Vascularized Bone Tissue Engineering Research Articles

Hypoxia as a stimulus for tissue formation: The concept of organogenesis in microsurgically vascularized tissue engineering constructs

Axial vascularization of tissue constructs is essential to maintain an adequate blood supply for a stable regeneration of a clinically relevant tissue size. The versatility of the arterio-venous loop (AVL) has been previously shown in various small and large animal models as well as in clinical reports for bone regeneration. We have previously demonstrated the capability of the AVL to induce axial vascularization and to support the nourishment of tissue constructs in small animal models after applying high doses of ionizing radiation comparable to those applied for adjuvant radiotherapy after head and neck cancer. We hypothesize that this robust ability to induce regeneration after irradiation could be related to a state of hypoxia inside the constructs that triggers the HIF1 (hypoxia induced factor 1) - SDF1 (stromal derived factor 1) axis leading to chemotaxis of progenitor cells and induction of tissue regeneration and vascularization. We analyzed the expression of HIF1 and SDF1 via immunofluorescence in axially vascularized bone tissue engineering constructs in Lewis rats 2 and 5 weeks after local irradiation with 9Gy or 15Gy. We also analyzed the expression of various genes for osteogenic differentiation (collagen 1, RUNX, alkaline phosphatase and osteonectin) via real time PCR analysis. The expression of HIF1 and SDF1 was enhanced two weeks after irradiation with 15Gy in comparison to non-irradiated constructs. The expression of osteogenic markers was enhanced at the 5-weeks time point with significant results regarding collagen, alkaline phosphatase and osteonectin. These results indicate that the hypoxia within the AVL constructs together with an enhanced SDF1 expression probably play a role in promoting tissue differentiation. The process of tissue generation triggered by hypoxia in the vicinity of a definite vascular axis with enhanced tissue differentiation over time resembles hereby the well-known concept of organogenesis in fetal life.

Fabrication of vascularized tissue-engineered bone models using triaxial bioprinting.

Bone tissue is a highly vascularized tissue. When constructing tissue-engineered bone models, both the osteogenic and angiogenic capabilities of the construct should be carefully considered. However, fabricating a vascularized tissue-engineered bone to promote vascular formation and bone generation, while simultaneously establishing nutrition channels to facilitate nutrient exchange within the constructs, remains a significant challenge. Triaxial bioprinting, which not only allows the independent encapsulation of different cell types while simultaneously forming nutrient channels, could potentially emerge as a strategy for fabricating vascularized tissue-engineered bone. Moreover, bioinks should also be applied in combination to promote both osteogenesis and angiogenesis. In this study, employing triaxial bioprinting, we used a blend bioink of gelatin methacryloyl (GelMA), sodium alginate (Alg), and different concentrations of nano beta-tricalcium phosphate (nano β-TCP) encapsulated MC3T3-E1 preosteoblasts as the outer layer, a mixed bioink of GelMA and Alg loaded with human umbilical vein endothelial cells (HUVEC) as the middle layer, and gelatin as a sacrificial material to form nutrient channels in the inner layer to fabricate vascularized bone constructs simulating the microenvironment for bone and vascular tissues. The results showed that the addition of nano β-TCP could adjust the mechanical, swelling, and degradation properties of the constructs. Biological assessments revealed the cell viability of constructs containing different concentrations of nano β-TCP was higher than 90% on day 7, The cell-laden constructs containing 3% (w/v) nano β-TCP exhibited better osteogenic (higher Alkaline phosphatase activity and larger Osteocalcin positive area) and angiogenic (the gradual increased CD31 positive area) potential. Therefore, using triaxial bioprinting technology and employing GelMA, Alg, and nano β-TCP as bioink components could fabricate vascularized bone tissue constructs, offering a novel strategy for vascularized bone tissue engineering.

3D Printed Piezoelectric BaTiO3/Polyhydroxybutyrate Nanocomposite Scaffolds for Bone Tissue Engineering.

