Jing Hu

The effect of strontium-substituted hydroxyapatite coating on implant fixation in ovariectomized rats

This study was designed to investigate the effects of strontium-substituted hydroxyapatite coatings with 10 mol% Ca(2+) replaced by Sr(2+) (10% SrHA) on implant fixation in ovariectomized (OVX) rats. Coatings of HA and 10% SrHA were prepared on the surface of titanium implant using sol-gel dip methods, and then characterized by Scanning Electron Microscope, Atomic Force Microscope, X-ray diffraction, X-ray Photoemission Spectroscopy, and an automatic scratch tester. Twelve weeks after bilateral ovariectomy, twenty OVX rats accepted implant insertion in the proximal tibiae, half with HA-coated implants and the other half with 10% SrHA coated implants. After 12-week healing period, 10% SrHA coated implants revealed improved osseointegration compared to HA, with the bone area ratio and bone-to-implant contact increased by 70.9% and 49.9% in histomorphometry, the bone volume ratio and percent osseointegration by 73.7% and 45.2% in micro-CT evaluation, and the maximal push-out force and ultimate shear strength by 107.2% and 132.9% in push out test. These results demonstrated that 10% SrHA coatings could enhance implant osseointegration in OVX rats, and suggested the feasibility of using SrHA coatings to improve implant fixation in osteoporotic bone.

The effect of surface immobilized bisphosphonates on the fixation of hydroxyapatite-coated titanium implants in ovariectomized rats

Immobilized bisphosphonates (BPs) have been introduced to improve implant fixation, however, no information could be found about the efficiency of this approach in osteoporotic bone. This study was designed to evaluate the bone response to surface immobilized BPs on implants inserted in tibiae of ovariectomized (OVX) rats. Three months after bilateral ovariectomy, 40 rats were randomly assigned into four groups for implantation of hydroxyapatite-coated titanium implants with or without immobilized BPs: (1) control group (without BP treatments); (2) pamidronate (PAM) group (1mg/ml of PAM immersing); (3) ibandronate group (1mg/ml of ibandronate immersing); and (4) zoledronic acid (ZOL) group (1mg/ml of ZOL immersing). After implantation periods of 3 months, the peri-implant-bone density, trabecular microstructure, bone-implant interface and mechanical fixation of implants were evaluated by dual energy X-ray absorptiometry, micro-computed tomography, histology and push-out test. We found that three BPs triggered pronounced bone-implant integration and early bone formation around implants in OVX rats, with a rank order of ZOL>ibandronate>PAM. These results provide new evidence that immobilized BPs have positive effects on implant fixation in osteoporotic bone, in addition to their well-documented potency to inhibit implant loosening in normal bone.

Effect of combined local treatment with zoledronic acid and basic fibroblast growth factor on implant fixation in ovariectomized rats

Osteoporosis is a skeletal disorder characterized by low bone mass and deterioration of bone microarchitecture resulting in bone fragility, which impairs fixation of the implants. Zoledronic acid (ZOL) is a potential inhibitor of osteoclast-mediated bone resorption and basic fibroblast growth factor (bFGF) is a growth factor that stimulates osteoblast-mediated bone formation, and these drugs could enhance fixation of implants under osteoporotic conditions. In this study, 40 ovariectomized (OVX) rats were randomly divided into 4 groups (n=10 for each group) and underwent bilateral tibiae implantation using hydroxyapatite (HA)-coated titanium implant: Control group (distilled water immersing before implantation), ZOL group (1 mg/ml of ZOL immersing), bFGF group (20 microg/ml of bFGF immersing), and ZOL+bFGF group (1 mg/ml of ZOL and 20 microg/ml of bFGF immersing). At 3 months after implantation, all animal were sacrificed and the tibiae were harvested for histology, micro-CT examinations and biomechanical testing. Bone area and contact, determined by histomorphometric analysis, were 2.7-fold and 1.8-fold in the ZOL-treated implants, 1.9-fold and 1.8-fold in the bFGF-treated implants, 3.6-fold and 2.3-fold in the both-treated implants compared with controls (p<0.01). Such significant effects were further confirmed by microstructure parameters, the bone volume ratio and the percentage osteointegration were significantly increased by ZOL treatment (3.0-fold and 1.8-fold), bFGF treatment (1.2-fold and 1.9-fold) and ZOL+bFGF treatment (3.3-fold and 2.7-fold) (p<0.001). In addition, push-out test showed that the maximum force and the corresponding interfacial shear strength of the implants treated by ZOL, bFGF and ZOL+bFGF was 8.4-fold and 8.6-fold, 3.8-fold and 3.7-fold, 10.8-fold and 10.7-fold of the control levels, respectively (p<0.05). The combined treatment was better than either treatment alone for force, but was not different from ZOL alone for interfacial strength. The significant correlation between biomechanical and micro-CT parameters demonstrates the role of microstructure assessments in predicting mechanical fixation of implants (p<0.01). Our study suggests that locally applied ZOL or bFGF may improve implant fixation in the ovariectomized rats, and that combined treatment has more beneficial effects on osseointegration, peri-implant bone formation and maximum force than either intervention alone.

