Cellular Alkaline Phosphatase Research Articles

Abstract 2920: In vitro assays for investigating the effects of cancer therapeutics on bone

Abstract Bone microenvironment is an important factor in regulating bone metastasis. When cancer cells home to bone they secrete factors that stimulate osteoclast activity leading to increased bone resorption. Stimulatory factors released from the resorbed bone matrix in turn promote proliferation of cancer cells and this process, referred as the vicious cycle, often leads to osteolytic lesions in bone when osteoclastic bone resorption exceeds osteoblastic bone formation. The vicious cycle observed in osteolytic disease occurs also in osteoblastic metastases, and in addition cancer cells produce osteoblast-stimulating factors. Cancers with bone metastases often include both osteolytic and osteoblastic lesions. Our aim was to establish in vitro cell culture models to study the effects of cancer therapeutics on osteoblast and osteoclast differentiation and activity. In the osteoclast differentiation assay, human osteoclast precursor cells (Lonza) were cultured on bovine bone slices for 7 days. Denosumab was added in the cultures at day 0, and tartrate-resistant acid phosphatase isoform 5b (TRACP 5b) activity was measured from the culture medium collected at day 7. Osteoclast activity was studied by allowing osteoclasts resorb bone after 7 days’ maturation period for additional 3 days in the presence of odanacatib. The amount of C-terminal cross-linked telopeptides of type I collagen (CTX-I) was measured in the culture medium collected at day 10 to quantitate osteoclast activity. Denosumab and odanacatib showed strong concentration dependent inhibition of osteoclast differentiation and activity, respectively. A mouse osteoblast progenitor cell line KS483 (Pharmatest Services) was used to study differentiation and activity of osteoblasts with test compounds bone morphogenetic protein 2 (BMP-2) and estradiol (E2). In the osteoblast differentiation assay, the activity of cellular alkaline phosphatase (ALP), a marker of osteoblast differentiation, was measured at day 8. In the osteoblast activity assay, the cells were cultured for 13 days. N-terminal propeptide of type I procollagen (PINP) secreted into the culture medium was determined at day 11 to indicate the effects on organic bone formation and calcium deposition was determined at the end of the study as a marker of inorganic bone formation. BMP-2 and E2 stimulated osteoblast differentiation and activity indicated by the increase in ALP activity, PINP and calcium levels. We conclude that these culture systems can be used for studying the effects of cancer therapeutics and identifying new potential compounds affecting the disease process of bone metastases with different mechanisms of action. Citation Format: Jenni H. Mäki-Jouppila, Mervi Ristola, Jukka Rissanen, Katja Fagerlund. In vitro assays for investigating the effects of cancer therapeutics on bone [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2920.

Icariin promotes bone marrow mesenchymal stem cells osteogenic differentiation via the mTOR/autophagy pathway to improve ketogenic diet-associated osteoporosis

BackgroundIcariin, a traditional Chinese medicine, has demonstrated anti-osteoporotic properties in ovariectomized mice. However, its effectiveness in preventing bone loss induced by ketogenic diet (KD), which mimics osteoporosis in human, remains unexplored. This study aims to investigate icariin’s impact on KD-induced bone loss in mice.MethodsThirty mice were divided into: sham, KD, and KD + icariin groups. Post a 12-week intervention, evaluation including bone microstructures, serum concentrations of tartrate-resistant acid phosphatase (TRAP) and bone-specific alkaline phosphatase (ALP), and femoral tissue expression levels of osteocalcin (OCN) and TRAP. The expression levels of mammalian target of rapamycin (mTOR), ALP, peroxisome proliferator-activated receptor gamma (PPAR-γ), phosphorylated mTOR (p-mTOR), and the autophagy adaptor protein (p62) were also analyzed. Alizarin granule deposition and cellular ALP levels were measured following the induction of bone marrow mesenchymal stem cells (BMSCs) into osteogenesis.ResultsThe study found that KD significantly impaired BMSCs' osteogenic differentiation, leading to bone loss. Icariin notably increased bone mass, stimulated osteogenesis, and reduced cancellous bone loss. In the KD + icariin group, measures such as bone tissue density (TMD), bone volume fraction (BV/TV), trabecular number (Tb.N), and trabecular thickness (Tb.Th) were significantly higher than in the KD group. Additionally, bone trabecular separation (Tb.Sp) was markedly lower in the KD + icariin group. Moreover, icariin increased OCN and ALP levels while suppressing PPAR-γ, TRAP, p62, and p-mTOR. In cellular studies, icariin encouraged osteogenic development in BMSCs under KD conditions.ConclusionsIcariin effectively counteracts bone thinning and improves bone microstructure. Its mechanism likely involves stimulating BMSCs osteogenic differentiation and inhibiting bone resorption, potentially through mTOR downregulation. These findings suggest icariin's potential as an alternative treatment for KD-induced bone loss.