Bone defects are a significant health problem worldwide. Novel treatment approaches in the tissue engineering field rely on the use of biomaterial scaffolds to stimulate and guide the regeneration of damaged tissue that cannot repair or regrow spontaneously. This work aimed at developing and characterizing new piezoelectric scaffolds to provide electric bio-signals naturally present in bone and vascular tissues. Mixing and extrusion were used to obtain nanocomposites made of polyhydroxybutyrate (PHB) as a matrix and barium titanate (BaTiO3) nanoparticles as a filler, at BaTiO3/PHB compositions of 5/95, 10/90, 15/85 and 20/80 (w/w%). The morphological, thermal, mechanical and piezoelectric properties of the nanocomposites were studied. Scanning electron microscopy analysis showed good nanoparticle dispersion within the polymer matrix. Considerable increases in the Young's modulus, compressive strength and the piezoelectric coefficient d31 were observed with increasing BaTiO3 content, with d31 = 37 pm/V in 20/80 (w/w%) BaTiO3/PHB. 3D printing was used to produce porous cubic-shaped scaffolds using a 90° lay-down pattern, with pore size ranging in 0.60-0.77 mm and good mechanical stability. Biodegradation tests conducted for 8 weeks in saline solution at 37 °C showed low mass loss (∼4%) for 3D printed scaffolds. The results obtained in terms of piezoelectric, mechanical and chemical properties of the nanocomposite provide a new promising strategy for vascularized bone tissue engineering.

3D-Bioprinting Strategies Based on In Situ Bone-Healing Mechanism for Vascularized Bone Tissue Engineering.

Over the past decades, a number of bone tissue engineering (BTE) approaches have been developed to address substantial challenges in the management of critical size bone defects. Although the majority of BTE strategies developed in the laboratory have been limited due to lack of clinical relevance in translation, primary prerequisites for the construction of vascularized functional bone grafts have gained confidence owing to the accumulated knowledge of the osteogenic, osteoinductive, and osteoconductive properties of mesenchymal stem cells and bone-relevant biomaterials that reflect bone-healing mechanisms. In this review, we summarize the current knowledge of bone-healing mechanisms focusing on the details that should be embodied in the development of vascularized BTE, and discuss promising strategies based on 3D-bioprinting technologies that efficiently coalesce the abovementioned main features in bone-healing systems, which comprehensively interact during the bone regeneration processes.

Strontium-calcium phosphate hybrid cement with enhanced osteogenic and angiogenic properties for vascularised bone regeneration.

Vascularized bone tissue engineering is regarded as one of the optimal treatment options for large bone defects. The lack of angiogenic properties and unsatisfactory physicochemical performance restricts calcium phosphate cement (CPC) from application in vascularized bone tissue engineering. Our previous studies have developed a starch and BaSO4 incorporated calcium phosphate hybrid cement (CPHC) with improved mechanical strength and handling properties. However, the bioactivity-especially the angiogenic ability-is still absent and requires further improvement. Herein, based on the reported CPHC and the osteogenic and angiogenic properties of strontium (Sr) ions, a strontium-enhanced calcium phosphate hybrid cement (Sr-CPHC) was developed to improve both biological and physicochemical properties of CPC. Compared to CPC, the initial setting time of Sr-CPHC was prolonged from 2.2 min to 20.7 min. The compressive strength of Sr-CPHC improved from 11.21 MPa to 45.52 MPa compared with CPC as well. Sr-CPHC was biocompatible and showed promotion of alkaline phosphatase (ALP) activity, calcium nodule formation and osteogenic relative gene expression, suggesting high osteogenic-inductivity. Sr-CPHC also facilitated the migration and tube formation of human umbilical vein endothelial cells (HUVECs) in vitro and up-regulated the expression of the vascular endothelial growth factor (VEGF) and Angiopoietin-1 (Ang-1). In vivo evaluation showed marked new bone formation in a rat calvarial defect model with Sr-CPHC implanted. Sr-CPHC also exhibited enhancement of neovascularization in subcutaneous connective tissue in a rat subcutaneous implantation model. Thus, the Sr-CPHC with the dual effects of osteogenesis and angiogenesis shows great potential for clinical applications such as the repair of ischemic osteonecrosis and critical-size bone defects.

