RhBMP-2-loaded calcium silicate/calcium phosphate cement scaffold with hierarchically porous structure for enhanced bone tissue regeneration

Calcium phosphate cement scaffold (CPC) has been widely used as bone graft substitutes, but undesirable osteoinductivity and slow degradability greatly hamper their clinic application. To address these problems, a recombinant human bone morphogenetic protein-2 (rhBMP-2)-loaded calcium silicate/calcium phosphate cement scaffold (CSPC) with hierarchical pores was developed in this study. The CSPC scaffold with both interconnected macropores on the order of 200–500 μm and micropores of 2–5 μm was synthesized from CPC and calcium silicate (CS) by a NaCl particulate-leaching method. In vitro cell culture with C2C12 model cells, in vivo ectopic bone formation and rabbit femur cavity defect repair were performed to evaluate the osteogeneic capacity of the CSPC/rhBMP-2 scaffold. CPC, CSPC and CPC/rhBMP-2 scaffolds were parallelly investigated for comparison. The results demonstrated that the hierarchical macro/microporous structure, whether in presence of CS or rhBMP-2, highly favored the adhesion of C2C12 cells and bone in-growth into the CPC-based scaffolds. But, in comparison to the CPC-based scaffolds with CS or rhBMP-2 alone, the CSPC/rhBMP-2 scaffold strongly promoted osteogenic differentiation in vitro and osteogenetic efficacy in vivo. Further studies demonstrated that Si ions derived from CSPC contributed mainly to maintain the conformation of rhBMP-2 and thus stimulate the synergistic action of CS and rhBMP-2 in osteogenic differentiation and osteoinductivity. Additionally, the incorporation of CS was also beneficial for the dissolution of the scaffold. Those results suggest that the CSPC has superior properties for incorporation of rhBMP-2 and our developed CSPC/rhBMP-2 scaffold have great potential for future use in bone tissue regeneration.     

Bone regeneration using photocrosslinked hydrogel incorporating rhBMP-2 loaded 2-N, 6-O-sulfated chitosan nanoparticles

Although rhBMP-2 has excellent ability to accelerate the repair of normal bone defects, limitations of its application exist in the high cost and potential side effects. This study aimed to develop a composite photopolymerisable hydrogel incorporating rhBMP-2 loaded 2-N, 6-O-sulfated chitosan nanoparticles (PH/rhBMP-2/NPs) as the bone substitute to realize segmental bone defect repair at a low growth factor dose. Firstly rhBMP-2 loaded 2-N, 6-O-sulfated chitosan nanoparticles (rhBMP-2/NPs) were prepared and characterized by DLS and TEM. Composite materials, PH/rhBMP-2/NPs were developed and investigated by SEM-EDS as well as a series of physical characterizations. Using hMSCs as an in vitro cell model, composite photopolymerisable hydrogels incorporating NPs (PH/NPs) showed good cell viability, cell adhesion and time dependent cell ingrowth. In vitro release kinetics of rhBMP-2 showed a significantly lower initial burst release from the composite system compared with the growth factor-loaded particles alone or encapsulated directly within the hydrogel, followed by a slow release over time. The bioactivity of released rhBMP-2 was validated by alkaline phosphatase (ALP) activity as well as a mineralization assay. In in vivo studies, the PH/rhBMP-2/NPs induced ectopic bone formation in the mouse thigh. In addition, we further investigated the in vivo effects of rhBMP-2-loaded scaffolds in a rabbit radius critical defect by three dimensional micro-computed tomographic (μCT) imaging, histological analysis, and biomechanical measurements. Animals implanted with the composite hydrogel containing rhBMP-2-loaded nanoparticles underwent gradual resorption with more pronounced replacement by new bone and induced reunion of the bone marrow cavity at 12 weeks, compared with animals implanted with hydrogel encapsulated growth factors alone. These data provided strong evidence that the composite PH/rhBMP-2/NPs are a promising substitute for bone tissue engineering.     