Effect of composite resin monomer BisGMA on odontogenic differentiation of human dental pulp stem cells

Objective : To study the effect of composite resin monomer BisGMA on human dental pulp stem cell (hDPSCs) odontoblasts differentiation, and explore the effect of composite resin filling teeth on dentin formation. Material and Methods : separate and culture hDPSCs, identify cell phenotype by flow cytometry, and induce staining to assess multidirectional differentiation potential. The hDPSCs was stimulated with different concentrations of monomeric BisGMA, and the odontoblast differentiation capacity of each group was determined by CCK-8 assay, alkaline phosphatase activity detection, and q-PCR. Results: CCK-8 showed that the addition of 0.5 mM —— 8 mM BisGMA affected the cell increment, but no significant difference; the concentration of 0.5 mM —— 8 mM BisGMA reduced the cellular alkaline phosphatase activity and further decreased the activity with increasing concentration.The q-PCR results also showed that monomeric BisGMA inhibited the dentin-related gene OC (osteocalcin), DSPP (dentin sialophosphoprotein)Expression, and with the concentration increases, the inhibition is more obvious of. Conclusion : Composite resin monomer BisGMA has a negative effect on odontogenic differentiation of human dental pulp stem cells

Content of bone morphogenetic protein 2 in demineralized bone matrix prepared from different long bones and study of the osteogenic properties in vitro

To measure the concentration of bone morphogenetic protein 2 (BMP-2) in demineralized bone matrix (DBM) prepared from different long bones and to evaluate the osteoinductivity of different DBM on MC3T3-E1 cells. Different bones from the same cadaver donor were used as the initial materials for making DBM, which were divided into ulna group (uDBM), humerus group (hDBM), tibia group (tDBM), and femur group (fDBM) according to the origins, and boiled DBM (cDBM) was taken as the control group. The proteins of DBM were extracted by guanidine hydrochloride, and the concentrations of BMP-2 were determined by ELISA assay. Then the DBM were co-cultured with MC3T3-E1 cells, the proliferation of MC3T3-E1 cells was observed by cell counting kit 8 (CCK-8) assay. The osteogenic differentiation ability of MC3T3-E1 cells was qualitatively observed by alizarin red, alkaline phosphatase (ALP), and Van Gieson staining, and the osteogenic differentiation ability of MC3T3-E1 cells was quantitatively analyzed by ALP content. Linear regression was used to analyze the effect of BMP-2 concentration in DBM on ALP synthesis. There were significant differences in the concentration of BMP-2 among the DBM groups (P<0.05). The concentrations of BMP-2 in the lower limb long bone were higher than those in the upper limb long bone, and the concentration of BMP-2 in the fDBM group was about 35.5 times that in the uDBM group. CCK-8 assay showed that the cells in each group continued to proliferate within 5 days of co-culture, and the absorbance (A) values at different time points were in the order of cDBM group<uDBM group<hDBM group<tDBM group<fDBM group. After co-culture for 14 days, the expressions of ALP, calcified nodules, and collagen fibers in each group were consistent with the distribution of BMP-2 concentration in DBM. The order of ALP content from low to high was cDBM group<uDBM group<hDBM group<tDBM group<fDBM group, and the differences between the groups were significant (P<0.05). Linear regression analysis showed that =0.361+0.017x, the effect of BMP-2 concentration in DBM on cellular ALP content was significant (t=3.552, P=0.005); for every 1 ng/g increase in BMP-2 concentration, ALP content would increase by 0.017 [95%CI (0.006, 0.027)] U/100 mL. The concentration of natural BMP-2 in different long bones varies greatly, and the lower limb long bone is higher than the upper limb long bone. The harvested location of bone material was an important factor affecting the osteoinductivity of DBM.

The Effect of Apple-Derived Nanovesicles on the Osteoblastogenesis of Osteoblastic MC3T3-E1 Cells.

