Biological performance of novel phosphate-based glass microspheres for mesenchymal stem cell therapy in osteoporotic patients

In this study, degradable phosphate-based bulk or porous glass microspheres (BGMS or PGMS), with nominal molar compositions of P45-(45P2O5-16CaO-24MgO-11Na2O-4Fe2O3) and P40-(40P2O5-16CaO-24MgO-20Na2O), were evaluated for cytotoxicity, cytocompatibility and osteogenic potential for Mesenchymal stem cell (MSC)-based therapy in osteoporotic patients. Evaluations were performed using direct-contact and indirect-contact bone marrow derived human MSC (hMSC)-based experiments, in addition to material characterisations such as morphology, elemental composition and degradation behaviour, which were correlated to the hMSC experiments. Degradation of microspheres (MS) was measured using a novel method where Scanning Electron micrographs was used to assess the number of MS with surface damage (cracks and peeling effect), over 42 days of degradation in culture medium. Results showed that after 42 days, 2%, 46% and 29% of P45 BGMS, P40 BGMS and P40 PGMS, respectively, had cracks or peeling off surfaces. The results for direct-contact hMSC-experiments showed that P45 BGMS supported 1.4 times more hMSCs than P40 BGMS over 31 days of culture period. However, P45 BGMS were not osteoinductive, possibly due to hydrophobic nature of this glass and its slower dissolution rate. On the other hand, in comparison to P45 BGMS, hMSCs seeded on P40 BGMS showed up to 1.7 times higher alkaline phosphatase (ALP) activity on Day 7, up to 1.5 times more collagen and at least 6 times more Ca deposited in extracellular matrix, in addition to osteocalcin on Day 21 of culture, which strongly indicated the osteoinductive nature of P40 BGMS. This effect was also confirmed through indirect-contact experiments where there was higher collagen and Ca production by hMSCs was observed after 25 days of culture in P40 BGMS-conditioned medium as compared to control (no MS) or P45-conditioned medium. Elemental analysis using Energy Dispersive X-Ray Spectroscopic (EDS) analysis revealed that the Ca-based porogen used in the manufacturing of PGMS, may have been retained on the edges of the pores in PGMS. Therefore, an acid-washing step was introduced at the end of manufacturing process in order to remove the porogen and limit the possible cytotoxic effect of porogen and excess calcium. Characterisation results indicated that acid washing changed the physicality of these microspheres without changing their chemical composition. For example, mean and mode pore window sizes on the surface of PGMS increased from 2.63 μm to 2.73 μm and from 1.15 μm to 1.53 μm, respectively, and closed porosity decreased by 27%, as a result of acid washing. However, more detailed EDS analysis revealed that the Ca-based porogen was not being completely removed from PGMS even after acid washing and this may need further investigation. Cytotoxicity evaluations over 7 days of elution (indirect-contact hMSC experiments) suggested that there was marked improvement in hMSC membrane integrity and metabolic activity in PGMS neat extracts after acid washing. Moreover, direct-contact hMSC experiments also showed higher DNA content on acid washed (AW) P40 PGMS over 7 days of culture. Therefore, based on these results, it was hypothesised that acid washing may have opened up some of the pores and removed some of the glass fragments from PGMS surface, which may have been responsible for cytotoxicity in non-AW PGMS. Direct-contact experiments also showed that over 42-day culture period, there was up to 1.6 times higher hMSC numbers in AW P40 PGMS as compared to P40 BGMS. However, this increase was much lower than the expected range as there was more than 10-fold increase in surface area after the introduction of porosity. This was probably due to presence of <5 μm and <10 μm pore window sizes and interconnection sizes, respectively, in these microspheres, which allowed limited penetration of hMSCs into the porous structures. There was also evidence of at least 2 times more ALP activity up to day 42 of culture and up to 1.7 times more collagen production by day 21 of culture, in case of AW P40 PGMS as compared to P40 BGMS, which strongly indicated a positive effect of porosity on osteogenesis. Interestingly, there was also lower Ca and P deposited by hMSCs in porous microspheres, which was in line with the observations made through indirect-contact experiments, where there was lower collagen and Ca production by hMSCs in P40 PGMS-conditioned medium as compared to P40 BGMS-conditioned medium. This negative effect of PGMS was hypothesised due to excess release of glass fragments/particulates and calcium ions into the medium, possibly leading to cytotoxicity. Based on the results shown here, there is a potential of P40 BGMS and AW P40 PGMS for hMSC-based bone repair therapy. However, future work needs to be done in order to limit the delamination of glass surfaces and release of glass fragments/particulates from these MS, as a result of degradation