Vascularization and bone regeneration in a critical sized defect using 2-N,6-O-sulfated chitosan nanoparticles incorporating BMP-2

An ideal bone tissue engineering graft should have both excellent pro-osteogenesis and pro-angiogenesis to rapidly realize the bone regeneration in vivo. To meet this goal, 2-N,6-O-sulfated chitosan (26SCS) based nanoparticle (S-NP) was successfully developed and showed a dose-dependent enhancement on angiogenesis in vitro. For the repair of a critical sized defect in rabbit radius, we developed BMP-2 loaded S-NP (BMP-2/S-NP) with protein loading efficiency of 1.4 ± 0.2% and fabricated a gelatin sponge (G) based implant loaded with BMP-2/S-NP (BMP-2/S-NP/G). This implant exerted a delivery of BMP-2 with an initial burst release of 15.3 ± 4.1% in first 24 h and a gradual release for 21 days to 77.8 ± 3.6%. The in vitro ALP assay revealed that the activity of released BMP-2 from BMP-2/S-NP/G was maintained after 3-d and 7-d delivery and further enhanced after 14-d delivery compared with the original BMP-2. Furthermore, the in vivo effects of BMP-2/S-NP/G on the bone regeneration and vessel formation in the critical sized defect (18 mm) of rabbit radius were investigated by synchrotron radiation-based micro-computed tomography (SRμCT) imaging, three dimensional micro-computed tomographic (μCT) imaging, histological analysis, immunohistochemistry and biomechanical measurement. Based on the results, both peripheral vessel and new vessel formation were significantly increased by the BMP-2/S-NP/G treatment, along with the bridged defects at as early as 2 weeks, the healed defects at 8 weeks and the reunion of bone marrow cavity at 12 weeks. The results indicated that both controlled release of active BMP-2 and favorable vascularization at the defect site contributed by BMP-2/S-NP/G played a crucial role in accelerating and promoting bone augmentation. This study suggests that BMP-2/S-NP/G demonstrates promise for vascularization and bone regeneration in clinical case of large defect.     

The effect of metallic magnesium degradation products on osteoclast-induced osteolysis and attenuation of NF-κB and NFATc1 signaling

Wear particle-induced aseptic prosthetic loosening is one of the most common reasons for total joint arthroplasty (TJA). Extensive bone destruction (osteolysis) by osteoclasts plays an important role in wear particle-induced peri-implant loosening. Thus, strategies for inhibiting osteoclast function may have therapeutic benefit for prosthetic loosening. Here, we mimicked the process of magnesium (Mg) degradation in vivo and obtained Mg leach liquor (MLL) by immersing pure Mg in culture medium. For the first time, we demonstrated that MLL suppresses osteoclast formation, polarization, and osteoclast bone resorption in vitro. An in vivo assay demonstrated that MLL attenuates wear particle-induced osteolysis. Furthermore, we found that MLL significantly inhibits nuclear factor-κB (NF-κB) activation by retarding inhibitor-κB degradation and subsequent NF-κB nuclear translocation. We also found that MLL attenuates the expression of NFATc1 at both the protein and mRNA levels. These results demonstrate that MLL has anti-osteoclast activity in vitro and prevents wear particle-induced osteolysis in vivo. Collectively, our study suggests that metallic magnesium, one of the orthopedic implants with superior properties, has significant potential for the treatment of osteolysis-related diseases caused by excessive osteoclast formation and function.     