Osteoporosis is characterized by low bone mass and elevated structural deterioration of the bone tissue, resulting in bone weakness with an increased risk of fracture. Considering biological activities of various phytochemicals extracted from apples, we herein demonstrated the potential antiosteoporotic effects of apple-derived nanovesicles (apple NVs) using osteoblastic MC3T3-E1 cells. Apple NVs significantly stimulated the growth of MC3T3-E1 cells. The cellular alkaline phosphatase (ALP) activity was significantly upregulated in the 5 μg/mL apple NVs-treated group. In addition, the concentrarion of mineralized nodules was significantly increased in the apple NVs-treated groups. Furthermore, apple NVs increased the expression of the genes and proteins associated with osteoblast growth and differentiation, such as Runx2, ALP, OPN, and BMP2/4, which further activated ERK- and JNK-related mitogen-activated protein kinase signaling. These results demonstrate that apple NVs have a potential to prevent osteoporosis by promoting osteoblastogenesis in osteoblastic MC3T3-E1 cells through regulating the BMP2/Smad1 pathways.

Growth and Physiological Responses of Chrysotila roscoffensis (Coccolithales) to Different Phosphorus Resources

The growth and physiological process of microalgae interact with environmental nutrients. In the present study, we compared the growth and physiological characteristics of coccolithophore Chrysotila roscoffensis under the effects of phosphate at high and low concentrations, and of organic phosphorus of adenosine triphosphate (ATP) and Na2-glycerophosphate (SG). The growth, photosynthesis, calcification, alkaline phosphatase activity, and inorganic nutrient absorption rate were comparatively studied. The results showed that the culture with a low concentration of initial phosphate (6 μM) had a lower growth rate than that with a high concentration (45 μM). However, a relatively similar maximum cell density could be achieved. Equimolar inorganic phosphorus supported a higher initial growth rate than organic phosphorus. ATP was better than SG at supporting higher growth rates, higher photosynthetic activity, and higher cell density. Cellular alkaline phosphatase (AP) responded rapidly to nutrient variations with sharp changes of activities, independent of the initial P resources. Cellular calcification was at a higher level in groups with lower growth rates. Phosphate in low concentration in the medium was not absorbed during the early growth period while that in high concentration was rapidly absorbed. Instead, phosphate in low concentration was rapidly absorbed in the late stationary phase. The absorption of nitrate was affected by the initial P resources as well. The DIN/DIP ratio in the water varied significantly during the growth periods. The results indicated that C. roscoffensis had flexible physiological strategies in utilizing varied phosphorus resources, and high cell density maintenance of C. roscoffensis may play roles in nutrient conditions in the water. This study may help to extend the understanding of nutrient utilization strategy in microalgae and to apply reference in the application of Chrysotila species in the removal of nutrient pollution.

High-Fat Diet Increases Bone Loss by Inducing Ferroptosis in Osteoblasts.

Current research suggests that chronic high-fat dietary intake can lead to bone loss in adults; however, the mechanism by which high-fat diets affect the development of osteoporosis in individuals is unclear. As high-fat diets are strongly associated with ferroptosis, whether ferroptosis mediates high-fat diet-induced bone loss was the focus of our current study. By dividing the mice into a high-fat diet group, a high-fat diet + ferroptosis inhibitor group and a normal chow group, mice in the high-fat group were given a high-fat diet for 12 weeks. The mice in the high-fat diet + ferroptosis inhibitor group were given 1 mg/kg Fer-1 per day intraperitoneally at the start of the high-fat diet. Microscopic CT scans, histological tests, and biochemical indicators of ferroptosis were performed on bone tissue from all three groups at the end of the modelling period. Mc3t3-E1 cells were also used in vitro and divided into three groups: high-fat medium group, high-fat medium+ferroptosis inhibitor group, and control group. After 24 hours of incubation in high-fat medium, Mc3t3-E1 cells were assayed for ferroptosis marker proteins and biochemical parameters, and osteogenesis induction was performed simultaneously. Cellular alkaline phosphatase content and expression of osteogenesis-related proteins were measured at day 7 of osteogenesis induction. The results showed that a high-fat diet led to the development of femoral bone loss in mice and that this process could be inhibited by ferroptosis inhibitors. The high-fat diet mainly affected the number of osteoblasts produced in the bone marrow cavity. The high-fat environment in vitro inhibited osteoblast proliferation and osteogenic differentiation, and significant changes in ferroptosis-related biochemical parameters were observed. These findings have implications for the future clinical treatment of bone loss caused by high-fat diets.