Fucoidan Inhibition of Osteosarcoma Cells Is Species and Molecular Weight Dependent.

Fucoidan is a brown algae-derived polysaccharide having several biomedical applications. This study simultaneously compares the anti-cancer activities of crude fucoidans from Fucus vesiculosus and Sargassum filipendula, and effects of low (LMW, 10-50 kDa), medium (MMW, 50-100 kDa) and high (HMW, >100 kDa) molecular weight fractions of S. filipendula fucoidan against osteosarcoma cells. Glucose, fucose and acid levels were lower and sulphation was higher in F. vesiculosus crude fucoidan compared to S. filipendula crude fucoidan. MMW had the highest levels of sugars, acids and sulphation among molecular weight fractions. There was a dose-dependent drop in focal adhesion formation and proliferation of cells for all fucoidan-types, but F. vesiculosus fucoidan and HMW had the strongest effects. G1-phase arrest was induced by F. vesiculosus fucoidan, MMW and HMW, however F. vesiculosus fucoidan treatment also caused accumulation in the sub-G1-phase. Mitochondrial damage occurred for all fucoidan-types, however F. vesiculosus fucoidan led to mitochondrial fragmentation. Annexin V/PI, TUNEL and cytochrome c staining confirmed stress-induced apoptosis-like cell death for F. vesiculosus fucoidan and features of stress-induced necrosis-like cell death for S. filipendula fucoidans. There was also variation in penetrability of different fucoidans inside the cell. These differences in anti-cancer activity of fucoidans are applicable for osteosarcoma treatment

Role of geometrical cues in bone marrow-derived mesenchymal stem cell survival, growth and osteogenic differentiation

Mesenchymal stem cells play a vital role in bone formation process by differentiating into osteoblasts, in a tissue that offers not a flat but a discontinuous three-dimensional (3D) topography in vivo. In order to understand how geometry may be affecting mesenchymal stem cells, this study explored the influence of 3D geometry on mesenchymal stem cell-fate by comparing cell growth, viability and osteogenic potential using monolayer (two-dimensional, 2D) with microsphere (3D) culture systems normalised to surface area. The results suggested lower cell viability and reduced cell growth in 3D. Alkaline phosphatase activity was higher in 3D; however, both collagen and mineral deposition appeared significantly lower in 3D, even after osteogenic supplementation. Also, there were signs of patchy mineralisation in 3D with or without osteogenic supplementation as early as day 7. These results suggest that the convex surfaces on microspheres and inter-particulate porosity may have led to variable cell morphology and fate within the 3D culture. This study provides deeper insights into geometrical regulation of mesenchymal stem cell responses applicable for bone tissue engineering

Performance of multiphase scaffolds for bone repair based on two-photon polymerized poly(d,l-lactide-co-ɛ-caprolactone), recombinamers hydrogel and nano-HA