Osteoinductivity potential of rhBMP-2 associated with two carriers in different dosages

The objective of this study was to evaluate bone formation after application of different doses of recombinant human bone morphogenetic protein-2 (rhBMP-2) combined with monoolein or poloxamer gels, in critical bone defects of rats. Forty-five Wistar rats were divided into nine treatment groups with five animals each: I: application of 1 μg rhBMP-2 + monoolein; II: 3 μg rhBMP-2 + monoolein; III: 7 μg rhBMP-2 + monoolein; IV: 1 μg rhBMP-2 + poloxamer; V: 3 μg rhBMP-2 + poloxamer; VI: 7 μg rhBMP-2 + poloxamer; VII: monoolein only; VIII: poloxamer only; and IX: critical bone defect only. A critical-sized defect of 6 mm diameter was produced in the left parietal bone and it was filled with gels of the above mentioned treatments. After 2 weeks, the calvarial bones were removed for histological processing. Bone formation in the groups that received poloxamer gel and rhBMP-2 was not significantly different from the control group (IX). Groups receiving monoolein and rhBMP-2 (1 and 3 μg) and those that received only the carriers (VII and VIII) had less bone formation in relation to the control. The association of rhBMP-2 to both poloxamer and monoolein did not exhibit any significant differentiation in bone formation in comparison with the control group.     

Effects of compatibility of deproteinized antler cancellous bone with various bioactive factors on their osteogenic potential

Combinations of calcium phosphate scaffolds and bioactive factors are promising niche-mimetic solutions for repairing large-sized bone defects. However, the importance of compatibility between scaffolds and bioactive factors on their osteogenic outcomes has been largely ignored. This study aimed to investigate the compatibility of calcinated antler cancellous bone (CACB) scaffolds with various bioactive factors including icariin (ICA), velvet antler polypeptides (VAP) or recombinant human bone morphogenetic protein-2 (rhBMP-2) as well as their combinational osteogenic potential in vitro and in vivo. Scanning electron microscopy and fourier transform infrared spectroscopy confirmed the uniform distribution and chemical stability of the reagents on CABC. In vitro release profiles showed relative steady release of ICA from ICA/CACB, burst VAP release from VAP/CACB, and minimal rhBMP-2 release from rhBMP-2/CACB composites. When compared with VAP and rhBMP-2, incorporation of ICA within CACB resulted in most increased cell attachment, proliferation, alkaline phosphatase activity, osteogenic gene expression, and mineralization of rat bone marrow mesenchymal stem cells. In rabbit mandible critical-sized defects, the most extensive osteogenesis and neovascularization were observed in the ICA/CACB group. Differences between the VAP/CACB and rhBMP-2/CACB groups were not apparent. Interestingly, low pro-inflammatory (TNF-α, IL-6) and high anti-inflammatory (IL-10) mRNA levels were observed at scaffold implantation sites which were in close association with amount of new bone formation. These findings highlight that the compatibility between scaffolds and bioactive factors should been taken into account when considering the formula of optimized bone defect repair.     

Enhanced regeneration of rabbit mandibular defects through a combined treatment of electrical stimulation and rhBMP-2 application

We evaluated the new bone regeneration of a rabbit mandibular defect using hBMSCs under electrical stimulation combined with rhBMP-2 in this study. An inner scaffold prepared by setting a collagen sponge with hBMSCs and hydrogel was placed into a polycaprolactone (PCL) outer box, and an electrical stimulation device was installed between the inner scaffold and the outer box. There were three experimental groups depending on electrical stimulation and application of rhBMP-2. The experimental group was divided into the following three groups. Group 1, in which rhBMP-2 (5 μg/defect) was added to hydrogel and electrical stimulation was not applied; Group 2, in which rhBMP-2 (5 μg/defect) was added as in Group 1 and electrical stimulation was applied; and Group 3, in which electrical stimulation was applied and rhBMP-2 (5 μg/defect) was injected directly into defect site. The delivered electrical stimulation was charge-balanced bi-phasic electric current pulses, and electrical stimulation was conducted for 7 days. The stimulation parameters of the bi-phasic electrical current set at an amplitude of 20 μA, a duration of 100 μs and a frequency of 100 Hz. Four weeks after surgery, new bone formation in each group was evaluated using radiography, histology, and micro-computed tomography (μCT). Groups 2 and 3 exhibited a significant increase in new bone formation compared to Group 1, while Group 3 showed the highest level of new bone regeneration. In a comparison between two groups, Group 2 showed a higher bone volume (BV) by 260 % (p < 0.01) compared with Group 1, and Group 3 showed a higher BV by 442 % (p < 0.01) compared with Group 1. The trend of the bone surface density (ratio of new bone to the real defect volume, BS/TV), trabecular number, and connectivity was identical to that of the BV. The total bone mineral density (BMD) of Groups 2 and 3 showed values higher by the ratios of 103 % (p < 0.01) and 107.5 % (p < 0.01) compared with Group 1, respectively. Part BMD for Groups 2 and 3 showed higher values by the ratios of 104.9 % (p < 0.01) and 122.4 % (p < 0.01) compared with Group 1, respectively. These results suggest that the combined treatment of electrical stimulation, hBMSCs, a collagen sponge, hydrogel, and rhBMP-2 was effective for bone regeneration of large-size mandibular defects. The application of rhBMP-2 with an injection following electrical stimulation demonstrated better efficiency as regards bone regeneration.     