The homing of bone marrow MSCs to non-osseous sites for ectopic bone formation induced by osteoinductive calcium phosphate

Osteoinductive biomaterials are promising for bone repair. There is no direct proof that bone marrow mesenchymal stem cells (BMSCs) home to non-osseous sites and participate in ectopic bone formation induced by osteoinductive bioceramics. The objective of this study was to use a sex-mismatched beagle dog model to investigate BMSC homing via blood circulation to participate in ectopic bone formation via osteoinductive biomaterial. BMSCs of male dogs were injected into female femoral marrow cavity. The survival and stable chimerism of donor BMSCs in recipients were confirmed with polymerase chain reaction (PCR) and fluorescence in situ hybridization (FISH). Biphasic calcium phosphate (BCP) granules were implanted in dorsal muscles of female dogs. Y chromosomes were detected in samples harvested from female dogs which had received male BMSCs. At 4 weeks, cells with Y-chromosomes were distributed in the new bone matrix throughout the BCP granule implant. At 6 weeks, cells with Y chromosomes were present in newly mineralized woven bone. TRAP positive osteoclast-like cells were observed in 4-week implants, and the number of such cells decreased from 4 to 6 weeks. These results show that osteoprogenitors were recruited from bone marrow and homed to ectopic site to serve as a cell source for calcium phosphate-induced bone formation. In conclusion, BMSCs were demonstrated to migrate from bone marrow through blood circulation to non-osseous bioceramic implant site to contribute to ectopic bone formation in a canine model. BCP induced new bone in muscles without growth factor delivery, showing excellent osteoinductivity that could be useful for bone tissue engineering.

Angiogenesis and osteogenesis enhanced by bFGF ex vivo gene therapy for bone tissue engineering in reconstruction of calvarial defects.

Reconstruction of bone defects by tissue engineered substitutes requires coordinated coupling between osteogenesis and angiogenesis. Basic fibroblast growth factor (bFGF or FGF-2) is a protein which acts actively in osteogenesis and angiogenesis during skeletal healing and development. It is hypothesized that BMSCs transfected with bFGF can directly stimulate regeneration of vascular tissue, and subsequently enhance osseous formation and remodeling after implantation of the tissue engineered bone. This study was designed to examine the impact of bFGF-BMSCs, seeded on nano-hydroxyapatite/polyamide66 (n-HA/PA66) composite scaffold, to enhance angiogenesis and osteogenesis in a calvarial critical-sized defect model in rats. To investigate the vascularization and bone formation of tissue engineered bone, the substrate was removed and processed for immunohistochemical, scanning electron microscopic examinations (SEM), reverse transcriptase-polymerase chain reaction (RT-PCR), dual energy X-ray absorptiometry (DEXA), microvessels counting, and new bone volume assay. The results demonstrate that bFGF mediated ex vivo gene transfer based on BMSCs can accelerate vascularization and bone regeneration on these composite scaffolds. The n-HA/PA66 scaffold combined with the bFGF-BMSCs may mimic the natural process of osteogenesis during repair of defect by tissue engineered bone.