Alkaline Phosphatase Activity of Serum Affects Osteogenic Differentiation Cultures

Fetal bovine serum (FBS) is a widely used supplement in cell culture medium, despite its known variability in composition, which greatly affects cellular function and consequently the outcome of studies. In bone tissue engineering, the deposited mineralized matrix is one of the main outcome parameters, but using different brands of FBS can result in large variations. Alkaline phosphatase (ALP) is present in FBS. Not only is ALP used to judge the osteogenic differentiation of bone cells, it may affect deposition of mineralized matrix. The present study focused on the enzymatic activity of ALP in FBS of different suppliers and its contribution to mineralization in osteogenic differentiation cultures. It was hypothesized that culturing cells in a medium with high intrinsic ALP activity of FBS will lead to higher mineral deposition compared to media with lower ALP activity. The used FBS types were shown to have significant differences in enzymatic ALP activity. Our results indicate that the ALP activity of the medium not only affected the deposited mineralized matrix but also the osteogenic differentiation of cells as measured by a changed cellular ALP activity of human-bone-marrow-derived mesenchymal stromal cells (hBMSCs). In media with low inherent ALP activity, the cellular ALP activity was increased and played the major role in the mineralization process, while in media with high intrinsic ALP activity contribution from the serum, less cellular ALP activity was measured, and the ALP activity of the medium also contributed to mineral formation substantially. Our results highlight the diverse effects of ALP activity intrinsic to FBS on osteogenic differentiation and matrix mineralization and how FBS can determine the experimental outcomes, in particular for studies investigating matrix mineralization. Once again, the need to replace FBS with more controlled and known additives is highlighted.

Pterostilbene promotes the osteogenic differentiation of MC3T3-E1 cells inhibited by H2O2 by activating the AMPK signaling pathways

Osteoporosis is one of the common degenerative skeletal diseases whose essential mechanism is the imbalance between bone resorption and formation. Currently, the close coordination of signaling pathways at genetic level during bone development is a hot topic of osteoporosis research. The present study is focused on whether pterostilbene protects against hydrogen peroxide (H2O2)-induced osteoblastic cell apoptosis, oxidative stress, and reveals the related underlying mechanisms. MC3T3-E1 osteoblastic cells were cultured and treated with different concentrations of H2O2 (0–1 mM) along with or without pterostilbene, and MTT assay or Annexin V-FITC/propidium iodide staining was applied for measuring cell viability and apoptosis. The in vitro cellular antioxidant analysis was performed using 2,7-dichlorodihydrofluorescein diacetate assay and enzyme-linked-immunosorbent serologic assay against glutathione peroxidase and malondialdehyde. In addition, cellular alkaline phosphatase activity was executed by Alizarin Red S staining. Western blot assay was conducted to determine the expression levels of osteogenic-related markers, including type I collagen, osteopontin, runt-related transcription factor 2, and the 5' adenosine monophosphate-activated protein kinase (AMPK) signaling pathways related proteins. The key finding was that pterostilbene could attenuate the H2O2-induced cellular apoptosis and oxidative stress. Pterostilbene also promoted osteogenic differentiation in H2O2-treated MC3T3-E1 cells through activation of the AMPK pathway. In conclusion, pterostilbene blocked the H2O2-induced MC3T3-E1 cells dysfunction, indicating its potential to be a promising medication for treating osteoporosis.

Mutual chemical effect of autograft and octacalcium phosphate implantation on enhancing intramembranous bone regeneration

ABSTRACT This study examined the effect of a mixture of octacalcium phosphate (OCP) and autologous bone on bone regeneration in rat calvaria critical-sized defect (CSD). Mechanically mixed OCP and autologous bone granules (OCP+Auto), approximately 500 to 1000 μm in diameter, and each individual material were implanted in rat CSD for 8 weeks, and subjected to X-ray micro-computed tomography (micro-CT), histology, tartrate-resistant acid phosphatase (TRAP) staining, and histomorphometry for bone regeneration. Osteoblastic differentiation from mesenchymal stem cells (D1 cells) was examined in the presence of non-contacting materials by alkaline phosphatase (ALP) activity for 21 days. The material properties and medium composition before and after the incubation were determined by selected area electron diffraction (SAED) under transmission electron microscopy (TEM), Fourier transform infrared (FT-IR) spectroscopy, scanning electron microscopy (SEM), and chemical analysis. The results showed that while bone formation coupled with TRAP-positive osteoclastic resorption and cellular ALP activity were the highest in the Auto group, a positive effect per OCP weight or per autologous bone weight on ALP activity was found. Although the OCP structure was maintained even after the incubation (SAED), micro-deposits were grown on OCP surfaces (TEM). Fibrous tissue was also exposed on the autologous bone surfaces (SEM). Through FT-IR absorption, it was determined that bone mineral-like characteristics of the phosphate group increased in the OCP + Auto group. These findings were interpreted as a structural change from OCP to the apatitic phase, a conclusion supported by the medium degree of saturation changes. The results demonstrate the mutual chemical effect of mixing OCP with autologous bone as an active bone substitute material.

