Xinquan Jiang

Antibacterial activity and increased bone marrow stem cell functions of Zn-incorporated TiO2 coatings on titanium

In this work, zinc was incorporated into TiO2 coatings on titanium by plasma electrolytic oxidation to obtain the implant with good bacterial inhibition ability and bone-formability. The porous and nanostructured Zn-incorporated TiO2 coatings are built up from pores smaller than 5 μm and grains 20-100 nm in size, in which the element Zn exists as ZnO. The results obtained from the antibacterial studies suggest that the Zn-incorporated TiO2 coatings can greatly inhibit the growth of both Staphylococcus aureus and Escherichia coli, and the ability to inhibit bacteria can be improved by increasing the Zn content in the coatings. Moreover, the in vitro cytocompatibility evaluation demonstrates that the adhesion, proliferation and differentiation of rat bone marrow stem cells (bMSC) on Zn-incorporated coatings are significantly enhanced compared with Zn-free coating and commercially pure Ti plate, and no cytotoxicity appeared on any of the Zn-incorporated TiO2 coatings. Moreover, bMSC express higher level of alkaline phosphatase activity on Zn-incorporated TiO2 coatings and are induced to differentiate into osteoblast cells. The better antibacterial activity, cytocompatibility and the capability to promote bMSC osteogenic differentiation of Zn-incorporated TiO2 coatings may be attributed to the fact that Zn ions can be slowly and constantly released from the coatings. In conclusion, innovative Zn-incorporated TiO2 coatings on titanium with excellent antibacterial activity and biocompatibility are promising candidates for orthopedic and dental implants.

Mandibular Repair in Rats with Premineralized Silk Scaffolds and BMP-2-modified bMSCs

Premineralized silk fibroin protein scaffolds (mSS) were prepared to combine the osteoconductive properties of biological apatite with aqueous-derived silk scaffold (SS) as a composite scaffold for bone regeneration. The aim of present study was to evaluate the effect of premineralized silk scaffolds combined with bone morphogenetic protein-2 (BMP-2) modified bone marrow stromal cells (bMSCs) to repair mandibular bony defects in a rat model. bMSCs were expanded and transduced with adenovirus AdBMP-2, AdLacZ gene in vitro. These genetically modified bMSCs were then combined with premineralized silk scaffolds to form tissue-engineered bone. Mandibular repairs with AdBMP-2 transduced bMSCs/mSS constructs were compared with those treated with AdLacZ-transduced bMSCs/mSS constructs, native (nontransduced) bMSCs/mSS constructs and mSS alone. Eight weeks after post-operation, the mandibles were explanted and evaluated by radiographic observation, micro-CT, histological analysis and immunohistochemistry. The presence of BMP-2 gene enhanced tissue-engineered bone in terms of the most new bone formed and the highest local bone mineral densities (BMD) found. These results demonstrated that premineralized silk scaffold could serve as a potential substrate for bMSCs to construct tissue-engineered bone for mandibular bony defects. BMP-2 gene therapy and tissue engineering techniques could be used in mandibular repair and bone regeneration.

The synergistic effect of hierarchical micro/nano-topography and bioactive ions for enhanced osseointegration

Both surface chemistry and topography have significant influence on good and fast osseointegration of biomedical implants; the main goals in orthopeadic, dental and maxillofacial surgeries. A surface modification strategy encompassing the use of bioactive trace elements together with surface micron/nano-topographical modifications was employed in this study in an attempt to enhance the osseointegration of Ti alloy (Ti-6Al-4V), a commonly used implant. Briefly, we developed strontium-substituted hardystonite (Sr-HT) ceramic coating with a hierarchical topography where the nanosized grains were superimposed in the micron-rough coating structure. Its ability to induce new bone formation was evaluated by an in vivo animal model (beagle dogs). Hardystonite (HT), classic hydroxyapatite (HAp) coated and uncoated Ti-alloy implants were parallelly investigated for comparison. In addition, we investigated the effects of surface topography and the dissolution products from the coatings on the in vitro bioactivity using canine bone marrow mesenchymal stem cells (BMMSCs) cultured on the implant surface as well as using extracts of the coated implants. Micro-CT evaluation, histological observations, biomechanical test (push-out test) and sequential fluorescent labeling and histomorphometrical analysis consistently demonstrated that our developed Sr-HT-coated Ti-alloy implants have the highest osseointegration, while the uncoated implants had the lowest. The osseointegration ability of HAp-coated Ti alloy was inferior to that seen for HT- and Sr-HT-coated Ti alloy. We demonstrated that the dissolution products, particularly strontium (Sr) from the Sr-HT-coated implants, enhanced the ALP activity and in vitro mineralization ability, while the micro/nano-topography was more related to the promotion of cell adhesion. Those results suggest that our developed Sr-HT coatings have the potential for future use as coatings for orthopedic/dental and maxillofacial devices.