Peri-implant reactivity and osteoinductive potential of immobilized rhBMP-2 on titanium carriers

Recombinant human BMP-2 (rhBMP-2) was immobilized non-covalently and covalently as a monolayer on plasma vapour deposited (PVD) porous commercially pure titanium surfaces in amounts of 5–8 μg cm^?2, providing a ca. 10-fold increase vs. previously reported values [37]. Dissociation of the immobilized [¹²⁵I]rhBMP-2 from the surface occurred in a two-phase exponential decay: a first rapid phase (ca. 15% of immobilized BMP-2) with a half-life of 1–2 days and a second slow sustained release phase (ca. 85% of immobilized BMP-2) with a half-life of 40–60 days. Dissociation rate constants of sustained release of k_?1 = 1.3–1.9 × 10^?7 s^?1 were determined, allowing an estimation of the binding constants (K_A) for the adsorbed rhBMP-2 monolayer, to be around 10¹² M^?1. The rhBMP-2-coated surfaces showed a high level of biological activity, as demonstrated by in vitro epifluorescence tests for alkaline phosphatase with MC3T3-E1 cells and in vivo experiments. In vivo osteoinductivity of rhBMP-2-coated implants was investigated in a gap-healing model in the trabecular bone of the distal femur condylus of sheep. Healing occurred without inflammation or capsule formation. The calculated concentration of released rhBMP-2 in the 1 mm gap ranged from 20 to 98 nM – well above the half-maximal response concentration (K_0.5) for inducing alkaline phosphatase in MC3T3-E1 cells. After 4, 9 and 12 weeks the bone density (BD) and bone-to-implant contact (BIC) of the explanted implants were assessed histomorphometrically. Implants with immobilized rhBMP-2 displayed a significant (2- to 4-fold) increase in BD and BIC values vs. negative controls after 4–9 weeks. Integration of implants by trabecular bone was achieved after 4 weeks, indicating a mean “gap-filling rate” of ～250 μm week^?1. Integration of implants by cortical bone was observed after 9 weeks. Control implants without rhBMP-2 were not osseointegrated. This study demonstrates the feasibility of enhancing peri-implant osseointegration and gap bridging by immobilized rhBMP-2 on implant surfaces which may serve as a model for future clinical applications.     