Human adipose-derived stem cell spheroids incorporating platelet-derived growth factor (PDGF) and bio-minerals for vascularized bone tissue engineering

Stem cells with mineralized materials have been used for bone regeneration; however, engineering the complex vascularized structure of the natural bone remains a challenge. Here, we developed platelet-derived growth factor (PDGF) and bio-mineral coated fibers which were then assembled with human adipose-derived stem cells (hADSCs) to form spheroids as building blocks for vascularized bone regeneration. The PDGF incorporated within the spheroid increased the proliferation of hADSCs, which was characterized by Ki-67 staining and DNA contents. Furthermore, the PDGF enhanced not only osteogenic differentiation, but also endothelial differentiation of hADSCs; the cells within the spheroids showed significantly greater gene expression by 2.46 ± 0.14 fold for osteocalcin (OCN) and by 12.85 ± 3.36 fold for von Willebrand factor (vWF) than those without PDGF. Finally, at two months following transplantation of PDGF-incorporated spheroids onto in vivo mouse calvarial defect, the regenerated bone area (42.48 ± 10.84%) was significantly enhanced and the greatest number of capillaries and arterioles with indication of transplanted hADSCs were observed. Moreover, millimeter-scale in vitro tissue prepared by fused assembly of the spheroids exhibited greater mRNA expression-associated to endothelial lineage. Taken together, these findings indicate that stem cell spheroids incorporating PDGF and bio-minerals could be used as a module for successful vascularized bone regeneration.

Injectable hydrogel delivering bone morphogenetic protein-2, vascular endothelial growth factor, and adipose-derived stem cells for vascularized bone tissue engineering

We aimed to evaluate the integration of stem cells with injectable microspheres loaded with growth factors using a chitosan (CS) scaffold to promote healthy bone regeneration. We prepared and injected CS scaffolds containing adipose-derived stem cell (ADSC)-, vascular endothelial growth factor (VEGF)-, and bone morphogenetic protein-2 (BMP-2)-loaded nanohydroxyapatite (nHA)/poly lactic-co-glycolic acid microspheres (PLGAs) into critical-sized mandibular bone defects in rabbits. This injectable hydrogel displayed excellent physicochemical properties and degradability and achieved sustainable release and bioactivity preservation for both BMP-2 and VEGF. Computed tomography imaging and gross and histological observations indicated that bone defect cavities in the control group did not narrow and were filled with granulation tissue. Limited new bone formation for the first 12 weeks post-operation was observed in the defect cavities of the VEGF group; the scaffold degraded gradually and was replaced with fibrous connective tissue. In the BMP-2 or BMP-2/VEGF groups, bone defect cavities narrowed with time and healed completely after 12 weeks; scaffolds gradually degraded and infiltrated tightly into the surrounding tissues. Overall, the BMP-2/VEGF group demonstrated greater new bone formation, faster healing, and better callus remodeling. Simultaneous application of VEGF and BMP-2, therefore, promoted ossification and vascularization in critical-sized rabbit mandibular bone defects.

Spatial Immobilization of Autologous Growth Factors to Control Vascularization in Bone Tissue Engineering

The intimate crosstalk, between endothelial and bone cells is essential for the reconstruction of bone defects. Indeed, a successful bone repair is greatly dependent on the formation of new blood vessels, to ensure the supply of nutrients and excretion of metabolites. Bone morphogenetic proteins (BMPs) and vascular endothelial growth factor (VEGF) are involved on cell differentiation and bone vascularization to develop viable bone tissue. Herein it is hypothesized that autologous BMP-2 and VEGF bound in a parallel arrangement over a single nanofibrous substrate (NFM) can lead to a successful osteogenic and angiogenic differentiation of mesenchymal stem cells. An engineered biofunctional system was developed comprising anti-BMP-2 and anti-VEGF antibodies immobilized over the same NFMs in a parallel pattern design, in an attempt to recreate the vasculature of a bone tissue. The osteogenic and angiogenic potential of this engineered biofunctional system was demonstrated by culturing human bone marrow-derived mesenchymal stem cells (hBM-MSCs) during 21 days without exogenous induction. A chick chorioallantoic membrane (CAM) assay showed that the engineered biofunctional system comprising autologous BMP-2 and VEGF bound induced an increased angiogenic response. The angiogenic ability of this system, together with the osteogenic inductor BMP-2 enable obtaining an effective vascularized bone tissue engineering approach.

