Haiying Yu

Improved tissue-engineered bone regeneration by endothelial cell mediated vascularization

Natural bone growth greatly depends on the precedent vascular network that supplies oxygen and essential nutrients and removes metabolites. Likewise, it is crucial for tissue-engineered bone to establish a vascular network that temporally precedes new bone formation, and spatially originates from within the graft. In order to recapitulate physiological skeletal development, we have developed a complex bone graft to repair rat bone defects. We have demonstrated that endothelial cells and osteoblasts (identified by cell morphology, quantification of specific marker antigens, calcium deposition and capillary-like growth) were able to differentiate and expand from donor rat bone marrow mononuclear cell populations. The biocompatibilities of poly-epsilon-caprolactone (PCL)-hydroxyapatite (HA) composites used for graft fabrication were evaluated at different component ratios to identify the optimal and support of cellular viability and functions for endothelial cells and osteoblasts. Using point-injection and low-pressure techniques, seeded endothelial cells and osteoblasts were able to assemble into microvascular networks and form bony matrix in grafts. The exogenous origination of these cells and their contribution to the vascularization and osteogenesis was confirmed using sex-mismatch implantation and Y chromosome tracking. By pre-seeding with endothelial cells, the resulting vascularization was able to promote osteogenesis, prevent ischemic necrosis and improve the mechanical properties in engineered bone tissue. Taken together, the results indicated that the integration of complex cell populations with composite scaffold materials provided an effective technique to improve osteogenesis in engineered bone graft. These findings suggest that hybrid grafts have great potential for clinical use to treat large bone defects.

Promotion of osteogenesis in tissue-engineered bone by pre-seeding endothelial progenitor cells-derived endothelial cells

In addition to a biocompatible scaffold and an osteogenic cell population, tissue‐engineered bone requires an appropriate vascular bed to overcome the obstacle of nutrient and oxygen transport in the 3D structure. We hypothesized that the addition of endothelial cells (ECs) may improve osteogenesis and prevent necrosis of engineered bone via effective neovascularization. Osteoblasts and ECs were differentiated from bone marrow of BALB/c mice, and their phenotypes were confirmed prior to implantation. Cylindrical porous polycaprolactone (PCL)‐hydroxyapatite (HA) scaffolds were synthesized. ECs were seeded on scaffolds followed by seeding of osteoblasts in the EC‐OB group. In the OB group, scaffolds were only seeded with osteoblasts. The cell‐free scaffolds were denoted as control group. A 0.4‐cm‐long segmental femur defect was established and replaced with the grafts. The grafts were evaluated histologically at 6 weeks postimplantation. In comparison with the OB group, the EC‐OB group resulted in a widely distributed capillary network, osteoid generated by osteoblasts and absent ischemic necroses. Pre‐seeding scaffold with ECs effectively promoted neovascularization in grafts, prevented the ischemic necrosis, and improved osteogenesis. The integration of bone marrow‐derived ECs and osteoblasts in porous scaffold is a useful strategy to achieve engineered bone.

Effect of porosity and pore size on microstructures and mechanical properties of poly-ε-caprolactone-hydroxyapatite composites

The influence of variant pore-size and porosity on the microstructure and the mechanical properties of poly-epsilon-caprolactone (PCL) and hydroxyapatite (HA) composite were examined for an optimal scaffold in bone tissue engineering. Three various amounts of sodium chloride (NaCl, as porogens) with two distinct particle size ranges (212-355 mum and 355-600 mum) were blended into PCL and HA mixture, followed by a leaching technique to generate PCL-HA scaffolds with various pores and porosity. The porosities of the scaffolds were correlated with the porogen size and concentration. The morphological properties of the resulting scaffolds were assessed by micro-computerized tomography (muCT), scanning electron microscopy (SEM), and energy dispersive X-ray analysis (EDX). Extensive PCL-HA pore interconnections with thinner pore walls were present in scaffolds with higher concentration (4:1 w/w) and larger particulate of porogen used in the fabrication process. Embedding of HA particles in the scaffold resulted in rough surfaces on the composites. Instron actuator testing indicated that the tensile strengths and Youngs moduli of scaffolds were influenced by both the porosities and pore sizes of the scaffold. It was apparent that increasing the concentration of porogen compromised the mechanical properties; and a larger porogen particle size led to increased tensile strength but a reduction in Youngs modulus. Overall, the data indicated that modification of the concentration and particle size of porogen altered the porous features and mechanical strength of HA-PCL scaffolds. This provided means to manipulate the properties of biocompatible cell-supporting scaffolds for use as bone graft substitutes.

