.(310 400±6917)pixel;t=2.10,P=0.07].Conclusion EPCs as seed cells combined with BMSCs can promote vascularization and osteogenesis of tissue-engineered bone in Beagle dogs.

Dorsal Root Ganglion Maintains Stemness of Bone Marrow Mesenchymal Stem Cells by Enhancing Autophagy through the AMPK/mTOR Pathway in a Coculture System.

Our previous studies found that sensory nerve tracts implanted in tissue-engineered bone (TEB) could result in better osteogenesis. To explore the mechanism of the sensory nerve promoting osteogenesis in TEB in vitro, a transwell coculture experiment was designed between dorsal root ganglion (DRG) cells and bone marrow mesenchymal stem cells (BMSCs). BMSC proliferation was determined by CCK8 assay, and osteo-, chondro-, and adipogenic differentiation were assessed by alizarin red, alcian blue, and oil red staining. We found that the proliferation and multipotent differentiation of BMSCs were all enhanced in the coculture group compared to the BMSCs group. Crystal violet staining showed that the clone-forming ability of BMSCs in the coculture group was also enhanced and mRNA levels of Sox2, Nanog, and Oct4 were significantly upregulated in the coculture group. Moreover, the autophagy level of BMSCs, regulating their stemness, was promoted in the coculture group, mediated by the AMPK/mTOR pathway. In addition, AMPK inhibitor compound C could significantly downregulate the protein expression of LC3 and the mRNA level of stemness genes in the coculture group. Finally, we found that the NK1 receptor antagonist, aprepitant, could partly block this effect, which indicated that substance P played an important role in the effect. Together, we conclude that DRG could maintain the stemness of BMSCs by enhancing autophagy through the AMPK/mTOR pathway in a transwell coculture system, which may help explain the better osteogenesis after implantation of the sensory nerve into TEB.

A novel strategy for in vivo angiogenesis and osteogenesis: magnetic micro-movement in a bone scaffold

Angiogenesis and osteogenesis in tissue-engineered bone are the key factors in the clinical application of tissue-engineering technology to repair large bone defects. In vivo cells that are farther than 200 μm from capillaries cannot survive due to lack of nutrients and oxygen, and thus, the tissue-engineered bone is not suitable for repairing large bone defects. In this study, we constructed a novel artificial bone scaffold loaded with superparamagnetic plasmid gene microspheres. Magnetic micro-movement of the magnetic microspheres in the scaffold was generated by an oscillating magnetic field and a static magnetic field to promote the release of plasmid genes from microspheres for transfection of surrounding cells, resulting in protein expression of vascular endothelial growth factor, thus promoting angiogenesis and osteogenesis in the scaffold, internal vascularization of the artificial bone scaffold and repair of large bone defects.

The effects of different vascular carrier patterns on the angiogenesis and osteogenesis of BMSC-TCP-based tissue-engineered bone in beagle dogs.

Bone repair using tissue-engineered bone (TEB) in a large defect or accompanied by a poor recipient vascular bed is a long-standing challenge. Surgical vascular carrier patterns of vascular bundle (VB) and arteriovenous loop (AV loop) have been shown to improve the vascularization and repair capacity of TEB. However, the effects of these different vascular carrier patterns on angiogenesis and osteogenesis in TEB have never been evaluated. Here, TEB was constructed with bone marrow mesenchymal stem cells (BMSCs) and β-TCP and prevascularized by the VB or AV loop technique in beagle dogs. The vascularization and bone formation in TEB were quantitatively compared using Microfil perfusion, histological examination and CT and micro-CT analyses. The distribution and constitution of the neovasculature were analysed to determine the underlying mechanism of angiogenesis. The results showed that prevascularized TEB generated bone tissue faster and more homogeneously than untreated TEB. The VB technique was found to strike a better balance between bone regeneration and β-TCP scaffold degradation than the AV loop strategy, which resulted in more vascularization but less bone yield, due to faster degradation of the β-TCP scaffold. This study indicates that a suitable triangular relationship, composed of bone regeneration, scaffold degradation and vasculature, is critical to TEB construction. Copyright © 2015 John Wiley & Sons, Ltd.

The effects of separating inferior alveolar neurovascular bundles on osteogenesis of tissue-engineered bone and vascularization.