Dextran-based hydrogel with enhanced mechanical performance via covalent and non-covalent cross-linking units carrying adipose-derived stem cells toward vascularized bone tissue engineering.

Hydrogels for biomedical applications were limited toward bone tissue engineering due to the poor mechanical performance. Tough hydrogels with strong and elastic features have received extensive attention, the application of which, however, was limited by their degradation. The present study introduced an approach to enhance mechanical properties of hydrogel while ensuring its degradation. Carboxyl dextran (Dex) was grafting modified by poly (ε-caprolactone) (PCL), sequentially followed by being cross-linked through polyethyleneglycol 400 (PEG400) to yield a gel with covalent cross-linking units in DMSO. The gel was underwent solvent displacement in H2 O to induce hydrophobic association of PCL to form non-covalent cross-linking units. The tough Dex-g-PCL hydrogel showed maximum strain of Dex-g-PCL hydrogel was 90% ± 6%, with the corresponding stress of 2.7 ± 0.2 MPa, which was significantly enhanced when comparing to dextran hydrogel (maximum strain 65% ± 5%, with the corresponding stress of 0.225 ± 0.06 MPa). Most hydrogel degraded after 12 w in vivo with only a little residues. Adipose-derived stem cells (ASCs) proliferated well after being seeded in hydrogel to form micro-mass at 14 days post-seeding. In vitro and in vivo angiogenesis, as well as in vitro osteogenesis illustrated the potential of the Dex-g-PCL hydrogel carrying ASCs toward vascularized bone tissue engineering. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1120-1131, 2019.

S53P4 Bioactive Glass Inorganic Ions for Vascularized Bone Tissue Engineering by Dental Pulp Pluripotent-Like Stem Cell Cocultures.

In this study, we proposed for the first time the use of inorganic ions dissolved from BaG in a cell coculture system to induce vascularized bone formation in vitro. For that, we used dental pulp pluripotent-like stem cells from a single individual source obtained in a minimally invasive extraction manner. Moreover, we carried out all the experiments under xeno-free conditions, allowing the extrapolation of the results to the development of clinically orientated applications. Overall, these results would provide a new promising system to promote the success and survival of bone tissue engineering constructs after implantation.

Novel standardized massive bone defect model in rats employing an internal eight-hole stainless steel plate for bone tissue engineering.

Massive bone defects are a challenge in orthopaedic research. Defective regeneration leads to bone atrophy, non-union of bone, and physical morbidity. Large animals are important models, however, production costs are high, nursing is complex, and evaluation methods are limited. A suitable laboratory animal model is required to explore the underlying molecular mechanism and cellular process of bone tissue engineering. We designed a stainless steel plate with 8 holes; the middle 2 holes were used as a guide to create a standardized critical size defect in the femur of anaesthetized rats. The plate was fixed to the bone using 6 screws, serving as an inner fixed bracket to secure a tricalcium phosphate implant seeded with green fluorescent protein-positive rat bone marrow mesenchymal stem cells within the defect. In some animals, we also grafted a vessel bundle into the lateral side of the implant, to promote vascularized bone tissue engineering. X-ray, microcomputed tomography, and histological analyses demonstrated the stainless steel plate resulted in a stable large segmental defect model in the rat femur. Vascularization significantly increased bone formation and implant degradation. Moreover, survival and expansion of green fluorescent protein-positive seeded cells could be clearly monitored in vivo at 1, 4, and 8weeks postoperation via fluorescent microscopy. This standardized large segmental defect model in a small animal may help to advance the study of bone tissue engineering. Furthermore, availability of antibodies and genetically modified rats could help to dissect the precise cellular and molecular mechanisms of bone repair.