Multiphase hybrids were fabricated from poly(d,l-lactide-co-ɛ-caprolactone) (PLCL) copolymer scaffolds impregnated with silk-elastin-like recombinamers (SELRs) hydrogel containing 2 wt% hydroxyapatite nanoparticles (nHA). The PLCL scaffolds, triply-periodic minimal surface geometry, were manufactured using two-photon stereolithography. In vitro degradation studies were conducted on PLCL scaffolds in inflamed tissue mimic media (pH ~ 4.5–6.5) or phosphate buffered saline (PBS) at 37 °C. Compression test revealed instant shape recovery of PLCL scaffolds after compression to 70% strain, ideal for arthroscopic delivery. Degradation of these scaffolds was accelerated in acidic media, where mass loss and compressive properties at day 56 were about 2–6 times lower than the scaffolds degraded in PBS. No significant difference was seen in the compressive properties between PLCL scaffolds and the hybrids due to the order of magnitude difference between the hydrogels and the PLCL scaffolds. Moreover, degradation properties of the hybrids did not significantly change by inclusion of SELR+/−nHA nanocomposite hydrogels. The hybrids lost approximately 40% and 84% of their initial weight and mechanical properties, respectively after 112 days of degradation. Cytotoxicity assessment revealed no cytotoxic effects of PLCL or PLCL-SELR+/−2%nHA scaffolds on bone marrow-derived human Mesenchymal Stem Cells. These findings highlight the potential of these hybrid constructs for bone and cartilage repair

Long-Term Culture of Stem Cells on Phosphate-Based Glass Microspheres: Synergistic Role of Chemical Formulation and 3D Architecture

Phosphate-based glasses (PBGs) are biomaterials that degrade under physiological conditions and can be modified to release various ions depending on end applications. This study utilized slow-degrading (P45:45P2O5-16CaO-24MgO-11Na2O10 4Fe2O3, mol %) and comparatively faster degrading (P40:40P2O5-16CaO-24MgO-20Na2O, mol %) PBG microspheres with or without porosity, to evaluate the combined effect of chemical formulation and geometry on human mesenchymal stem cells (MSCs), a clinically relevant cell source for orthopedic applications. Scanning electron microscopy showed 2, 46, and 29% of P45 bulk (P45-B), P40 bulk (P40-B), and P40 porous (P40-P) microspheres, respectively, that had cracks or peeling off surfaces after 42 days of incubation in culture medium. Cytotoxicity assessment showed that glass debris released into the culture medium may interact with cells and affect their survival. Direct-contact cell experiments up to 42 days showed that P45-B microspheres did not sustain viable long-term cell cultures and did not facilitate extracellular matrix formation. On the other hand, P40-B microspheres enhanced alkaline phosphatase activity, calcium deposition, and collagen and osteocalcin production in MSCs. Introduction of porosity in P40 glass further enhanced these parameters and proliferation at later time points. The small pore windows

Traditional multiwell plates and petri dishes limit the evaluation of the effects of ultrasound on cells in vitro

Ultrasound accelerates healing in fractured bone; however, the mechanisms responsible are poorly understood. Experimental setups and ultrasound exposures vary or are not adequately characterized across studies, resulting in inter-study variation and difficulty in concluding biological effects. This study investigated experimental variability introduced through the cell culture platform used. Continuous wave ultrasound (45 kHz; 10, 25 or 75 mW/cm2, 5 min/d) was applied, using a Duoson device, to Saos-2 cells seeded in multiwell plates or Petri dishes. Pressure field and vibration quantification and finite-element modelling suggested formation of complex interference patterns, resulting in localized displacement and velocity gradients, more pronounced in multiwell plates. Cell experiments revealed lower metabolic activities in both culture platforms at higher ultrasound intensities and absence of mineralization in certain regions of multiwell plates but not in Petri dishes. Thus, the same transducer produced variable results in different cell culture platforms. Analysis on Petri dishes further revealed that higher intensities reduced vinculin expression and distorted cell morphology, while causing mitochondrial and endoplasmic reticulum damage and accumulation of cells in sub-G1 phase, leading to cell death. More defined experimental setups and reproducible ultrasound exposure systems are required to study the real effect of ultrasound on cells for development of effective ultrasound-based therapies not just limited to bone repair and regeneration

Traditional multiwell plates and petri dishes limit the evaluation of the effects of ultrasound on cells in vitro