Reduction of ectopic bone growth in critically-sized rat mandible defects by delivery of rhBMP-2 from kerateine biomaterials

Absorbable collagen sponges (ACS) are used clinically as carriers of recombinant human bone morphogenetic protein 2 (rhBMP-2) to promote bone regeneration. ACS exhibit ectopic bone growth due to delivery of supraphysiological levels of rhBMP-2, which is particularly problematic in craniofacial bone injuries for both functional and esthetic reasons. We hypothesized that hydrogels from the reduced form of keratin proteins (kerateine) would serve as a suitable alternative to ACS carriers of rhBMP-2. The rationale for this hypothesis is that keratin biomaterials degrade slowly in vivo, have modifiable material properties, and have demonstrated capacity to deliver therapeutic agents. We investigated kerateine hydrogels and freeze-dried scaffolds as rhBMP-2 carriers in a critically-sized rat mandibular defect model. ACS, kerateine hydrogels, and kerateine scaffolds loaded with rhBMP-2 achieved bridging in animals by 8 weeks as indicated by micro-computed tomography. Kerateine scaffolds achieved statistically increased bone mineral density compared to ACS and kerateine hydrogels, with levels reaching those of native bone. Importantly, both kerateine hydrogels and kerateine scaffolds had significantly less ectopic bone growth than ACS sponges at both 8 and 16 weeks post-operatively. These studies demonstrate the suitability of keratins as rhBMP-2 carriers due to equal regenerative capacity with reduced ectopic growth compared to ACS.     

Enhanced bone tissue regeneration by antibacterial and osteoinductive silica-HACC-zein composite scaffolds loaded with rhBMP-2

Next-generation orthopedic implants with both osteoinductivity and antibacterial ability are greatly needed. In the present study, biodegradable rhBMP-2 loaded zein-based scaffolds with a macroporous structure were synthesized, and SBA-15 nanoparticles and hydroxypropyltrimethyl ammonium chloride chitosan (HACC) were incorporated into the scaffolds to produce an anti-infective composite scaffold for delivery of osteogenic factors that facilitate the functional repair of bone defects. The silica/HACC/zein scaffolds developed here showed bioactivity, biocompatibility, and effective antibacterial activity. Confocal laser scanning microscopy (CLSM) was used to quantitatively measure the bactericidal efficacy with respect to bacterial adhesion. Results showed that the sample zein-HACC-S20 exhibited long-lasting antibacterial activity against Escherichia coli and Staphylococcus aureus up to 5 d. At a low dosage of rhBMP-2 (ca. 80 μg), the scaffolds released rhBMP-2 protein efficiently at a relatively slow rate, even after 27 d. An ALP activity and ECM mineralization assay showed that the zein-HACC-S20 scaffolds exhibited significant early osteogenic differentiation by generating enhanced ALP product on day 14 and ECM mineralization on day 21. In a mouse model of thigh muscle pouches, zein-S20 and zein-HACC-S20 groups resulted in obvious bone formation and gave more extensive mineralization to the implants than silica free groups, indicating effective bone induction in vivo. In a rabbit model of critical-sized radius bone defects (20 mm in length and 5 mm in diameter), the bone defects were almost fully repaired and bone marrow cavity recanalization was detectable by 3D micro-CT technique and histological analysis after 12 weeks. In this way, the zein-HACC-S20 scaffolds were proven to significantly promote the bone repair. They also demonstrated considerable promise for tissue engineering. Silica/HACC/zein scaffolds with both antibacterial activity and the ability to induce osteogenesis have immense potential in orthopedics and other biomedical applications.     

Pharmacokinetics and osteogenic potential of PEGylated NELL-1 in vivo after systemic administration

Osteoporosis is a skeletal disorder attributable to an imbalance in osteoblast and osteoclast activity. NELL-1, a secretory protein that promotes osteogenesis while suppressing osteoclastic activity, holds potential as an osteoporosis therapy. Recently, we demonstrated that PEGylation of NELL-1 significantly improves its thermostability while preserving its bioactivity in vitro. However, the effect of PEGylation on the pharmacokinetics and osteogenic potential of NELL-1 in vivo have yet to be investigated. The present study demonstrated that PEGylation of NELL-1 significantly increases the elimination half-life time of the protein from 5.5 h to 15.5 h while distributing more than 2–3 times the amount of protein to bone tissues (femur, tibia, vertebrae, calvaria) in vivo when compared to naked NELL-1. In addition, microCT and DXA analyses demonstrated that systemic NELL-PEG therapy administered every 4 or 7 days significantly increases not only femoral and lumbar BMD and percent bone volume, but also new bone formation throughout the overall skeleton after four weeks of treatment. Furthermore, immunohistochemistry revealed increased osteocalcin expression, while TRAP staining showed reduced osteoclast numbers in NELL-PEG groups. Our findings suggest that the PEGylation technique presents a viable and promising approach to further develop NELL-1 into an effective systemic therapeutic for the treatment of osteoporosis.     