Co-culture of Human Dental Pulp Stem Cells and Endothelial Cells Using Porous Biopolymer Microcarriers: A Feasibility Study for Bone Tissue Engineering.

Delivery of stem cells with osteogenesis while enabling angiogenesis is important for vascularized bone tissue engineering. Here a three-dimensional (3D) co-culture system of dental pulp stem cells (DPSCs) and endothelial cells (ECs) was designed using porous microcarriers, and the feasibility of applying to bone tissue engineering was investigated in vitro. Highly porous spherical microcarriers made of degradable biopolymers were prepared with sizes of hundreds of micrometers. The microcarriers loaded with DPSCs were co-cultured with ECs embedded in a hydrogel of type I collagen. An optimal co-culture medium that preserves the viability of ECs while stimulating the osteogenic differentiation of DPSCs was found to be a 10:1 of osteogenic medium:endothelial medium. The co-cultured constructs of DPSCs/ECs showed significantly higher level of alkaline phosphatase activity than the mono-cultured cells. Moreover, the expressions of genes related with osteogenesis and angiogenesis were significantly up-regulated by the co-cultures with respect to the mono-cultures. Results imply the interplay between ECs and DPSCs through the designed 3D co-culture models. The microcarrier-enabled co-cultured cell system is considered to be useful as an alternative tool for future vascularized bone tissue engineering.

Novel hiPSC-based tri-culture for pre-vascularization of calcium phosphate scaffold to enhance bone and vessel formation

Vascularization of tissue-engineered bone is a critical step in maintaining cell viability and advancing cell performance in vivo. In this study, a novel tri-culture system was developed to elicit pre-vascularization of calcium phosphate cement (CPC) scaffold in which human induced pluripotent stem cell-derived mesenchymal stem cells (hiPSMSCs) were seeded together with human umbilical vein endothelial cells (HUVECs) and pericytes. In a two-step methodology design, we first performed osteoinduction of the seeded hiPSMSCs on the CPC scaffold and then incorporated HUVECs and pericytes to the hiPSMSC-colonized CPC scaffold under a favorable culturing condition, with an objective to form a stable and functional capillary-like vascular network that sustained the engineered osseous tissue. The angiogenic and osteogenic effects of various culture strategies were studied and compared in nude rat model with cranial bone defects: (1) CPC scaffold alone (CPC control); (2) Pericyte-seeded CPC (CPC-pericytes); (3) HUVEC-seeded CPC (CPC-HUVECs); (4) hiPSMSC-seeded CPC (CPC-hiPSMSCs); (5) HUVECs co-cultured with hiPSMSCs on CPC (bi-culture group) and (6) HUVECs and pericytes co-cultured with hiPSMSCs on CPC (tri-culture group). After 12weeks, tri-culture group showed the highest amount of new bone (new bone area fraction of (45.3±2.7) %, p<0.05) and vessel formation (new blood vessel density of (50.7±3.8) vessels/mm2, p<0.05) in all groups. Our results demonstrated that the tri-culture strategy was effective in promoting angiogenesis and osteogenesis for bone tissue engineering.

Microengineered platforms for co-cultured mesenchymal stem cells towards vascularized bone tissue engineering.