The effect of osteoprotegerin gene modification on wear debris-induced osteolysis in a murine model of knee prosthesis failure

Using an in vivo adeno-associated virus (AAV)-mediated gene transfer technique, this study evaluated the therapeutic effects of an osteoprotegerin (OPG) transgene against orthopaedic wear debris-induced osteolysis in a long-term murine model. A titanium pin was surgically implanted into proximal tibia of Balb/c mice to mimic a weight-bearing knee arthroplasty, followed by an intra-articular challenge with Ti particles to provoke periprosthetic inflammation and osteolysis. rAAV-hOPG or AAV-LacZ vectors were injected into the prosthetic joint at 3 weeks post-op. The tissues were harvested at 2, 4, 12 and 24 weeks after transduction for histological and molecular analyses. Successful transgene expression at the local site was confirmed by real-time PCR and ELISA. Inflammatory pseudo-membranes were ubiquitously present at the interface between the Ti implant and the surrounding bone in both LacZ and virus-free control groups, while soft tissue was only observed sporadically at the bone-implant interface in the OPG group. A significant reduction in TRAP+ osteoclast numbers was observed in the OPG treatment group. MicroCT assessment indicated a marked reversal in the loss of peri-implant bone mineral density (BMD) in the OPG-transduced group, when compared with the LacZ and virus-free controls. Further, OPG gene modification appeared to reduce local bone collagen loss by a mean of 40%. Real-time PCR examination confirmed that in vivo OPG gene transfer dramatically influenced the periprosthetic tissue gene expression profiles by diminishing the mRNA expression of TNF, IL-1, CPK and RANKL. There were no transgene-associated toxic effects apparent during the experiment, and the PCR detection of transgenes in remote organs such as lungs, kidneys, liver, and muscle of contralateral limb were consistently negative. Overall, rAAV-mediated OPG gene transfer effectively reversed Ti-particle-induced bone resorption in this experimental model. The therapeutic effects may be due to the blockage of local osteoclastogenesis and possibly the down-regulation of RANKL expression.

Cell-based osteoprotegerin therapy for debris-induced aseptic prosthetic loosening on a murine model

Exogenous osteoprotegerin (OPG) gene modification appears a therapeutic strategy for osteolytic aseptic loosening. The feasibility and efficacy of a cell-based OPG gene delivery approach were investigated using a murine model of knee prosthesis failure. A titanium pin was implanted into mouse proximal tibia to mimic a weight-bearing knee arthroplasty, followed by titanium particles challenge to induce periprosthetic osteolysis. Mouse fibroblast-like synoviocytes were transduced in vitro with either AAV-OPG or AAV-LacZ before transfused into the osteolytic prosthetic joint 3 weeks post surgery. Successful transgene expression at the local site was confirmed 4 weeks later after killing. Biomechanical pullout test indicated a significant restoration of implant stability after the cell-based OPG gene therapy. Histology revealed that inflammatory pseudo-membranes existed ubiquitously at bone–implant interface in control groups, whereas only observed sporadically in OPG gene-modified groups. Tartrate-resistant acid phosphatase+osteoclasts and tumor necrosis factor α, interleukin-1β, CD68+ expressing cells were significantly reduced in periprosthetic tissues of OPG gene-modified mice. No transgene dissemination or tumorigenesis was detected in remote organs and tissues. Data suggest that cell-based ex vivo OPG gene therapy was comparable in efficacy with in vivo local gene transfer technique to deliver functional therapeutic OPG activities, effectively halted the debris-induced osteolysis and regained the implant stability in this model.

