Ya-Fei Feng

Evaluation of Biological Properties of Electron Beam Melted Ti6Al4V Implant with Biomimetic Coating In Vitro and In Vivo

Background High strength porous titanium implants are widely used for the reconstruction of craniofacial defects because of their similar mechanical properties to those of bone. The recent introduction of electron beam melting (EBM) technique allows a direct digitally enabled fabrication of patient specific porous titanium implants, whereas both their in vitro and in vivo biological performance need further investigation. Methods In the present study, we fabricated porous Ti6Al4V implants with controlled porous structure by EBM process, analyzed their mechanical properties, and conducted the surface modification with biomimetic approach. The bioactivities of EBM porous titanium in vitro and in vivo were evaluated between implants with and without biomimetic apatite coating. Results The physical property of the porous implants, containing the compressive strength being 163 - 286 MPa and the Young’s modulus being 14.5–38.5 GPa, is similar to cortical bone. The in vitro culture of osteoblasts on the porous Ti6Al4V implants has shown a favorable circumstance for cell attachment and proliferation as well as cell morphology and spreading, which were comparable with the implants coating with bone-like apatite. In vivo, histological analysis has obtained a rapid ingrowth of bone tissue from calvarial margins toward the center of bone defect in 12 weeks. We observed similar increasing rate of bone ingrowth and percentage of bone formation within coated and uncoated implants, all of which achieved a successful bridging of the defect in 12 weeks after the implantation. Conclusions This study demonstrated that the EBM porous Ti6Al4V implant not only reduced the stress-shielding but also exerted appropriate osteoconductive properties, as well as the apatite coated group. The results opened up the possibility of using purely porous titanium alloy scaffolds to reconstruct specific bone defects in the maxillofacial and orthopedic fields.

Tantalum coating on porous Ti6Al4V scaffold using chemical vapor deposition and preliminary biological evaluation

Porous tantalum (Ta), produced via chemical vapor deposition (CVD) of commercially pure Ta onto a vitreous carbon, is currently available for use in orthopedic applications. However, the relatively high manufacturing cost and the incapability to produce customized implant using medical image data have limited its application to gain widespread acceptance. In this study, Ta film was deposited on porous Ti6Al4V scaffolds using CVD technique. Digital microscopy and scanning electron microscopy indicated that the Ta coating evenly covered the entire scaffold structure. X-ray diffraction analysis showed that the coating consisted of α and β phases of Ta. Goat mesenchymal stem cells were seeded and cultured on the Ti6Al4V scaffolds with and without coating. The tetrazolium-based colorimetric assay exhibited better cell adhesion and proliferation on Ta-coated scaffolds compared with uncoated scaffolds. The porous scaffolds were subsequently implanted in goats for 12weeks. Histological analysis revealed similar bone formation around the periphery of the coated and uncoated implants, but bone ingrowth is better within the Ta-coated scaffolds. To demonstrate the ability of producing custom implant for clinical applications via this technology, we designed and fabricated a porous Ti6Al4V scaffold with segmental mandibular shape derived from patient computerized tomography data.

Influence of Architecture of β-Tricalcium Phosphate Scaffolds on Biological Performance in Repairing Segmental Bone Defects

Background Although three-dimensional (3D) β-tricalcium phosphate (β-TCP) scaffolds serve as promising bone graft substitutes for the segmental bone defect treatment, no consensus has been achieved regarding their optimal 3D architecture. Methods In this study, we has systematically compared four types of β-TCP bone graft substitutes with different 3D architectures, including two types of porous scaffolds, one type of tubular scaffolds and one type of solid scaffolds, for their efficacy in treating segmental bone defect in a rabbit model. Results Our study has demonstrated that when compared to the traditional porous and solid scaffolds, tubular scaffolds promoted significantly higher amount of new bone formation in the defect regions as shown by X-ray, micro CT examinations and histological analysis, restored much greater mechanical properties of the damaged bone evidenced by the biomechanical testing, and eventually achieved the complete union of segmental defect. Moreover, the implantation of tubular scaffolds enhanced the neo-vascularization at the defect region with higher bone metabolic activities than others, as indicated by the bone scintigraphy assay. Conclusions This study has further the current knowledge regarding the profound influence of overall 3D architecture of β-TCP scaffolds on their in vivo defect healing performance and illuminated the promising potential use of tubular scaffolds as effective bone graft substitute in treating large segmental bone defects.