Bone defects are common disease requiring thorough treatments since the bone is a complex vascularized tissue that is composed of multiple cell types embedded within an intricate extracellular matrix (ECM). For past decades, tissue engineering using cells, proteins, and scaffolds has been suggested as one of the promising approaches for effective bone regeneration. Recently, many researchers have been interested in designing effective platform for tissue regeneration by orchestrating factors involved in microenvironment around tissues. Among factors affecting bone formation, vascularization during bone development and after minor insults via endochondral and intramembranous ossification is especially critical for the long-term support for functional bone. In order to create vascularized bone constructs, the interactions between human mesenchymal stem cells (MSCs) and endothelial cells (ECs) have been investigated using both direct and indirect co-culture studies. Recently, various culture methods including micropatterning techniques, three dimensional scaffolds, and microfluidics have been developed to create micro-engineered platforms that mimic the nature of vascularized bone formation, leading to the creation of functional bone structures. This review focuses on MSCs co-cultured with endothelial cells and microengineered platforms to determine the underlying interplay between co-cultured MSCs and vascularized bone constructs, which is ultimately necessary for adequate regeneration of bone defects.

Dimethyloxaloylglycine Promotes the Angiogenic Activity of Mesenchymal Stem Cells Derived from iPSCs via Activation of the PI3K/Akt Pathway for Bone Regeneration.

The vascularization of tissue-engineered bone is a prerequisite step for the successful repair of bone defects. Hypoxia inducible factor-1α (HIF-1α) plays an essential role in angiogenesis-osteogenesis coupling during bone regeneration and can activate the expression of angiogenic factors in mesenchymal stem cells (MSCs). Dimethyloxaloylglycine (DMOG) is an angiogenic small molecule that can inhibit prolyl hydroxylase (PHD) enzymes and thus regulate the stability of HIF-1α in cells at normal oxygen tension. Human induced pluripotent stem cell-derived MSCs (hiPSC-MSCs) are promising alternatives for stem cell therapy. In this study, we evaluated the effect of DMOG on promoting hiPSC-MSCs angiogenesis in tissue-engineered bone and simultaneously explored the underlying mechanisms in vitro. The effectiveness of DMOG in improving the expression of HIF-1α and its downstream angiogenic genes in hiPSC-MSCs demonstrated that DMOG significantly enhanced the gene and protein expression profiles of angiogenic-related factors in hiPSC-MSCs by sustaining the expression of HIF-1α. Further analysis showed that DMOG-stimulated hiPSC-MSCs angiogenesis was associated with the phosphorylation of protein kinase B (Akt) and with an increase in VEGF production. The effects could be blocked by the addition of the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002. In a critical-sized calvarial defect model in rats, DMOG-treated hiPSC-MSCs showed markedly improved angiogenic capacity in the tissue-engineered bone, leading to bone regeneration. Collectively, the results indicate that DMOG, via activation of the PI3K/Akt pathway, promotes the angiogenesis of hiPSC-MSCs in tissue-engineered bone for bone defect repair and that DMOG-treated hiPSC-MSCs can be exploited as a potential therapeutic tool in bone regeneration.

Comment on “Microsurgical Techniques Used to Construct the Vascularized and Neurotized Tissue Engineered Bone”