Ultrasound accelerates healing in fractured bone; however, the mechanisms responsible are poorly understood. Experimental setups and ultrasound exposures vary or are not adequately characterized across studies, resulting in inter-study variation and difficulty in concluding biological effects. This study investigated experimental variability introduced through the cell culture platform used. Continuous wave ultrasound (45 kHz; 10, 25 or 75 mW/cm2, 5 min/d) was applied, using a Duoson device, to Saos-2 cells seeded in multiwell plates or Petri dishes. Pressure field and vibration quantification and finite-element modelling suggested formation of complex interference patterns, resulting in localized displacement and velocity gradients, more pronounced in multiwell plates. Cell experiments revealed lower metabolic activities in both culture platforms at higher ultrasound intensities and absence of mineralization in certain regions of multiwell plates but not in Petri dishes. Thus, the same transducer produced variable results in different cell culture platforms. Analysis on Petri dishes further revealed that higher intensities reduced vinculin expression and distorted cell morphology, while causing mitochondrial and endoplasmic reticulum damage and accumulation of cells in sub-G1 phase, leading to cell death. More defined experimental setups and reproducible ultrasound exposure systems are required to study the real effect of ultrasound on cells for development of effective ultrasound-based therapies not just limited to bone repair and regeneration

Antiinflammatory Therapy with Canakinumab for Atherosclerotic Disease

Background: Experimental and clinical data suggest that reducing inflammation without affecting lipid levels may reduce the risk of cardiovascular disease. Yet, the inflammatory hypothesis of atherothrombosis has remained unproved. Methods: We conducted a randomized, double-blind trial of canakinumab, a therapeutic monoclonal antibody targeting interleukin-1β, involving 10,061 patients with previous myocardial infarction and a high-sensitivity C-reactive protein level of 2 mg or more per liter. The trial compared three doses of canakinumab (50 mg, 150 mg, and 300 mg, administered subcutaneously every 3 months) with placebo. The primary efficacy end point was nonfatal myocardial infarction, nonfatal stroke, or cardiovascular death. RESULTS: At 48 months, the median reduction from baseline in the high-sensitivity C-reactive protein level was 26 percentage points greater in the group that received the 50-mg dose of canakinumab, 37 percentage points greater in the 150-mg group, and 41 percentage points greater in the 300-mg group than in the placebo group. Canakinumab did not reduce lipid levels from baseline. At a median follow-up of 3.7 years, the incidence rate for the primary end point was 4.50 events per 100 person-years in the placebo group, 4.11 events per 100 person-years in the 50-mg group, 3.86 events per 100 person-years in the 150-mg group, and 3.90 events per 100 person-years in the 300-mg group. The hazard ratios as compared with placebo were as follows: in the 50-mg group, 0.93 (95% confidence interval [CI], 0.80 to 1.07; P = 0.30); in the 150-mg group, 0.85 (95% CI, 0.74 to 0.98; P = 0.021); and in the 300-mg group, 0.86 (95% CI, 0.75 to 0.99; P = 0.031). The 150-mg dose, but not the other doses, met the prespecified multiplicity-adjusted threshold for statistical significance for the primary end point and the secondary end point that additionally included hospitalization for unstable angina that led to urgent revascularization (hazard ratio vs. placebo, 0.83; 95% CI, 0.73 to 0.95; P = 0.005). Canakinumab was associated with a higher incidence of fatal infection than was placebo. There was no significant difference in all-cause mortality (hazard ratio for all canakinumab doses vs. placebo, 0.94; 95% CI, 0.83 to 1.06; P = 0.31). Conclusions: Antiinflammatory therapy targeting the interleukin-1β innate immunity pathway with canakinumab at a dose of 150 mg every 3 months led to a significantly lower rate of recurrent cardiovascular events than placebo, independent of lipid-level lowering. (Funded by Novartis; CANTOS ClinicalTrials.gov number, NCT01327846.

