Doctor of Philosophy, Sichuan University (2008)
Yunzhi Peter Yang, Postdoctoral Faculty Sponsor
Osteogenetic microenvironment is a complex constitution in which extracellular matrix (ECM) molecules, stem cells and growth factors each interact to direct the coordinate regulation of bone tissue development. Importantly, angiogenesis improvement and revascularization are critical for osteogenesis during bone tissue regeneration processes. In this study, we developed a three-dimensional (3D) multi-scale system model to study cell response to growth factors released from a 3D biodegradable porous calcium phosphate (CaP) scaffold. Our model reconstructed the 3D bone regeneration system and examined the effects of pore size and porosity on bone formation and angiogenesis. The results suggested that scaffold porosity played a more dominant role in affecting bone formation and angiogenesis compared with pore size, while the pore size could be controlled to tailor the growth factor release rate and release fraction. Furthermore, a combination of gradient VEGF with BMP2 and Wnt released from the multi-layer scaffold promoted angiogenesis and bone formation more readily than single growth factors. These results demonstrated that the developed model can be potentially applied to predict vascularized bone regeneration with specific scaffold and growth factors.
View details for DOI 10.1016/j.biomaterials.2013.03.015
View details for Web of Science ID 000319630000008
View details for PubMedID 23566802
The use of biodegradable beta-tricalcium phosphate (?-TCP) scaffolds holds great promise for bone tissue engineering. However, the effects of ?-TCP on bone and endothelial cells are not fully understood. This study aimed to investigate cell proliferation and differentiation of mono- or co-cultured human-bone-marrow-derived mesenchymal stem cells (hBMSCs) and human-umbilical-vein endothelial cells (HUVECs) on a three-dimensional porous, biodegradable ?-TCP scaffold. In co-culture studies, the ratios of hBMSCs:HUVECs were 5:1, 1:1 and 1:5. Cellular morphologies of HUVECs, hBMSCs and co-cultured HUVECs/hBMSCs on the ?-TCP scaffolds were monitored using confocal and scanning electron microscopy. Cell proliferation was monitored by measuring the amount of double-stranded DNA (dsDNA) whereas hBMSC and HUVEC differentiation was assessed using the osteogenic and angiogenic markers, alkaline phosphatase (ALP) and PECAM-1 (CD31), respectively. Results show that HUVECs, hBMSCs and hBMSCs/HUVECs adhered to and proliferated well on the ?-TCP scaffolds. In monoculture, hBMSCs grew faster than HUVECs on the ?-TCP scaffolds after 7 days, but HUVECs reached similar levels of proliferation after 14 days. In monoculture, ?-TCP scaffolds promoted ALP activities of both hBMSCs and HUVECs when compared to those grown on tissue culture well plates. ALP activity of cells in co-culture was higher than that of hBMSCs in monoculture. Real-time polymerase chain reaction results indicate that runx2 and alp gene expression in monocultured hBMSCs remained unchanged at days 7 and 14, but alp gene expression was significantly increased in hBMSC co-cultures when the contribution of individual cell types was not distinguished.
View details for DOI 10.1016/j.actbio.2012.08.008
View details for Web of Science ID 000313376900047
View details for PubMedID 22902820
Extracellular matrix (ECM) serves a key role in cell migration, attachment, and cell development. Here we report that ECM derived from human umbilical vein endothelial cells (HUVEC) promoted osteogenic differentiation of human bone marrow mesenchymal stem cells (hMSC). We first produced an HUVEC-derived ECM on a three-dimensional (3D) beta-tricalcium phosphate (?-TCP) scaffold by HUVEC seeding, incubation, and decellularization. The HUVEC-derived ECM was then characterized by SEM, FTIR, XPS, and immunofluorescence staining. The effect of HUVEC-derived ECM-containing ?-TCP scaffold on hMSC osteogenic differentiation was subsequently examined. SEM images indicate a dense matrix layer deposited on the surface of struts and pore walls. FTIR and XPS measurements show the presence of new functional groups (amide and hydroxyl groups) and elements (C and N) in the ECM/?-TCP scaffold when compared to the ?-TCP scaffold alone. Immunofluorescence images indicate that high levels of fibronectin and collagen IV and low level of laminin were present on the scaffold. ECM-containing ?-TCP scaffolds significantly increased alkaline phosphatase (ALP) specific activity and up-regulated expression of osteogenesis-related genes such as runx2, alkaline phosphatase, osteopontin and osteocalcin in hMSC, compared to ?-TCP scaffolds alone. This increased effect was due to the activation of MAPK/ERK signaling pathway since disruption of this pathway using an ERK inhibitor PD98059 results in down-regulation of these osteogenic genes. Cell-derived ECM-containing calcium phosphate scaffolds is a promising osteogenic-promoting bone void filler in bone tissue regeneration.
View details for DOI 10.1016/j.biomaterials.2012.06.061
View details for Web of Science ID 000308269600010
View details for PubMedID 22795852
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.