Strontium ranelate treatment enhances hydroxyapatite-coated titanium screws fixation in osteoporotic rats

Increased bone turnover with excessive bone resorption and decreased bone formation is known to impair implant fixation. Strontium ranelate is well known as an effective antiosteoporotic agent by its dual effect of antiresorbing and bone‐forming activity. This study was designed to evaluate the effect of systemic strontium ranelate (SR) treatment on fixation of hydroxyapatite (HA)‐coated titanium screws in ovariectomized (OVX) rats. Twelve weeks after being OVX (nu2009=u200930) or sham (nu2009=u200910) operated, 40 female Sprague–Dawley rats received unilateral implants in the proximal tibiae. The OVX rats were randomly divided into the following groups: OVX, OVXu2009+u2009SRL (“L” refers to low SR dose of 500 mg/kg/day), OVXu2009+u2009SRH (“H” refers to high SR dose of 1000 mg/kg/day).Twelve weeks after treatment, bone blocks with implants were evaluated with micro‐CT and biomechanical push‐out tests. Compared to OVX animals, SR treatment increased the bone volume ratio by 51.5% and 1.1‐fold, the percentage osteointegration by 1.0‐fold and 1.9‐fold in micro‐CT evaluation, and the maximal force by 1.9‐fold and 3.3‐fold in biomechanical push‐out test, for the low and high dose of SR, respectively. Significant correlation between micro‐CT and biomechanical properties demonstrated that trabecular parameters played an important role in predicting the biomechanical properties of implant fixation. Our findings suggest that SR treatment can dose‐dependently improve HA‐coated screw fixation in OVX rats and facilitate the stability of the implant in the osteoporotic bone.

The effect of hydrofluoric acid treatment on titanium implant osseointegration in ovariectomized rats

This study aimed to investigate the effects of hydrofluoric acid (HF) treatment of grit-blasted Ti implants on osseointegration in ovariectomized (OVX) rats. After blasting with aluminium oxide particles, half implants were treated with 0.2 vol.% HF, and the other half were kept non-modified as control. The topographical and chemical changes of implant surface were determined by Scanning Electron Microscope, Atomic Force Microscope, and X-ray Photoemission Spectroscopy. 12 Weeks after bilateral ovariectomy, each rat accepted two implants in distal femora, with the control implant on the left and the fluoride-modified on the right. As a result, fluoride modification induced markedly changed surface topography and chemical composition. 12 Weeks after implant insertion, the fluoride-modified implants showed improved osseointegration compared to control, with the bone area ratio and bone-to-implant contact increased by 0.9- and 1.4-fold in histomorphometry, the bone volume ratio and percent osseointegration by 0.8- and 1.3-fold in micro-CT evaluation, and the maximal push-out force and ultimate shear strength by 1.2- and 2.0-fold in biomechanical test. These promising results indicated that HF treatment of Ti surface improved implant osseointegration in OVX rats, and suggested the feasibility of using fluoride modification to improve Ti implant osseointegration in osteoporotic bone.

Effect of the anti-diabetic drug metformin on bone mass in ovariectomized rats

Recent studies have reported bone loss in the patients with diabetes and a direct osteogenic effect of metformin on osteoblast-like cells in culture. In this study, we investigated the action of metformin on bone mass in ovariectomized (OVX) rats. Three months after either a sham surgery or bilateral ovariectomy, thirty-two female Sprague-Dawley rats were randomly assigned into four groups: (1) Sham group; (2) OVX group; (3) OVX+metformin (50mg/kg/day) group; and (4) OVX+metformin (100mg/kg/day) group. After 2 months of oral administration with or without metformin, tibiae were harvested for dual energy X-ray absorptiometry, micro-computed tomography (micro-CT) and histology analysis, while the bone marrow cells from tibiae were collected for measurement of the mRNAs expressions for three osteoblast genes and estrogen receptors alpha by the use of real-time RT-PCR. We found that the impaired bone density and quality induced by bilateral ovariectomy were significantly improved by the treatment of metformin (both 50 and 100mg/kg/day), and this action could be partly mediated by regulating bone marrow cells development through induction of mechanisms regulating osteoblast markers core binding factor a1 and LDL receptor-related protein 5. These findings provide new evidence that the anti-diabetic drug metformin has a direct inhibition effect on bone loss in OVX rats, in addition to its well-documented osteogenic potency in vitro.

bFGF-Modified BMMSCs enhance bone regeneration following distraction osteogenesis in rabbits