Diphlorethohydroxycamalol isolated from Ishige okamurae prevents H2O2-induced oxidative damage via BMP2/Runx2 signaling in osteoblastic MC3T3-E1 cells

Accumulating evidence has shown an association between osteoporosis and oxidative damage. In the present study, the protective effects of diphlorethohydroxycarmalol (DPHC) isolated from the brown algae Ishige okamurae against H2O2-induced oxidative damage via bone morphogenetic protein 2 (BMP2)/ runt-related transcription factor 2 (Runx2) signaling were investigated using MC3T3-E1 osteoblastic cells. DPHC counteracted the reduction in cell viability caused by H2O2 exposure and protected against H2O2-induced dysfunction, demonstrated by improved cellular alkaline phosphatase (ALP) activity and calcium deposition. In addition, treatment with 0.05–0.2 mM DPHC elevated the protein expression of osteoblast differentiation factors type 1 collagen, ALP, p-Smad1/5, Osterix, BMP2, and Runx2, in response to H2O2-induced oxidative damage. Importantly, DPHC decreased the expression levels of receptor activator of nuclear factor kappa-B ligand, which promotes bone resorption, and inhibited the H2O2-induced generation of reactive oxygen species. Taken together, the results suggest that DPHC counteracts the effects of oxidative stress in osteoblastic cells and has the potential to be effective in preventing and alleviating osteoporosis.

Optimal parameters for the enhancement of human osteoblast-like cell proliferation in vitro via shear stress induced by high-frequency mechanical vibration

Introduction: Biomechanical stimulation of cultured human osteoblast-like cells, which is based on controlled mechanical vibration, has been previously indicated, but the exact mechanical parameters that are effective for cells' proliferation enhancement are still elusive due to the lack of direct data recordings from the stimulated cells in culture. Therefore, we developed a low friction tunable system that enables recording of a narrow range of mechanical parameters, above the infrasonic spectrum, that applied uniformly to human osteoblast-like cells in monolayer culture, aiming to identify a range of mechanical parameters that are effective to enhance osteoblast proliferation in vitro. Methods: Human osteoblast-like cells in explant monolayer culture samples were exposed to mechanical vibration in the 10-70Hz range of frequencies for two minutes, in four 24 hours intervals. Cell numbers in culture, cellular alkaline phosphatase activity (a marker of cell maturation), and lactate dehydrogenase activity in culture media (representing cell death) were measured after the mechanical stimulation protocol application and compared statistically to the control cell cultures kept in static conditions. The cell proliferation was deduced from cell number in culture and cell death measurements. Results: We found that 50-70 Hz of vibration frequency protocol (10-30 μm of maximal displacement amplitude, 0.03g of peak-to-peak acceleration) is optimal for enhancing cells' proliferation(p&lt;0.05), with a parallel decrease of their maturation (p&lt;0.01). Discussion: We detected the optimal mechanical parameters of excitation protocol for induction of osteoblast proliferation in vitro by a mechanical platform, which can be used as a standardized method in the research of mechanotransduction in human osteoblast.

Activation of Human Osteoblasts via Different Bovine Bone Substitute Materials With and Without Injectable Platelet Rich Fibrin in vitro.

IntroductionThe aim of the in vitro study was to compare the effect of four bovine bone substitute materials (XBSM) with and without injectable platelet-reach fibrin for viability and metabolic activity of human osteoblasts (HOB) as well as expression of alkaline phosphatase (ALP), bone morphogenetic protein 2 (BMP-2), and osteonectin (OCN).Materials and MethodsCerabone® (CB), Bio-Oss® (BO), Creos Xenogain® (CX) and MinerOss® X (MO) ± i-PRF were incubated with HOB. At day 3, 7, and 10, cell viability and metabolic activity as well as expression of ALP, OCN, and BMP-2, was examined.ResultsFor non-i-PRF groups, the highest values concerning viability were seen for CB at all time points. Pre-treatment with i-PRF increased viability in all groups with the highest values for CB-i-PRF after 3 and 7 and for CX-i-PRF after 10 days. For metabolic activity, the highest rate among non-i-PRF groups was seen for MO at day 3 and for CB at day 7 and 10. Here, i-PRF groups showed higher values than non-i-PRF groups (highest values: CB + i-PRF) at all time points. There was no difference in ALP-expression between groups. For OCN expression in non-i-PRF groups, CB showed the highest values after day 3, CX after day 7 and 10. Among i-PRF-groups, the highest values were seen for CX + i-PRF. At day 3, the highest BMP-2 expression was observed for CX. Here, for i-PRF groups, the highest increase was seen for CX + i-PRF at day 3. At day 7 and 10, there was no significant difference among groups.ConclusionXBSM sintered under high temperature showed increased HOB viability and metabolic activity through the whole period when compared to XBSM manufactured at lower temperatures. Overall, the combination of XBSM with i-PRF improved all cellular parameters, ALP and BMP-2 expression at earlier stages as well as OCN expression at later stages.