Enhanced osteoporotic bone regeneration by strontium-substituted calcium silicate bioactive ceramics

The regeneration capacity of the osteoporotic bones is generally lower than that of the normal bones. Current methods of bone defect treatment for osteoporosis are not always satisfactory. Recent studies have shown that the silicate based biomaterials can stimulate osteogenesis and angiogenesis due to the silicon (Si) ions released from the materials, and enhance bone regeneration in vivo. Other studies showed that strontium (Sr) plays a distinct role on inhibiting bone resorption. Based on the hypothesis that the combination of Si and Sr may have synergetic effects on osteoporotic bone regeneration, the porous Sr-substituted calcium silicate (SrCS) ceramic scaffolds combining the functions of Sr and Si elements were developed with the goals to promote osteoporotic bone defect repair. The effects of the ionic extract from SrCS on osteogenic differentiation of bone marrow mesenchymal stem cells derived from ovariectomized rats (rBMSCs-OVX), angiogenic differentiation of human umbilical vein endothelial cells (HUVECs) were investigated. The in vitro results showed that Sr and Si ions released from SrCS enhanced cell viability, alkaline phosphatase (ALP) activity, and mRNA expression levels of osteoblast-related genes of rBMSCs-OVX and expression of vascular endothelial growth factor (VEGF) without addition of extra osteogenic and angiogenic reagents. The activation in extracellular signal-related kinases (ERK) and p38 signaling pathways were observed in rBMSCs-OVX cultured in the extract of SrCS, and these effects could be blocked by ERK inhibitor PD98059, and P38 inhibitor SB203580, respectively. Furthermore, the ionic extract of SrCS stimulated HUVECs proliferation, differentiation and angiogenesis process. The in vivo experiments revealed that SrCS dramatically stimulated bone regeneration and angiogenesis in a critical sized OVX calvarial defect model, and the enhanced bone regeneration might be attributed to the modulation of osteogenic differentiation of endogenous mesenchymal stem cells (MSCs) and the inhibition of osteoclastogenesis, accompanying with the promotion of the angiogenic activity of endothelial cells (ECs).

miR-30 Family Members Negatively Regulate Osteoblast Differentiation

Background: microRNAs (miRNAs) are closely related to osteogenesis. Results: miR-30 family members (miR-30a, -30b, -30c, and -30d) mediate the inhibition of osteogenesis by targeting Smad1 and Runx2. Conclusion: miR-30 family members are key negative regulators of BMP-2-mediated osteogenic differentiation. Significance: These findings may provide new insights into understanding the regulatory role of miRNAs in the process of osteogenic differentiation. miRNAs are endogenously expressed 18- to 25-nucleotide RNAs that regulate gene expression through translational repression by binding to a target mRNA. Recently, it has been indicated that miRNAs are closely related to osteogenesis. Our previous data suggested that miR-30 family members might be important regulators during the biomineralization process. However, whether and how they modulate osteogenic differentiation have not been explored. In this study, we demonstrated that miR-30 family members negatively regulate BMP-2-induced osteoblast differentiation by targeting Smad1 and Runx2. Evidentially, overexpression of miR-30 family members led to a decrease of alkaline phosphatase activity, whereas knockdown of them increased the activity. Then bioinformatic analysis identified potential target sites of the miR-30 family located in the 3′ untranslated regions of Smad1 and Runx2. Western blot analysis and quantitative RT-PCR assays demonstrated that miR-30 family members inhibit Smad1 gene expression on the basis of repressing its translation. Furthermore, dual-luciferase reporter assays confirmed that Smad1 is a direct target of miR-30 family members. Rescue experiments that overexpress Smad1 and Runx2 significantly eliminated the inhibitory effect of miR-30 on osteogenic differentiation and provided strong evidence that miR-30 mediates the inhibition of osteogenesis by targeting Smad1 and Runx2. Also, the inhibitory effects of the miR-30 family were validated in mouse bone marrow mesenchymal stem cells. Therefore, our study uncovered that miR-30 family members are key negative regulators of BMP-2-mediated osteogenic differentiation.