We read with great interest the review article by Fan et al. [1] that discusses a range of microsurgical techniques used for vascularization and neurotization in bone tissue engineering. Not only the lack of vascularization but also the lack of neurotization within tissue engineered bone can lead to insufficient ossification resulting in treatment failure. Since we have been working in the research area of vascularized bone tissue engineering for many years, we are aware of the problem of generating an optimal prevascularized scaffold for large and poorly vascularized bone defects. One of the main obstacles remains an adequate vascularization and thus survival of implanted cells or, with regard to the manuscript by Fan et al., of nerve tracts, especially in the center of scaffolds. As mentioned by Fan et al. in a range of studies it could be demonstrated that sensory nerve innervation can contribute to bone integrity and bone formation [2–4]. Chen et al. demonstrate that sensory nerves could enhance the osteogenesis of tissue engineered bone. Sensory nerves or blood vessels were implanted with a scaffold directly in the defect site [3]. Fan et al. emphasize that an optimal support with oxygen and nutrients is of fundamental importance for the survival of the neurocytes. For optimization and constructing the vascularized and neurotized tissue engineered bone simultaneously both blood vessel and the nerve tract should be implanted together in the bone graft [1]. Another way for engineering vascularized tissue is the prevascularization of constructs distant to the injury site in unharmed areas of the body using the intrinsic vascularization with an arteriovenous (AV) loop that was firstly described by Erol and Spira [5]. After a sufficient prevascularization period osteogenic cells such as osteoblasts or nerve tracts can be implanted without being lost through inadequate supply of oxygen or nutrients. Tissue engineered constructs can afterwards be microsurgically transplanted in the defect site independent of the local vasculature. In a previously performed study in the rat AV loop model space holders were placed in the scaffold during the prevascularization period, leaving holes for subsequent injection of CFDA-labeled osteoblasts. A significantly higher number of CFDA-positive cells and a higher expression of bone specific markers were detected in the prevascularized scaffolds compared to control, indicating this technique to be well suited to support bone formation and the probable survival of implanted cells. Nevertheless, moving from bench to bedside, the large animal sheep AV loop model was established, and we were able to generate intrinsically vascularized tissue in a clinically relevant size (volume 15 cm³). For faster vascularization and consequently better nutritional support of implanted cells, we modified the AV loop model using a perforated chamber. Combining extrinsic and intrinsic vascularization, enhancement of angiogenesis could be demonstrated in the small and large animal [6] (Figure 1). Connection between both vascular pathways allows the microsurgical transplantation of the vascularized bone construct. Based on the results of our studies, we have previously used the AV loop successfully for the reconstruction of large bone defects in two patients [7]. Figure 1 Arteriovenous loop model sheep. ((a)-(b)) Implantation of a primary stable bone substitute within a perforated titanium chamber allowing combined intrinsic-extrinsic vascularization of the scaffold. (c) After explantation vessels were perfused with a ... To pave the way for further preclinical testing, the efficient implantation of a sensor nerve tract by Fan et al. should be proofed in a large animal and combined with the intrinsic-extrinsic large animal AV loop model. For minimizing hypoxic-dependent cell death, the nerve tract could be implanted after an adequate prevascularization period or close to AV loop vessels. We agree with Fan et al. that further studies must be performed to find efficient therapies for large and complicated bone defects, in order to make wide clinical application possible.

Stimulations of strontium-doped calcium polyphosphate for bone tissue engineering to protein secretion and mRNA expression of the angiogenic growth factors from endothelial cells in vitro

The vascularization of tissue-engineered bone is the key problem needed solving before application of tissue-engineered bone in clinical practice. Meanwhile, endothelial cells are the major and important source of seed cells in bone tissue engineering, and significant on promoting vascularization in tissue-engineered bone. Vascularization (namely angiogenesis) is a process mainly controlled by several angiogenic growth factors (VEGF, bFGF and MMP-2) which can be secreted by endothelial cells. Therefore, the research on the stimulations of SCPP to the secretion of the angiogenic growth factors from endothelial cells is very important. This study was performed to determine the ability of strontium-doped calcium polyphosphate (SCPP) to induce angiogenesis by detecting the protein secretion levels and mRNA expression of VEGF, bFGF and MMP-2 from cultured endothelial cells. As a control, we also researched the effect of HA on the mRNA expressions and protein secretion of angiogenic growth factors from cultured endothelial cells. We cultured endothelial cells with SCPP scaffolds containing various concentration of strontium and HA. The results obtained in the MTT and SEM tests indicated that endothelial cells on SCPP scaffold exhibited higher proliferation rate and were easy to get a good spread than them on CPP, the best state of growth and proliferation of cells could be observed on 8%SCPP. The results of ELISA demonstrated that the protein levels of VEGF, bFGF and MMP-2 from cultured endothelial cells increased with the increasing Sr doped in calcium polyphosphate in SCPP groups, the peaks appeared on 8%SCPP. All SCPP groups showed a better ability to stimulate the protein secretion of VEGF, bFGF and MMP-2 from endothelial cells relative to CPP group and HA group. The results of RT-PCR suggested that the 8%SCPP group exhibited a significantly higher mRNA expression of VEGF, bFGF and MMP-2 relative to CPP group and HA group. In conclusion, the results of this study demonstrated that 8%SCPP had obvious promotion for secretion and mRNA expression of angiogenic growth factors from cultured endothelial cells.

