High concentrations of polyelectrolyte complex nanoparticles decrease activity of osteoclasts

Fracture treatment in osteoporotic patients is still challenging. Osteoporosis emerges when there is an imbalance between bone formation and resorption in favor of resorption by osteoclasts. Thus, new implantmaterials for osteoporotic fracture treatment should promote bone formation and reduce bone resorption. Nanoparticles can serve as drug delivery systems for growth factors like Brain-Derived Neurotrophic Factor (BDNF), which stimulated osteoblast differentiation. Therefore, polyelectrolyte complex nanoparticles (PEC-NPs) consisting of poly(l-lysine) (PLL) and cellulose sulfate (CS), with or without addition of BDNF, were used to analyze their effect on osteoclasts in vitro. Live cell images showed that osteoclast numbers decreased after application of high PLL/CS PEC-NPs concentrations independent of whether BDNF was added or not. Real-time RT-PCR revealed that relative mRNA expression of cathepsin K and calcitonin receptor significantly declined after incubation of osteoclasts with high concentrations of PLL/CS PEC-NPs. Furthermore, Enzyme-Linked Immunosorbent Assay indicated that tartrate-resistant acidic phosphatase 5b activity was significantly reduced in the presence of high PLL/CS PEC-NPs concentrations. Consistent with these results, the pit formation analysis showed that less hydroxyapatite was resorbed by osteoclasts after incubation with high concentrations of PLL/CS PEC-NPs. BDNF had no influence on osteoclasts. We conclude that highly concentrated PLL/CS PEC-NPs dosages decreased osteoclastogenesis and osteoclasts activity. Moreover, BDNF might be a promising growth factor for osteoporotic fracture treatment since it did not increase osteoclast activity. © 2019 by the authors

Podoplanin immunopositive lymphatic vessels at the implant interface in a rat model of osteoporotic fractures

Insertion of bone substitution materials accelerates healing of osteoporotic fractures. Biodegradable materials are preferred for application in osteoporotic patients to avoid a second surgery for implant replacement. Degraded implant fragments are often absorbed by macrophages that are removed from the fracture side via passage through veins or lymphatic vessels. We investigated if lymphatic vessels occur in osteoporotic bone defects and whether they are regulated by the use of different materials. To address this issue osteoporosis was induced in rats using the classical method of bilateral ovariectomy and additional calcium and vitamin deficient diet. In addition, wedge-shaped defects of 3, 4, or 5 mm were generated in the distal metaphyseal area of femur via osteotomy. The 4 mm defects were subsequently used for implantation studies where bone substitution materials of calcium phosphate cement, composites of collagen and silica, and iron foams with interconnecting pores were inserted. Different materials were partly additionally functionalized by strontium or bisphosphonate whose positive effects in osteoporosis treatment are well known. The lymphatic vessels were identified by immunohistochemistry using an antibody against podoplanin. Podoplanin immunopositive lymphatic vessels were detected in the granulation tissue filling the fracture gap, surrounding the implant and growing into the iron foam through its interconnected pores. Significant more lymphatic capillaries were counted at the implant interface of composite, strontium and bisphosphonate functionalized iron foam. A significant increase was also observed in the number of lymphatics situated in the pores of strontium coated iron foam. In conclusion, our results indicate the occurrence of lymphatic vessels in osteoporotic bone. Our results show that lymphatic vessels are localized at the implant interface and in the fracture gap where they might be involved in the removal of lymphocytes, macrophages, debris and the implants degradation products. Therefore the lymphatic vessels are involved in implant integration and fracture healing

Small changes in bone structure of female a7 nicotinic acetylcholine receptor knockout mice