Biological performance of novel phosphate-based glass microspheres for mesenchymal stem cell therapy in osteoporotic patients

In this study, degradable phosphate-based bulk or porous glass microspheres (BGMS or PGMS), with nominal molar compositions of P45-(45P2O5-16CaO-24MgO-11Na2O-4Fe2O3) and P40-(40P2O5-16CaO-24MgO-20Na2O), were evaluated for cytotoxicity, cytocompatibility and osteogenic potential for Mesenchymal stem cell (MSC)-based therapy in osteoporotic patients. Evaluations were performed using direct-contact and indirect-contact bone marrow derived human MSC (hMSC)-based experiments, in addition to material characterisations such as morphology, elemental composition and degradation behaviour, which were correlated to the hMSC experiments. Degradation of microspheres (MS) was measured using a novel method where Scanning Electron micrographs was used to assess the number of MS with surface damage (cracks and peeling effect), over 42 days of degradation in culture medium. Results showed that after 42 days, 2%, 46% and 29% of P45 BGMS, P40 BGMS and P40 PGMS, respectively, had cracks or peeling off surfaces. The results for direct-contact hMSC-experiments showed that P45 BGMS supported 1.4 times more hMSCs than P40 BGMS over 31 days of culture period. However, P45 BGMS were not osteoinductive, possibly due to hydrophobic nature of this glass and its slower dissolution rate. On the other hand, in comparison to P45 BGMS, hMSCs seeded on P40 BGMS showed up to 1.7 times higher alkaline phosphatase (ALP) activity on Day 7, up to 1.5 times more collagen and at least 6 times more Ca deposited in extracellular matrix, in addition to osteocalcin on Day 21 of culture, which strongly indicated the osteoinductive nature of P40 BGMS. This effect was also confirmed through indirect-contact experiments where there was higher collagen and Ca production by hMSCs was observed after 25 days of culture in P40 BGMS-conditioned medium as compared to control (no MS) or P45-conditioned medium. Elemental analysis using Energy Dispersive X-Ray Spectroscopic (EDS) analysis revealed that the Ca-based porogen used in the manufacturing of PGMS, may have been retained on the edges of the pores in PGMS. Therefore, an acid-washing step was introduced at the end of manufacturing process in order to remove the porogen and limit the possible cytotoxic effect of porogen and excess calcium. Characterisation results indicated that acid washing changed the physicality of these microspheres without changing their chemical composition. For example, mean and mode pore window sizes on the surface of PGMS increased from 2.63 μm to 2.73 μm and from 1.15 μm to 1.53 μm, respectively, and closed porosity decreased by 27%, as a result of acid washing. However, more detailed EDS analysis revealed that the Ca-based porogen was not being completely removed from PGMS even after acid washing and this may need further investigation. Cytotoxicity evaluations over 7 days of elution (indirect-contact hMSC experiments) suggested that there was marked improvement in hMSC membrane integrity and metabolic activity in PGMS neat extracts after acid washing. Moreover, direct-contact hMSC experiments also showed higher DNA content on acid washed (AW) P40 PGMS over 7 days of culture. Therefore, based on these results, it was hypothesised that acid washing may have opened up some of the pores and removed some of the glass fragments from PGMS surface, which may have been responsible for cytotoxicity in non-AW PGMS. Direct-contact experiments also showed that over 42-day culture period, there was up to 1.6 times higher hMSC numbers in AW P40 PGMS as compared to P40 BGMS. However, this increase was much lower than the expected range as there was more than 10-fold increase in surface area after the introduction of porosity. This was probably due to presence of <5 μm and <10 μm pore window sizes and interconnection sizes, respectively, in these microspheres, which allowed limited penetration of hMSCs into the porous structures. There was also evidence of at least 2 times more ALP activity up to day 42 of culture and up to 1.7 times more collagen production by day 21 of culture, in case of AW P40 PGMS as compared to P40 BGMS, which strongly indicated a positive effect of porosity on osteogenesis. Interestingly, there was also lower Ca and P deposited by hMSCs in porous microspheres, which was in line with the observations made through indirect-contact experiments, where there was lower collagen and Ca production by hMSCs in P40 PGMS-conditioned medium as compared to P40 BGMS-conditioned medium. This negative effect of PGMS was hypothesised due to excess release of glass fragments/particulates and calcium ions into the medium, possibly leading to cytotoxicity. Based on the results shown here, there is a potential of P40 BGMS and AW P40 PGMS for hMSC-based bone repair therapy. However, future work needs to be done in order to limit the delamination of glass surfaces and release of glass fragments/particulates from these MS, as a result of degradation