Blood vessel formation in the tissue-engineered bone with the constitutively active form of HIF-1α mediated BMSCs

The successful clinical outcome of the implanted tissue-engineered bone is dependent on the establishment of a functional vascular network. A gene-enhanced tissue engineering represents a promising approach for vascularization. Our previous study indicated that hypoxia-inducible factor-1α (HIF-1α) can up-regulate the expression of vascular endothelial growth factor (VEGF) and stromal-derived factor 1 (SDF-1) in bone mesenchymal stem cells (BMSCs). The angiogenesis is a co-ordinated process that requires the participation of multiple angiogenic factors. To further explore the angiogenic effect of HIF-1α mediated stem cells, in this study, we systematically evaluated the function of HIF-1α in enhancing BMSCs angiogenesis in vitro and in vivo. A constitutively active form of HIF-1α (CA5) was inserted into a lentivirus vector and transduced into BMSCs, and its effect on vascularization and vascular remodeling was further evaluated in a rat critical-sized calvarial defects model with a gelatin sponge (GS) scaffold. The expression of the key angiogenic factors including VEGF, SDF-1, basic fibroblast growth factor (bFGF), placental growth factor (PLGF), angiopoietin 1 (ANGPT1), and stem cell factor (SCF) at both mRNAs and proteins levels in BMSCs were significantly enhanced by HIF-1α overexpression compared to the in vitro control group. In addition, HIF-1α-over expressing BMSCs showed dramatically improved blood vessel formation in the tissue-engineered bone as analyzed by photography of specimen, micro-CT, and histology. These data confirm the important role of HIF-1α in angiogenesis in tissue-engineered bone. Improved understanding of the mechanisms of angiogenesis may offer exciting therapeutic opportunities for vascularization, vascular remodeling, and bone defect repair using tissue engineering strategies in the future.

Apatite-coated silk fibroin scaffolds to healing mandibular border defects in canines

Tissue engineering has become a new approach for repairing bony defects. Highly porous osteoconductive scaffolds perform the important role for the success of bone regeneration. By biomimetic strategy, apatite-coated porous biomaterial based on silk fibroin scaffolds (SS) might provide an enhanced osteogenic environment for bone-related outcomes. To assess the effects of apatite-coated silk fibroin (mSS) biomaterials for bone healing as a tissue engineered bony scaffold, we explored a tissue engineered bony graft using mSS seeded with osteogenically induced autologous bone marrow stromal cells (bMSCs) to repair inferior mandibular border defects in a canine model. The results were compared with those treated with bMSCs/SS constructs, mSS alone, SS alone, autologous mandibular grafts and untreated blank defects. According to radiographic and histological examination, new bone formation was observed from 4 weeks post-operation, and the defect site was completely repaired after 12 months for the bMSCs/mSS group. In the bMSCs/SS group, new bone formation was observed with more residual silk scaffold remaining at the center of the defect compared with the bMSCs/mSS group. The engineered bone with bMSCs/mSS achieved satisfactory bone mineral densities (BMD) at 12 months post-operation close to those of normal mandible (p>0.05). The quantities of newly formed bone area for the bMSCs/mSS group was higher than the bMSCs/SS group (p<0.01), but no significant differences were found when compared with the autograft group (p>0.05). In contrast, bony defects remained in the center with undegraded silk fibroin scaffold and fibrous connective tissue, and new bone only formed at the periphery in the groups treated with mSS or SS alone. The results suggested that apatite-coated silk fibroin scaffolds combined with bMSCs could be successfully used to repair mandibular critical size border defects and the premineralization of these porous silk fibroin protein scaffolds provided an increased osteoconductive environment for bMSCs to regenerate sufficient new bone tissue.