Biocompatibility of Poly-ε-caprolactone- hydroxyapatite composite on mouse bone marrow- derived osteoblasts and endothelial cells

BackgroundTissue-engineered bone may be developed by seeding the cells capable of both osteogenesis and vascularization on biocompatible composite scaffolds. The current study investigated the performance of mice bone marrow-derived osteogenic cells and endothelial cells as seeded on hydroxyapatite (HA) and poly-ε-caprolactone (PCL) composite scaffolds.MethodsMononuclear cells were induced to osteoblasts and endothelial cells respectively, which were defined by the expression of osteocalcin, alkaline phosphatase (ALP), and deposits of calcium-containing crystal for osteoblasts, or by the expression of vascular endothelial growth factor receptor-2 (VEGFR-2) and von Willebrand factor (vWF), and the formation of a capillary network in Matrigel™ for endothelial cells. Both types of cell were seeded respectively on PCL-HA scaffolds at HA to PCL weight ratio of 1:1, 1:4, or 0:1 and were evaluated using scanning electron microscopy, ALP activity (of osteoblasts) and nitric oxide production (of endothelial cells) plus the assessment of cell viability.ResultsThe results indicated that HA led to a positive stimulation of osteoblasts viability and ALP activity, while HA showed less influence on endothelial cells viability. An elevated nitric oxide production of endothelial cells was observed in HA-containing group.ConclusionSupplement of HA into PCL improved biocompatible for bone marrow-derived osteoblasts and endothelial cells. The PCL-HA composite integrating with two types of cells may provide a useful system for tissue-engineered bone grafts with vascularization.

High VEGF with rapid growth and early metastasis in a mouse osteosarcoma model

A murine model of osteosarcoma was developed to investigate the association between the expression of VEGF and the progression of osteosarcoma. Two human osteosarcoma cell lines with distinct VEGF expressions were introduced into proximal tibiae of immuno-deficient SCID mice, either by direct injection through the cortical bone or surgical exposing and drilling on the tibial metaphysis to seed tumor cells. Bone tumors were obvious on microCT within 4 weeks following osteosarcoma cell inoculation through surgical delivery. In contrast, direct injection without drilling often resulted in periosteal tumors. Although neoplasms were developed regardless of VEGF levels, orthotopic tumors derived from high VEGF-expressing cells were detected 2 weeks earlier on CT images than the ones from VEGF negative cells. At sacrifice, high VEGF tumors were distinctively larger in size and more frequently invaded the adjacent bone tissue. Multiple metastatic lesions were found in all the lung tissues at 8 weeks from high VEGF group, whereas only 1 of 7 VEGF negative tumors exhibited pulmonary metastasis. Overall, this model developed with the surgical tumor cell delivery results in histological and radiographic features more consistent with primary osteosarcoma. Interestingly, VEGF expression correlates with the early establishment, rapid tumor growth, and the development of pulmonary metastasis.

Polymethylmethacrylate and titanium alloy particles activate peripheral monocytes during periprosthetic inflammation and osteolysis

We investigated the interactions of particulate PMMA or titanium alloy, patient blood monocytes, and periprosthetic tissues using a SCID‐hu model of aseptic loosening. Periprosthetic tissues and bone chips obtained at revision surgery for loosening were transplanted into muscles of SCID mice. Peripheral blood monocytes (PBMCs) isolated from the same donors were fluorescently labeled and co‐cultured with PMMA or Ti‐6Al‐4V particles before intraperitoneal injection. Control mice with periprosthetic tissue or non‐inflammatory ligament xenografts received naive PBMCs transfusion. Mice were euthanized 2 weeks after PBMC transfusion. The human tissues were well accepted in SCID mice. Transfused fluorescent‐labeled PBMCs were markedly accumulated in transplanted periprosthetic tissues. Multinucleated osteoclast‐like cells were commonly seen within retrieved xenograft tissue, and focal bone erosions were ubiquitous. Total cell densities and CD68+ cells within the xenograft were significantly increased in mice transfused with PMMA and Ti‐provoked PBMCs compared to the naïve PBMC animals (p < 0.05). Immunohistochemical staining identified much stronger positive IL‐1 and TNF stains in xenografts from either PMMA or Ti‐stimulated monocytes transfusion groups (p < 0.05). TRAP+ cells were found around bone chips in both activated‐PBMCs groups, although markedly more aggregated TRAP+ cells in the PMMA‐challenged group than in the titanium group (p < 0.05). MicroCT assessment confirmed the significant decrease of bone mineral density in chips interacted with activated‐monocytes/osteoclasts. In conclusion, PMMA or titanium particles readily activate peripheral monocytes and promote the cell trafficking to the debris‐containing prosthetic tissues. Particles‐provoked PBMCs participated in and promoted the local inflammatory process, osteoclastogenesis, and bone resorption.