To evaluate the effects of autologous blood vessels and nerves on vascularization. A dog model of tissue-engineered bone vascularization was established by constructing inferior alveolar neurovascular bundles through the mandibular canal. Sixteen 12-month-old healthy beagles were randomly divided into two groups (n=8). Group A retained inferior alveolar neurovascular bundles, and Group B retained inferior alveolar nerves. Bone marrow mesenchymal stem cells were injected into β-tricalcium phosphate to prepare internal tissue-engineered bone scaffold. A personalized titanium mesh was then prepared by rapid prototyping and fixed by external titanium scaffold. Two dogs in each group were sacrificed on the 30th, 45th, 60th and 90th postoperative days respectively. The bone was visually examined, scanned by CT, and subjected to HE staining, immunohistochemical staining, vascular casting and PCR to detect the changes in osteogenesis and vascularization. The two groups had similar outcomes in regard to osteogenesis and vascularization (P>0.05): both showed remarkable regenerative capacities. The model of tissue-engineered bone vascularization is potentially applicable in clinical practice to allow satisfactory osteogenesis and vascularization.

Different effects of implanting sensory nerve or blood vessel on the vascularization, neurotization, and osteogenesis of tissue-engineered bone in vivo.

To compare the different effects of implanting sensory nerve tracts or blood vessel on the osteogenesis, vascularization, and neurotization of the tissue-engineered bone in vivo, we constructed the tissue engineered bone and implanted the sensory nerve tracts (group SN), blood vessel (group VB), or nothing (group Blank) to the side channel of the bone graft to repair the femur defect in the rabbit. Better osteogenesis was observed in groups SN and VB than in group Blank, and no significant difference was found between groups SN and VB at 4, 8, and 12 weeks postoperatively. The neuropeptides expression and the number of new blood vessels in the bone tissues were increased at 8 weeks and then decreased at 12 weeks in all groups and were highest in group VB and lowest in group Blank at all three time points. We conclude that implanting either blood vessel or sensory nerve tract into the tissue-engineered bone can significantly enhance both the vascularization and neurotization simultaneously to get a better osteogenesis effect than TEB alone, and the method of implanting blood vessel has a little better effect of vascularization and neurotization but almost the same osteogenesis effect as implanting sensory nerve.

9C-03 ランダムパルス列電気刺激による培養再生骨の石灰化促進(OS-10 ティッシュエンジニアリング(1))

9C-03 ランダムパルス列電気刺激による培養再生骨の石灰化促進(OS-10 ティッシュエンジニアリング(1))

Osteogenesis oftissue-engineered bone implanted with grafts of vascular and sensory nerve bundles

：Objective To investigatethe osteogenesis of the tissue-engineered bone implanted with grafts of vascular andsensory nerve bundles. Methods Models of left femoral defects of 1.5 cm were fabricated in36 New Zealand white rabbits which were randomized into 3 groups. In Group A, thetissue-engineered bone which had been constructed with autologous bone marrow mesenchymalstem cells plus β-tricalcium phosphate (β-TCP) was implanted into the defects. In group B, thetissue-engineered bone plus graft of vascular bundle was implanted into the femoraldefects. In group C, the tissue-engineered bone plus graft of sensory nerve bundle wasimplanted into the bony defects. Bone formation throughout the defects was assessedradiographically 1, 3 and 6 months after implantation, and at the same time 4 rabbits fromeach group were sacrificed to evaluate bone formation by gross and histologicalobservations. Results The bone formation in groups B and C was better than in group A(P 0.05).New bone formation occurred mostly around the vessels and chiefly in the form ofenchondral ossification. Conclusion Implantation of vascular and sensory nerve bundlesinto tissue-engineered bone may enhance the esteogenesis of tissue-engineered bone inreconstruction of large bony defects. Key words: Rabbit; Bone defect; Tissue engineering; Vascularbundle; Sensory nerve bundle

501 ひずみ誘導型液体流動刺激による培養再生骨の石灰化促進(OS10-1:組織再生のバイオメカニクス(1),OS10:組織再生のバイオエンジニアリング)

501 ひずみ誘導型液体流動刺激による培養再生骨の石灰化促進(OS10-1:組織再生のバイオメカニクス(1),OS10:組織再生のバイオエンジニアリング)