View details for DOI 10.1089/ten.teb.2012.0012
View details for Web of Science ID 000309516500003
View details for PubMedID 22765012
The purpose of this study was to develop and characterize a chitosan gel/gelatin microsphere (MSs) dual delivery system for sequential release of bone morphogenetic protein-2 (BMP-2) and insulin-like growth factor-1 (IGF-1) to enhance osteoblast differentiation in vitro. We made and characterized the delivery system based on its degree of cross-linking, degradation, and release kinetics. We also evaluated the cytotoxicity of the delivery system and the effect of growth factors on cell response using pre-osteoblast W-20-17 mouse bone marrow stromal cells. IGF-1 was first loaded into MSs, and then the IGF-1-containing MSs were encapsulated into the chitosan gel which contained BMP-2. Cross-linking of gelatin with glyoxal via Schiff bases significantly increased thermal stability and decreased the solubility of the MSs, leading to a significant decrease in the initial release of IGF-1. Encapsulation of the MSs into the chitosan gel generated polyelectrolyte complexes by intermolecular interactions, which further affected the release kinetics of IGF-1. This combinational delivery system provided an initial release of BMP-2 followed by a slow and sustained release of IGF-1. Significantly greater alkaline phosphatase activity was found in W-20-17 cells treated with the sequential delivery system compared with other treatments (P<0.05) after a week of culture.
View details for DOI 10.1016/j.actbio.2012.01.009
View details for Web of Science ID 000302989700012
View details for PubMedID 22293583
We investigated the effect of sustained release of bone morphogenetic protein-2 (BMP-2) from an injectable chitosan gel on osteoblastic differentiation in vitro. We first characterized the release profile of BMP-2 from the gels, and then examined the cellular responses of preosteoblast mouse stromal cells (W-20-17) and human embryonic palatal mesenchymal (HEPM) cells to BMP-2. The release profiles of different concentrations of BMP-2 exhibited sustained releases (41% for 2 ng/mL and 48% for 20 ng/mL, respectively) from the chitosan gels over a three-week period. Both cell types cultured in the chitosan gels were viable and significantly proliferated for 3 days (p < 0.05). Chitosan gels loaded with BMP-2 enhanced ALP activity of W-20-17 by 3.6-fold, and increased calcium mineral deposition of HEPM by 2.8-fold at 14 days of incubation, compared to control groups initially containing the same amount of BMP-2. In addition, schitosan gels loaded with BMP-2 exhibited significantly greater osteocalcin synthesis of W-20-17 at seven days, and of HEPM at both 7 and 14 days compared with the control groups (p<0.05). This study suggests that the enhanced effects of BMP-2 released from chitosan gels on cell differentiation and mineralization are species and cell type dependent.
View details for DOI 10.1002/jbm.b.31909
View details for PubMedID 21905214
Extracellular matrix (ECM) comprises a rich meshwork of proteins and proteoglycans, which not only contains biological cues for cell behavior, but is also a reservoir for binding growth factors and controlling their release. Here we aimed to create a suitable bony microenvironment with cell-derived ECM and biodegradable ?-tricalcium phosphate (?-TCP). More specifically, we investigated whether the ECM produced by bone marrow-derived mesenchymal stem cells (hBMSC) on a ?-TCP scaffold can bind bone morphogenetic protein-2 (BMP-2) and control its release in a sustained manner, and further examined the effect of ECM and the BMP-2 released from ECM on cell behaviors. The ECM was obtained through culturing the hBMSC on a ?-TCP porous scaffold and performing decellularization and sterilization. SEM, XPS, FTIR, and immunofluorescent staining results indicated the presence of ECM on the ?-TCP and the amount of ECM increased with the incubation time. BMP-2 was loaded onto the ?-TCP with and without ECM by immersing the scaffolds in the BMP-2 solution. The loading and release kinetics of the BMP-2 on the ?-TCP/ECM were significantly slower than those on the ?-TCP. The ?-TCP/ECM exhibited a sustained release profile of the BMP-2, which was also affected by the amount of ECM. This is probably because the ?-TCP/ECM has different binding mechanisms with BMP-2. The ?-TCP/ECM promoted cell proliferation. Furthermore, the BMP-2-loaded ?-TCP/ECM stimulated reorganization of the actin cytoskeleton, increased expression of alkaline phosphatase and calcium deposition by the cells compared to those without BMP-2 loading and the ?-TCP with BMP-2 loading.
View details for DOI 10.1016/j.biomaterials.2011.05.015
View details for Web of Science ID 000292904100018
View details for PubMedID 21632105
Porous ?-tricalcium phosphate (?-TCP) has been used for bone repair and replacement in clinics due to its excellent biocompatibility, osteoconductivity, and biodegradability. However, the application of ?-TCP has been limited by its brittleness. Here, we demonstrated that an interconnected porous ?-TCP scaffold infiltrated with a thin layer of poly (lactic-co-glycolic acid) (PLGA) polymer showed improved mechanical performance compared to an uncoated ?-TCP scaffold while retaining its excellent interconnectivity and biocompatibility. The infiltration of PLGA significantly increased the compressive strength of ?-TCP scaffolds from 2.90 MPa to 4.19 MPa, bending strength from 1.46 MPa to 2.41 MPa, and toughness from 0.17 MPa to 1.44 MPa, while retaining an interconnected porous structure with a porosity of 80.65%. These remarkable improvements in the mechanical properties of PLGA-coated ?-TCP scaffolds are due to the combination of the systematic coating of struts, interpenetrating structural characteristics, and crack bridging. The in vitro biological evaluation demonstrated that rat bone marrow stromal cells (rBMSCs) adhered well, proliferated, and expressed alkaline phosphatase (ALP) activity on both the PLGA-coated ?-TCP and the ?-TCP. These results suggest a new strategy for fabricating interconnected macroporous scaffolds with significantly enhanced mechanical strength for potential load-bearing bone tissue regeneration.
View details for PubMedID 21892228