Contrasting Effects of Vasculogenic Induction Upon Biaxial Bioreactor Stimulation of Mesenchymal Stem Cells and Endothelial Progenitor Cells Cocultures in Three-Dimensional Scaffolds Under In Vitro and In Vivo Paradigms for Vascularized Bone Tissue Engineering

Clinical translation of bone tissue engineering approaches for fracture repair has been hampered by inadequate vascularization required for maintaining cell survival, skeletal regeneration, and remodeling. The potential of vasculature formation within tissue-engineered grafts depends on various factors, including an appropriate choice of scaffold and its microarchitectural design for the support of tissue ingrowth and vessel infiltration, vasculogenic potential of cell types and mechanostimulation on cells to enhance cytokine expression. Here, we demonstrated the effect of biomechanical stimulation on vasculogenic and bone-forming capacity of umbilical-cord-blood endothelial progenitor cells (UCB-EPC) and human fetal bone marrow-derived mesenchymal stem cell (hfMSC) seeded within macroporous scaffolds and cocultured dynamically in a biaxial bioreactor. Dynamically cultured EPC/hfMSC constructs generated greater mineralization and calcium deposition consistently over 14 days of culture (1.7-fold on day 14; p<0.05). However, in vitro vessel formation was not observed as compared to an extensive EPC-vessel network formed under static culture on day 7. Subsequent subcutaneous implantations in NOD/SCID mice showed 1.4-fold higher human:mouse cell chimerism (p<0.001), with a more even cellular distribution throughout the dynamically cultured scaffolds. In addition, there was earlier evidence of vessel infiltration into the scaffold and a trend toward increased ectopic bone formation, suggesting improved efficacy and cellular survival through early vascularization upon biomechanical stimulation. The integrative use of bioreactor culture systems with macroporous scaffolds and cocultured osteogenic and vasculogenic cells promotes maturation of EPC/hfMSC-scaffold grafts necessary for vascularized bone tissue engineering applications.

Vascularized Bone Tissue Engineering: Approaches for Potential Improvement

Significant advances have been made in bone tissue engineering (TE) in the past decade. However, classical bone TE strategies have been hampered mainly due to the lack of vascularization within the engineered bone constructs, resulting in poor implant survival and integration. In an effort toward clinical success of engineered constructs, new TE concepts have arisen to develop bone substitutes that potentially mimic native bone tissue structure and function. Large tissue replacements have failed in the past due to the slow penetration of the host vasculature, leading to necrosis at the central region of the engineered tissues. For this reason, multiple microscale strategies have been developed to induce and incorporate vascular networks within engineered bone constructs before implantation in order to achieve successful integration with the host tissue. Previous attempts to engineer vascularized bone tissue only focused on the effect of a single component among the three main components of TE (scaffold, cells, or signaling cues) and have only achieved limited success. However, with efforts to improve the engineered bone tissue substitutes, bone TE approaches have become more complex by combining multiple strategies simultaneously. The driving force behind combining various TE strategies is to produce bone replacements that more closely recapitulate human physiology. Here, we review and discuss the limitations of current bone TE approaches and possible strategies to improve vascularization in bone tissue substitutes.

Research advances in vascularized bone tissue engineering

随着材料学和分子生物学的发展,运用组织工程技术修复标准骨缺损成为当前的研究热点[1].但在大体积骨缺损修复重建时,组织工程骨的血管化问题是制约其最终治疗效果的关键因素.骨组织工程研究中血管化问题的重要性日益彰显,而当组织工程骨的厚度超过100μm时,在体内构建能为细胞提供氧、营养及去除代谢废物的功能性血管会面临重大挑战[2].因此,血运重建被认为是骨组织工程继种子细胞、支架材料、细胞因子三大要素之后的第四大要素.现就血管化组织工程骨研究的最新进展综述。