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).     

Bioactivity and circulation time of PEGylated NELL-1 in mice and the potential for osteoporosis therapy

Osteoporosis is a progressive bone disease due to low osteoblast activity and/or high osteoclast activity. NELL-1 is a potential therapy for osteoporosis because it specifically increases osteoblast differentiation. However, similar to other protein drugs, the bioavailability of NELL-1 may be limited by its in vivo half-life and rapid clearance from body. The purpose of the present study is to prolong NELL-1 circulation time in vivo by PEGylation with three monomeric PEG sizes (5, 20, 40 kDa). While linear PEG 5k yielded the most efficient PEGylation and the most thermally stable conjugate, linear PEG 20k resulted in the conjugate with the highest M_w and longest in vivo circulation. Compared to non-modified NELL-1, all three PEGylated conjugates showed enhanced thermal stability and each prolonged the in vivo circulation time significantly. Furthermore, PEGylated NELL-1 retained its osteoblastic activity without any appreciable cytotoxicity. These findings motivate further studies to evaluate the efficacy of PEGylated NELL-1 on the prevention and treatment of osteoporosis.     

Does locally delivered Zoledronate influence peri-implant bone formation? – Spatio-temporal monitoring of bone remodeling in vivo

Bisphosphonates are known for their strong inhibitory effect on bone resorption. Their influence on bone formation however is less clear. In this study we investigated the spatio-temporal effect of locally delivered Zoledronate on peri-implant bone formation and resorption in an ovariectomized rat femoral model. A cross-linked hyaluronic acid hydrogel was loaded with the drug and applied bilaterally in predrilled holes before inserting polymer screws. Static and dynamic bone parameters were analyzed based on in vivo microCT scans performed first weekly and then biweekly. The results showed that the locally released Zoledronate boosted bone formation rate up to 100% during the first 17 days after implantation and reduced the bone resorption rate up to 1000% later on. This shift in bone remodeling resulted in an increase in bone volume fraction (BV/TV) by 300% close to the screw and 100% further away. The double effect on bone formation and resorption indicates a great potential of Zoledronate-loaded hydrogel for enhancement of peri-implant bone volume which is directly linked to improved implant fixation.     

Effects of soluble cobalt and cobalt incorporated into calcium phosphate layers on osteoclast differentiation and activation

Metal ions originating from mechanical debris and corrosive wear of prosthetic implant alloys accumulate in peri-implant soft tissues, bone mineral, and body fluids. Eventually, metal ions such as cobalt (II) (Co²⁺), which is a major component of cobalt–chromium-based implant alloys and a known activator of osteolysis, are incorporated into the mineral phase of bone. We hypothesize that the accumulation of Co²⁺ in the mineral could directly activate osteolysis by targeting osteoclasts. To test this hypothesis, we coated tissue culture plastic with a thin layer of calcium phosphate (CaP) containing added traces of Co²⁺, thereby mimicking the bone mineral accumulation of Co²⁺. Murine bone marrow osteoclasts formed in the presence of M-CSF and RANKL were cultured on these surfaces to examine the effects of Co²⁺ on osteoclast formation and resorptive activity. Treatment conditions with Co²⁺ involved incorporation into the CaP layer, adsorption to the mineral surface, or addition to culture media. Micromolar concentrations of Co²⁺ delivered to developing osteoclast precursors by all 3 routes increased both osteoclast differentiation and resorptive function. Compared to CaP layers without Co²⁺, we observed a maximal 75% increase in osteoclast numbers and a 2.3- to 2.7-fold increase in mineral resorption from the tissue culture wells containing 0.1 μm Co²⁺ and 0.1–10 μm Co²⁺, respectively. These concentrations are well within the range found in peri-implant tissues in vivo. This direct effect of Co²⁺ on osteoclasts appears to act independently of the particulate phagocytosis/inflammation-mediated pathways, thus enhancing osteolysis and aseptic implant loosening.     