BACKGROUND: Recently, analysis of bone from knockout mice identified muscarinic acetylcholine receptor subtype M3 (mAChR M3) and nicotinic acetylcholine receptor (nAChR) subunit a2 as positive regulator of bone mass accrual whereas of male mice deficient for a7-nAChR (a7KO) did not reveal impact in regulation of bone remodeling. Since female sex hormones are involved in fair coordination of osteoblast bone formation and osteoclast bone degradation we assigned the current study to analyze bone strength, composition and microarchitecture of female a7KO compared to their corresponding wild-type mice (a7WT). METHODS: Vertebrae and long bones of female 16-week-old a7KO (n = 10) and a7WT (n = 8) were extracted and analyzed by means of histological, radiological, biomechanical, cell- and molecular methods as well as time of flight secondary ion mass spectrometry (ToF-SIMS) and transmission electron microscopy (TEM). RESULTS: Bone of female a7KO revealed a significant increase in bending stiffness (p<0.05) and cortical thickness (p<0.05) compared to a7WT, whereas gene expression of osteoclast marker cathepsin K was declined. ToF-SIMS analysis detected a decrease in trabecular calcium content and an increase in C4H6N+ (p<0.05) and C4H8N+ (p<0.001) collagen fragments whereas a loss of osteoid was found by means of TEM. CONCLUSIONS: Our results on female a7KO bone identified differences in bone strength and composition. In addition, we could demonstrate that a7-nAChRs are involved in regulation of bone remodelling. In contrast to mAChR M3 and nAChR subunit a2 the a7-nAChR favours reduction of bone strength thereby showing similar effects as a7ß2-nAChR in male mice. nAChR are able to form heteropentameric receptors containing a- and ß-subunits as well as the subunits a7 can be arranged as homopentameric cation channel. The different effects of homopentameric and heteropentameric a7-nAChR on bone need to be analysed in future studies as well as gender effects of cholinergic receptors on bone homeostasis

2019 Proceedings of the 2nd International Conference on Trauma Surgery Technology in Giessen (Germany)

It is now for a second time that we can invite researchers to come to Giessen for an international exchange of the latest research and a discussion of ideas. This year again, the Deutsche Forschungsgemeinschaft (DFG) is sponsoring the event. The main topic for 2019 is 'Vibration in antibacterial and oncological therapy'. Many effects of mechanical vibration on tissue have been discovered so far. Clinical applications relying on vibration exist for a variety of conditions. The intracellular processes, however, are still largely not understood. And reproducibility remains a matter of potential for improvement. DFG funds for the 3rd conference in 2020 have already been approved for a focus on multifunctional trauma surgery implants.Deutsche Forschungsgemeischaft (DFG), German

High Concentrations of Polyelectrolyte Complex Nanoparticles Decrease Activity of Osteoclasts

Fracture treatment in osteoporotic patients is still challenging. Osteoporosis emerges when there is an imbalance between bone formation and resorption in favor of resorption by osteoclasts. Thus, new implant materials for osteoporotic fracture treatment should promote bone formation and reduce bone resorption. Nanoparticles can serve as drug delivery systems for growth factors like Brain-Derived Neurotrophic Factor (BDNF), which stimulated osteoblast differentiation. Therefore, polyelectrolyte complex nanoparticles (PEC-NPs) consisting of poly(l-lysine) (PLL) and cellulose sulfate (CS), with or without addition of BDNF, were used to analyze their effect on osteoclasts in vitro. Live cell images showed that osteoclast numbers decreased after application of high PLL/CS PEC-NPs concentrations independent of whether BDNF was added or not. Real-time RT-PCR revealed that relative mRNA expression of cathepsin K and calcitonin receptor significantly declined after incubation of osteoclasts with high concentrations of PLL/CS PEC-NPs. Furthermore, Enzyme-Linked Immunosorbent Assay indicated that tartrate-resistant acidic phosphatase 5b activity was significantly reduced in the presence of high PLL/CS PEC-NPs concentrations. Consistent with these results, the pit formation analysis showed that less hydroxyapatite was resorbed by osteoclasts after incubation with high concentrations of PLL/CS PEC-NPs. BDNF had no influence on osteoclasts. We conclude that highly concentrated PLL/CS PEC-NPs dosages decreased osteoclastogenesis and osteoclasts activity. Moreover, BDNF might be a promising growth factor for osteoporotic fracture treatment since it did not increase osteoclast activity