Sustained oxidative stress causes late acute renal failure via duplex regulation on p38 MAPK and Akt phosphorylation in severely burned rats.

Background Clinical evidence indicates that late acute renal failure (ARF) predicts high mortality in severely burned patients but the pathophysiology of late ARF remains undefined. This study was designed to test the hypothesis that sustained reactive oxygen species (ROS) induced late ARF in a severely burned rat model and to investigate the signaling mechanisms involved. Materials and Methods Rats were exposed to 100°C bath for 15 s to induce severe burn injury (40% of total body surface area). Renal function, ROS generation, tubular necrosis and apoptosis, and phosphorylation of MAPK and Akt were measured during 72 hours after burn. Results Renal function as assessed by serum creatinine and blood urea nitrogen deteriorated significantly at 3 h after burn, alleviated at 6 h but worsened at 48 h and 72 h, indicating a late ARF was induced. Apoptotic cells and cleavage caspase-3 in the kidney went up slowly and turned into significant at 48 h and 72 h. Tubular cell ROS production shot up at 6 h and continuously rose during the 72-h experiment. Scavenging ROS with tempol markedly attenuated tubular apoptosis and renal dysfunction at 72 h after burn. Interestingly, renal p38 MAPK phosphorylation elevated in a time dependent manner whereas Akt phosphorylation increased during the first 24 h but decreased at 48 h after burn. The p38 MAPK specific inhibitor SB203580 alleviated whereas Akt inhibitor exacerbated burn-induced tubular apoptosis and renal dysfunction. Furthermore, tempol treatment exerted a duplex regulation through inhibiting p38 MAPK phosphorylation but further increasing Akt phosphorylation at 72 h postburn. Conclusions These results demonstrate that sustained renal ROS overproduction induces continuous tubular cell apoptosis and thus a late ARF at 72 h after burn in severely burned rats, which may result from ROS-mediated activation of p38 MAPK but a late inhibition of Akt phosphorylation.

Time Related Changes of Mineral and Collagen and Their Roles in Cortical Bone Mechanics of Ovariectomized Rabbits.

As cortical bone has a hierarchical structure, the macroscopic bone strength may be affected by the alterations of mineral crystal and collagen, which are main components of cortical bone. Limited studies focused on the time related alterations of these two components in osteoporosis, and their contributions to bone mechanics at tissue level and whole-bone level. Therefore, the purpose of this study was to elucidate the time related changes of mineral and collagen in cortical bone of ovariectomized (OVX) rabbits, and to relate these changes to cortical bone nanomechanics and macromechanics. 40 Rabbits (7-month-old) were randomly allocated into two groups (OVX and sham). OVX group received bilateral ovariectomy operation. Sham group received sham-OVX operation. Cortical bone quality of five rabbits in each group were assessed by DXA, μCT, nanoindentation, Fourier transform infrared (FTIR) spectroscopy and biomechanical tests (3-point bending of femoral midshaft) at pre-OVX, 4, 6, and 8 weeks after OVX. As time increased from pre-OVX to 8 weeks, the mineral to matrix ratio decreased with time, while both collagen crosslink ratio and crystallinity increased with time in OVX group. Elastic modulus and hardness measured by nanoindentation, whole-bone strength measured by biomechanical tests all decreased in OVX group with time. Bone material properties measured by FTIR correlated well with nano or whole-bone level mechanics. However, bone mineral density (BMD), structure, tissue-level and whole-bone mechanical properties did not change with age in sham group. Our study demonstrated that OVX could affect the tissue-level mechanics and bone strength of cortical bone. And this influence was attributed to the time related alterations of mineral and collagen properties, which may help us to design earlier interventions and more effective treatment strategies on osteoporosis.

Promotion of osteointegration under diabetic conditions by tantalum coating-based surface modification on 3-dimensional printed porous titanium implants.