Feasibility of islet magnetic resonance imaging using ferumoxytol in intraportal islet transplantation

There is a clinical need for an alternative labeling agent for magnetic resonance imaging (MRI) in islet transplantation. We aimed to evaluate the feasibility of islet MRI using ferumoxytol, which is the only clinically-available ultrasmall superparamagnetic iron oxide. We compared islet function and viability of control islets and islets labeled with ferumoxytol and/or a heparin-protamine complex (HPF). Efficacy of ferumoxytol labeling was assessed in both ex vivo and in vivo models. Labeling for 48 h with HPF, but not up to 800 μg/mL ferumoxytol, deranged ex vivo islet viability and function. The T2¹ relaxation time was optimal when islets were labeled with 800 μg/mL of ferumoxytol for 48 h. Prussian blue stain, iron content assay, transmission electron microscopy (TEM) supported internalization of ferumoxytol particles. However, the labeling intensity in the ex vivo MRI of islets labeled with ferumoxytol was much weaker than that of islets labeled with ferucarbotran. In syngeneic intraportal islet transplantation, there was a correlation between the total area of visualized islets and the transplanted islet mass. In conclusion, islet MRI using ferumoxytol was feasible in terms of in vitro and in vivo efficacy and safety. However, the weak labeling efficacy is still a hurdle for the clinical application.     

Early osseointegration of a strontium containing glass ceramic in a rabbit model

The most important property of a bone cement or a bone substitute in load bearing orthopaedic implants is good integration with host bone with reduced bone resorption and increased bone regeneration at the implant interface. Long term implantation of metal-based joint replacements often results in corrosion and particle release, initiating chronic inflammation leading onto osteoporosis of host bone. An alternative solution is the coating of metal implants with hydroxyapatite (HA) or bioglass or the use of bulk bioglass or HA-based composites. In the above perspective, the present study reports the in vivo biocompatibility and bone healing of the strontium (Sr)-stabilized bulk glass ceramics with the nominal composition of 4.5SiO₂–3Al₂O₃–1.5P₂O₅–3SrO–2SrF₂ during short term implantation of up to 12 weeks in rabbit animal model. The progression of healing and bone regeneration was qualitatively and quantitatively assessed using fluorescence microscopy, histological analysis and micro-computed tomography. The overall assessment of the present study establishes that the investigated glass ceramic is biocompatible in vivo with regards to local effects after short term implantation in rabbit animal model. Excellent healing was observed, which is comparable to that seen in response to a commercially available implant of HA-based bioglass alone.     

The stimulation of healing within a rat calvarial defect by mPCL–TCP/collagen scaffolds loaded with rhBMP-2