Tailoring the Nanostructured Surfaces of Hydroxyapatite Bioceramics to Promote Protein Adsorption, Osteoblast Growth, and Osteogenic Differentiation

To promote and understand the biological responses of the implant via nanostructured surface design is essential for the development of bioactive bone implants. However, the control of the surface topography of the bioceramics in nanoscale is a big challenge because of their brittle property. Herein, the hydroxyapatite (HAp) bioceramics with distinct nanostructured topographies were fabricated via hydrothermal treatment using α-tricalcium phosphate ceramic as hard-template under different reaction conditions. HAp bioceramics with nanosheet, nanorod and micro-nanohybrid structured surface in macroscopical size were obtained by controlling the composition of the reaction media. Comparing with the traditional sample with flat and dense surface, the fabricated HAp bioceramics with hierarchical 3D micro-nanotextured surfaces possessed higher specific surface area, which selectively enhanced adsorption of specific proteins including Fn and Vn in plasma, and stimulated osteoblast adhesion, growth, and osoteogenic differentiation. In particular, the biomimetic features of the hierarchical micro-nanohybrid surface resulted in the best ability for simultaneous enhancement of protein adsorption, osteoblast proliferation, and differentiation. The results suggest that the hierarchical micro-nanohybrid topography might be one of the critical factors to be considered in the design of functional bone grafts.

Effect of nano-structured bioceramic surface on osteogenic differentiation of adipose derived stem cells

Tissue engineering strategies to construct vascularized bone grafts potentially revolutionize the treatment of massive bone loss. The surface topography of the grafts plays critical roles on bone regeneration, while adipose derived stem cells (ASCs) are known for their capability to promote osteogenesis and angiogenesis when applied to bone defects. In the present study, the effects of hydroxyapatite (HAp) bioceramic scaffolds with nanosheet, nanorod, and micro-nano-hybrid (the hybrid of nanorod and microrod) surface topographies on attachment, proliferation and osteogenic differentiation, as well as the expression of angiogenic factors of rat ASCs were systematically investigated. The results showed that the HAp bioceramic scaffolds with the micro-/nano-topography surfaces significantly enhanced cell attachment and viability, alkaline phosphatase (ALP) activity, and mRNA expression levels of osteogenic markers and angiogenic factors of ASCs. More importantly, the biomimetic feature of the hierarchical micro-nano-hybrid surface topography showed the highest stimulatory effect. The activation in Akt signaling pathway was observed in ASCs cultured on HAp bioceramics with nanorod, and micro-nano-hybrid surface topographies. Moreover, these induction effects could be repressed by Akt signaling pathway inhibitor LY294002. Finally, the in vivo bone regeneration results of rat critical-sized calvarial defect models confirmed that the combination of the micro-nano-hybrid surface and ASCs could significantly enhance both osteogenesis and angiogenesis as compared with the control HAp bioceramic scaffold with traditional smooth surface. Our results suggest that HAp bioceramic scaffolds with micro-nano-hybrid surface can act as cell carrier for ASCs, and consequently combine with ASCs to construct vascularized tissue-engineered bone.

Repair of Critical-Sized Rat Calvarial Defects Using Genetically Engineered Bone Marrow-Derived Mesenchymal Stem Cells Overexpressing Hypoxia-Inducible Factor-1ᆇ§

The processes of angiogenesis and bone formation are coupled both temporally and spatially during bone repair. Bone marrow‐derived mesenchymal stem cells (BMSCs) have been effectively used to heal critical‐size bone defects. Enhancing their ability to undergo angiogenic and osteogenic differentiation will enhance their potential use in bone regeneration. Hypoxia‐inducible factor‐1α (HIF‐1α) has recently been identified as a major regulator of angiogenic‐osteogenic coupling. In this study, we tested the hypothesis that HIF‐1α gene therapy could be used to promote the repair of critical‐sized bone defects. Using lentivirus‐mediated delivery of wild‐type (HIF) or constitutively active HIF‐1α (cHIF), we found that in cultured BMSCs in vitro, HIF and cHIF significantly enhanced osteogenic and angiogenic mRNA and protein expression when compared with the LacZ group. We found that HIF‐1α‐overexpressing BMSCs dramatically improved the repair of critical‐sized calvarial defects, including increased bone volume, bone mineral density, blood vessel number, and blood vessel area in vivo. These data confirm the essential role of HIF‐1α modified BMSCs in angiogenesis and osteogenesis in vitro and in vivo. STEM CELLS 2011;29:1380–1390