Effects of BDNF and PEC Nanoparticles on Osteocytes

Bone substitute materials loaded with mediators that stimulate fracture healing are demanded in the clinical treatment in trauma surgery and orthopedics. Brain-derived neurotrophic factor (BDNF) enhances the proliferation and differentiation of mesenchymal stem cells into osteoblast. To load the implants with BDNF, a drug delivery system that allows the release of BDNF under spatiotemporal control would improve functionality. Polyelectrolyte complex nanoparticles (PECNP) have been reported as a suitable drug delivery system. The suitability of PECNP in contact with osteocytes as the main cell type of bone is not known so far. Thus, we aimed to verify that BDNF and PECNP loaded with BDNF (PECNP+BDNF) as well as pure PECNP have no negative effects on osteocytes in vitro. Therefore, the murine osteocyte cell line MLO-Y4 was treated with BDNF and PECNP+BDNF. The effects on proliferation were analyzed by the BrdU test (n = 5). The results demonstrated a significant increase in proliferation 24 h after BDNF application, whereas PECNP+BDNF did not lead to significant changes. Thus, we conclude that BDNF is an appropriate mediator to stimulate osteocytes. Since the addition of PECNP did not affect the viability of osteocytes, we conclude that PECNP are a suitable drug delivery system for bone implants. © 2020 by the authors

Effects of new beta-type Ti-40Nb implant materials, brain-derived neurotrophic factor, acetylcholine and nicotine on human mesenchymal stem cells of osteoporotic and non osteoporotic donors.

Treatment of osteoporotic fractures is still challenging and an urgent need exists for new materials, better adapted to osteoporotic bone by adjusted Young's modulus, appropriate surface modification and pharmaceuticals.Titanium-40-niobium alloys, mechanically ground or additionally etched and titanium-6-aluminium-4-vanadium were analyzed in combination with brain-derived neurotrophic factor, acetylcholine and nicotine to determine their effects on human mesenchymal stem cells in vitro over 21 days using lactate dehydrogenase and alkaline phosphatase assays, live cell imaging and immunofluorescence microscopy.Cell number of human mesenchymal stem cells of osteoporotic donors was increased after 14 d in presence of ground titanium-40-niobium or titanium-6-aluminium-4-vanadium, together with brain-derived neurotrophic factor. Cell number of human mesenchymal stem cells of non osteoporotic donors increased after 21 d in presence of titanium-6-aluminium-4-vanadium without pharmaceuticals. No significant increase was measured for ground or etched titanium-40-niobium after 21 d. Osteoblast differentiation of osteoporotic donors was significantly higher than in non osteoporotic donors after 21 d in presence of etched, ground titanium-40-niobium or titanium-6-aluminium-4-vanadium accompanied by all pharmaceuticals tested. In presence of all alloys tested brain-derived neurotrophic factor, acetylcholine and nicotine increased differentiation of cells of osteoporotic donors and accelerated it in non osteoporotic donors.We conclude that ground titanium-40-niobium and brain-derived neurotrophic factor might be most suitable for subsequent in vivo testing

Live cell images of hMSCs after 21 days of differentiation in osteogenic medium <i>in vitro</i>.

<p>Shown are hMSCs of osteoporotic (left) and non osteoporotic (right) donors in presence of etched (1^st row) or ground Ti-40Nb (2^nd row), Ti-6Al-4V (3^rd row) or without Ti (4^th row) in presence of BDNF (A), ACh (B), Nic (C) or without pharmaceuticals serving as controls (D). White arrows indicate mineral. The images show cells of different donors as typical representative of 4 independent experiments. Black regions at the margin of pictures show Ti alloys. Scale bar shown in A applies to all photographs in this figure.</p