Bone morphogenetic proteins (BMPs) have been widely investigated for their clinical use in bone repair and it is known that a suitable carrier matrix to deliver them is essential for optimal bone regeneration within a specific defect site. Fused deposited modeling (FDM) allows for the fabrication of medical grade poly ?-caprolactone/tricalcium phosphate (mPCL–TCP) scaffolds with high reproducibility and tailor designed dimensions. Here we loaded FDM fabricated mPCL–TCP/collagen scaffolds with 5 μg recombinant human (rh)BMP-2 and evaluated bone healing within a rat calvarial critical-sized defect. Using a comprehensive approach, this study assessed the newly regenerated bone employing micro-computed tomography (μCT), histology/histomorphometry, and mechanical assessments. By 15 weeks, mPCL–TCP/collagen/rhBMP-2 defects exhibited complete healing of the calvarium whereas the non-BMP-2-loaded scaffolds showed significant less bone ingrowth, as confirmed by μCT. Histomorphometry revealed significantly increased bone healing amongst the rhBMP-2 groups compared to non-treated scaffolds at 4 and 15 weeks, although the % BV/TV did not indicate complete mineralisation of the entire defect site. Hence, our study confirms that it is important to combine microCt and histomorphometry to be able to study bone regeneration comprehensively in 3D. A significant up-regulation of the osteogenic proteins, type I collagen and osteocalcin, was evident at both time points in rhBMP-2 groups. Although mineral apposition rates at 15 weeks were statistically equivalent amongst treatment groups, micro-compression and push-out strengths indicated superior bone quality at 15 weeks for defects treated with mPCL–TCP/collagen/rhBMP-2. Consistently over all modalities, the progression of healing was from empty defect < mPCL–TCP/collagen < mPCL–TCP/collagen/rhBMP-2, providing substantiating data to support the hypothesis that the release of rhBMP-2 from FDM-created mPCL–TCP/collagen scaffolds is a clinically relevant approach to repair and regenerate critically-sized craniofacial bone defects.     

osteoprotegerin-deficient mice develop early onset osteoporosis and arterial calcification

Osteoprotegerin (OPG) is a secreted protein that inhibits osteoclast formation. In this study the physiological role of OPG is investigated by generating OPG-deficient mice. Adolescent and adult OPG^−/− mice exhibit a decrease in total bone density characterized by severe trabecular and cortical bone porosity, marked thinning of the parietal bones of the skull, and a high incidence of fractures. These findings demonstrate that OPG is a critical regulator of postnatal bone mass. Unexpectedly, OPG-deficient mice also exhibit medial calcification of the aorta and renal arteries, suggesting that regulation of OPG, its signaling pathway, or its ligand(s) may play a role in the long observed association between osteoporosis and vascular calcification.     

Diverse effects of Porphyromonas gingivalis on human osteoclast formation

Porphyromonas gingivalis is associated with periodontitis, a chronic inflammatory disease of the tooth-supporting tissues. A major clinical symptom is alveolar bone loss due to excessive resorption by osteoclasts. P. gingivalis may influence osteoclast formation in diverse ways; by interacting directly with osteoclast precursors that likely originate from peripheral blood, or indirectly by activating gingival fibroblasts, cells that can support osteoclast formation. In the present study we investigated these possibilities.Conditioned medium from viable or dead P. gingivalis, or from gingival fibroblasts challenged with viable or dead P. gingivalis were added to human mononuclear osteoclast precursors. After 21 days of culture the number of multinucleated (≥3 nuclei) tartrate resistant acid phosphatase (TRACP)-positive cells was determined as a measure for osteoclast formation.Conditioned medium from viable P. gingivalis, and from fibroblasts with viable P. gingivalis stimulated osteoclast formation (1.6-fold increase p < 0.05). Conditioned medium from dead bacteria had no effect on osteoclast formation, whereas conditioned medium from fibroblasts with dead bacteria stimulated formation (1.4-fold increase, p < 0.05). Inhibition of P. gingivalis LPS activity by Polymyxin B reduced the stimulatory effect of conditioned medium. Interestingly, when RANKL and M-CSF were added to cultures, conditioned media inhibited osteoclast formation (0.6-0.7-fold decrease, p < 0.05).Our results indicate that P. gingivalis influences osteoclast formation in vitro in different ways. Directly, by bacterial factors, likely LPS, or indirectly, by cytokines produced by gingival fibroblasts in response to P. gingivalis. Depending on the presence of RANKL and M-CSF, the effect of P. gingivalis is either stimulatory or inhibitory.