Clinical evidence indicates a high failure rate for titanium implants (TiI) in diabetic patients, involving the overproduction of reactive oxygen species (ROS) at the implant/bone interface. Tantalum coating on titanium (TaTi) has exerted better tissue integration properties than TiI, but its biological performance under diabetic conditions remains elusive. To investigate whether TaTi may ameliorate diabetes-induced implant destabilization and the underlying mechanisms, primary rabbit osteoblasts cultured on 3-dimensional printed TiI and TaTi were exposed to normal serum (NS), diabetic serum (DS), DS+NAC (a potent ROS inhibitor), and DS+SB203580 (a specific p38 MAPK inhibitor). An in vivo study was performed on diabetic sheep implanted with TiI or TaTi. Diabetes induced mitochondrial-derived ROS overproduction and caused cellular dysfunction and apoptosis, together with the activation of p38 MAPK in osteoblasts on TiI surface. Importantly, TaTi significantly attenuated ROS production and p38 MAPK phosphorylation and exerted more osseointegrative cell behavior than TiI, as shown by improved osteoblast adhesion, increased cell proliferation and differentiation and decreased apoptosis. These results were confirmed in vivo by the enhanced bone healing efficacy of TaTi. Moreover, treatment with NAC or SB203580 on TiI not only inhibited the activation of p38 MAPK but also improved cell function and alleviated apoptotic injury, whereas TaTi combined with NAC or SB203580 failed to further improve osteoblast functional recovery compared with TaTi alone. These results demonstrated that the tantalum coating markedly improved diabetes-induced impaired osteogenesis of TiI, which may be attributed to the suppression of the ROS-mediated p38 MAPK pathway.

Systemic Treatment with Telmisartan Improves Femur Fracture Healing in Mice

Recent clinical studies indicated that angiotensin receptor blockers (ARBs) would decrease the risk of bone fractures in the elderly populations. There is little known about the role of the ARBs in the process of fracture healing. The purpose of the present study was to verify the hypothesis that systemic treatment with telmisartan has the ability to promote fracture healing. In this study, femur fractures were produced in 96 mature male BALB/c mice. Animals were treated with the ARBs telmisartan or vehicle. Fracture healing was analysed after 2, 5 and 10 weeks postoperatively using X-ray, biomechanical testing, histomorphometry, immunohistochemistry and micro-computed tomography (micro-CT). Radiological analysis showed the diameter of the callus in the telmisartan treated animals was significantly increased when compared with that of vehicle treated controls after two weeks of fracture healing. The radiologically observed promotion of callus formation was confirmed by histomorphometric analyses, which revealed a significantly increased amount of bone formation when compared with vehicle-treated controls. Biomechanical testing further showed a significantly greater peak torque at failure, and a higher torsional stiffness in telmisartan-treated animals compared with controls. There was an increased fraction of PCNA-positive cells and VEGF-positive cells in telmisartan-treated group compared with vehicle-treated controls. From the three-dimensional reconstruction of the bony callus, telmisartan-treated group significantly increased the values of BV/TV by 21.7% and CsAr by 26.0% compared to the vehicle-treated controls at 5 weeks post-fracture. In summary, we demonstrate in the current study that telmisartan could promote fracture healing in a mice model via increasing mechanical strength and improving microstructure. The most mechanism is probably by an increase of cell proliferation and neovascularization associated with a decreased VEGF expression in hypertrophic chondrocytes.

Preventive Effect of Crocin on Osteoporosis in an Ovariectomized Rat Model

The purpose of this study was to investigate the therapeutic effects of crocin on ovariectomy-induced osteoporosis in rats. Female Sprague-Dawley rats were randomly assigned to a sham-operated group (sham) and five ovariectomy (OVX) subgroups, that is, OVX with vehicle (OVX), OVX with 17β-estradiol (E 2, 25 μg/kg/day), and OVX with graded crocin doses (5, 10, or 20 mg/kg/day). Daily oral administration of E 2 or crocin started 4 weeks after OVX and lasted for 16 weeks. Our results showed that crocin dose-dependently inhibited the BMD reduction of L4 vertebrae and femurs caused by OVX and prevented the deterioration of trabecular microarchitecture, which were accompanied by a significant decrease in skeletal remodeling as evidenced by the lower levels of bone turnover markers. Furthermore, crocin reversed the oxidative stress status in both serum and bone tissue. The present study indicates that the administration of crocin at higher doses over a 16-week period can prevent OVX-induced osteoporosis in rats without hyperplastic effects on the uterus, which may, at least partially, be attributed to crocins antioxidative property. In brief, crocin is a natural alternative for postmenopausal osteoporosis treatment in elderly women.