Distraction osteogenesis (DO), which induces new bone formation along the vector of pull without requiring the use of bone graft, has become a valuable surgical method for patients with limb discrepancy or craniofacial microsomia. However, the long treatment period and possible fibrous union or nonunion hampers its further clinical application. Bone marrow mesenchymal stem cells (BMMSCs) have been widely used as a source of cell therapy or a vector for gene transfer. Basic fibroblast growth factor (bFGF) has high potential for promotion of bone regeneration. However, bFGF has a short half value period in vivo. In this study, osteodistraction was applied in craniofacial bone of rabbit to observe the effects of BMMSCs with or without bFGF gene transfected on bone regeneration in the distracted zone. Mandibular lengthening (10 mm) was performed in 42 New Zealand white rabbits using a rapid distraction rate (2 mm/day). The animals were then randomly divided into group A, group B and group C (n=14 for each group). At the end of distraction, physiological saline, autologous BMMSCs and BMMSCs transfected with bFGF were injected into the distraction gaps in groups A, B, and C, respectively. Eight weeks after DO, the rabbits were sacrificed, and the distracted mandibles were harvested and processed for radiography, dual energy x-ray absorptiometry (DXA), micro-CT, histology and three-point bend testing. Under a rapid distraction, immature or poor bone healing was observed in the distracted callus from group A. However, better bone formation and higher bone mineral density (BMD) and bone mineral content (BMC) in the distracted callus were observed in group B and group C, the latter showed excellent bone formation and highest BMD and BMC. Such finding was confirmed by histological and micro-CT examinations. The values of BT/TV in group C were highest and the micro-architecture presented more mature characteristics. The mechanical strength in group C was 1.63-fold and 1.28-fold greater than that in group A and B by three-point bending testing. The results of this study suggest that BMMSCs transplantation can promote bone formation in DO, and bFGF-modified BMMSCs were more effective in this enhancement.

Effect of zoledronate acid treatment on osseointegration and fixation of implants in autologous iliac bone grafts in ovariectomized rabbits

One main problem associated with alveolar bone augmentation in implant dentistry is resorption of grafted bone, which may be further compromised by systemic skeletal disorders such as osteoporosis. Zoledronate acid (ZOL) is the most potent bisphosphonate to treat osteoporosis and therefore it is hypothesized to be able to invert the negative effect of osteoporosis on osseointegration and fixation of dental implants in autologous bone grafts. In this study, 56 rabbits received bilateral ovariectomy (OVX) (40 rabbits) or sham operation (16 rabbits). Three months later, 8 animals from each group were sacrificed for bone mineral density (BMD) examination. Then the remaining animals underwent bilateral autologous iliac bone grafting with simultaneous implantation of titanium implants in tibiae and were divided into 5 groups (n=8): Sham, OVX, Loc-ZOL (local treatment), Sys-ZOL (systemic treatment) and Loc+Sys-ZOL (local plus systemic) group. At 3 months after implantation, all animals were sacrificed and specimens were harvested for examinations. Both BMD and histological examinations of femurs showed osteoporotic changes after ovariectomy, while systemic treatment with ZOL restored mineralized bone. Micro-CT examination demonstrated that OVX group showed significant decrease of mineralized bone and implant-bone contact when compared with sham control, whereas both systemic and local treatments of ZOL significantly increased mineralized bone and implant-bone contact in ovariectomized animals. However, the best effects were observed in Loc+Sys-ZOL group (combined use of ZOL) and most of bone indices were similar to (IBCR, p>0.05) or higher than (BV/TV, Conn.D and Tb.N) (p<0.01) those of the sham group, except Tb.Th, which was still significantly lower (p<0.01), and Tb.Sp, which was further decreased (p<0.01). The aforementioned effects were also confirmed by histomorphometric analysis of bone indices on implant-bone contact and mineralized bone. In addition, biomechanical testing further supported the beneficial effect of ZOL treatment and maximal removal torque of titanium implants was observed in Loc+Sys-ZOL group. In conclusion, our study suggests that both systemic and local treatments with ZOL can invert negative effect of osteoporosis and promote osseointegration and fixation of dental implants in autologous bone grafts under osteoporotic condition. Combined systemic and local use of ZOL exerts best effects when compared to their single use.