Insulin alleviates posttrauma cardiac dysfunction by inhibiting tumor necrosis factor-α-mediated reactive oxygen species production.

Objective:Clinical evidence indicates that intensive insulin treatment prevents the incidence of multiple organ failures in surgical operation and severe trauma, but the mechanisms involved remain elusive. This study was designed to test the hypothesis that insulin may exert anti-inflammatory and antioxidative effects and thus alleviate cardiac dysfunction after trauma. Design:Prospective, randomized experimental study. Setting:Animal research laboratory. Subjects:Sprague Dawley rats. Interventions:Anesthetized rats were subjected to 200 revolutions at a rate of 35 rpm in Noble-Collip drum to induce a nonlethal mechanical trauma and were randomized to receive vehicle, insulin, and insulin + wortmannin treatments. An in vitro study was performed on cultured cardiomyocytes subjected to sham-traumatic serum (SS), traumatic serum (TS), SS + tumor necrosis factor (TNF)-&agr;, SS + H2O2, TS + neutralizing anti-TNF-&agr; antibody, or TS + tempol treatments. Measurements and Main Results:Immediate cardiac dysfunction occurred 0.5 hr after trauma without significant cardiomyocyte necrosis and apoptosis, while serum TNF-&agr; and cardiac reactive oxygen species (ROS) production was increased. Importantly, incubation of cardiomyocytes with TS or SS + TNF-&agr; significantly increased ROS generation together with dampened cardiomyocyte contractility and Ca2+ transient, all of which were rescued by TNF-&agr; antibody. Administration of insulin inhibited TNF-&agr; and ROS overproduction and alleviated cardiac dysfunction 2 hours after trauma. Scavenging ROS with tempol also attenuated cardiac dysfunction after trauma, whereas insulin combined with tempol failed to further improve cardiac functional recovery compared with insulin treatment alone. Moreover, the aforementioned anti-TNF-&agr;, antioxidative, and cardioprotective effects afforded by insulin were almost abolished by the phosphatidylinositol 3-kinase inhibitor wortmannin. Conclusions:These results demonstrate for the first time that mechanical trauma induces a significant increase in TNF-&agr; and ROS production, resulting in immediate cardiac dysfunction. Early posttrauma insulin treatment alleviates cardiac dysfunction by inhibiting TNF-&agr;-mediated ROS production via a phosphatidylinositol 3-kinase/Akt-dependent mechanism.

Insulin improves osteogenesis of titanium implants under diabetic conditions by inhibiting reactive oxygen species overproduction via the PI3K-Akt pathway.

Clinical evidence indicates that insulin therapy improves implant survival rates in diabetic patients; however, the mechanisms responsible for this effect are unknown. Here, we test if insulin exerts anti-oxidative effects, thereby improving diabetes-associated impaired osteoblast behavior on titanium implants. To test this hypothesis, we cultured primary rabbit osteoblasts in the presence of titanium implants and studied the impact of treatment with normal serum (NS), diabetic serum (DS), DS + insulin, DS + tempol (a superoxide dismutase mimetic), DS + insulin + tempol, and DS + insulin + wortmannin. We analyzed cell function, apoptosis, and reactive oxygen species (ROS) production in osteoblasts following the various treatments. Treatment with DS induced osteoblast dysfunction, evidenced by impaired cell attachment and morphology, decreased cell proliferation and ALP activity, and decreased expression of osteogenesis-related genes. We also observed a significant increase in apoptosis. Importantly, treatment with DS resulted in increased production of ROS in osteoblasts. In contrast, treatment with insulin inhibited ROS production, alleviated cell dysfunction, and decreased apoptosis of osteoblasts on the implants. Scavenging ROS with tempol also attenuated cell dysfunction. Compared to insulin treatment alone, the combination of insulin and tempol failed to further improve osteoblast functional recovery. Moreover, the anti-oxidative and pro-osteogenic effects afforded by insulin were almost completely abolished by the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin. These results demonstrate, for the first time, that insulin treatment alleviates the impaired osteogenesis of titanium implants under diabetic conditions by inhibiting ROS overproduction via a PI3K/Akt-dependent mechanism. Both the anti-oxidative and metabolic properties of insulin should make it a viable therapeutic option to combat diabetic implant failure.