Triggering cultured human osteoblast-like cells’ maturation by an extremely low magnitude alternating electromagnetic field

Introduction: Alternating and pulsed electromagnetic magnetic fields (AEMF and PEMF) of different amplitudes and frequencies can induce metabolic and proliferative effects in osteoblasts, but there is no clearly directed tendency of these effects. I hypothesize that there are extremely low triggering parameters of alternating electromagnetic field (EMF) intensity, i.e., above the background magnetic field on earth but below the lowest AEMF and PEMF that have been investigated to date (above 0.07 mT and below 0.4 mT) that induce cellular response. Methods: Accordingly, human monolayer explant culture replica were exposed four times in 24-hour intervals to two minutes of 10 kHz AEMF or PEMF (10 Hz pulses at a basic 5 kHz frequency) with a maximal EMF intensity of 0.2 mT for both. Cell proliferation was estimated from microscopic cell counting and cell death by lactate dehydrogenase (LDH) specific activity in culture media (measured using a colorimetric method). The early marker of osteoblast maturation, cellular alkaline phosphatase (AP) specific activity, was measured using a colorimetric method (n=6 for all experiment conditions). Results: No difference was found in cell numbers in the culture samples exposed either to AEMF or PEMF and in the LDH’s specific activity in culture media in comparison to the unexposed controls (p&gt;0.05, for both). The cellular AP’s specific activity increased significantly only in cell cultures exposed to the 10 kHz AEMF (p=0.011). Conclusions: The triggering for human osteoblast activation for maturation by an extremely low AEMF (10 kHz) is at least 0.2 mT, which is distinct and below the previously found triggering range of a PEMF for proliferation induction. Therefore, application of these EMF parameters in a clinical setup by a separate finetuning of osteoblast proliferation and maturation might have a therapeutic value in enhancing damaged bone regeneration.

P6. A roughened 3D-printed surface enhances stem cell proliferation and osteoblast differentiation

BACKGROUND CONTEXT The advancement of manufacturing techniques, such as 3D-printing, has facilitated the design of complex and previously unattainable implant surface topographies. Research has shown surface properties, especially surface roughness, of an implant directly influence cellular response at the cell-material interface. Alkaline phosphatase (ALP), an early cellular marker of osteoblast differentiation, has been studied extensively to understand osteogenic cellular responses to a substrate. The identification of implant surfaces that promote a osteogenic environment may help identify those that are optimal for healing. PURPOSE The objective of this study was to investigate the effect of a roughened 3D-printed surface on the cellular proliferation and ALP expression of human bone-marrow derived mesenchymal stem cells (BM-MSCs). STUDY DESIGN/SETTING In vitro cell culture study. PATIENT SAMPLE N/A OUTCOME MEASURES Cellular proliferation was measured using a colorimetric, viability assay (alamarBlue™) to estimate the cell count on each substrate at days 1, 3 and 7. ALP activity was measured from cell lysate using a standard ALP assay on day 7. ALP activity was normalized to the total protein in each lysate sample measured using a bicinchoninic acid (BCA) assay also performed on day 7. METHODS Roughness (Ra) was characterized for each substrate in the study using a profilometer. BM-MSCs were seeded at a density of 10,000 cells/cm2 onto smooth polyetheretherkeytone (PEEK), smooth titanium-alloy (TAV), and a roughened, 3DP titanium-alloy (r3DP) substrates (n=6 per group) and cultured for 7 days. Cells were given 4 hours to attach, after which cell medium was changed and the substrates were moved to a separate well plate to remove any unattached cells. Assays were performed to quantify specific ALP cellular signaling, indicative of osteoblast differentiation. RESULTS The r3DP substrates had 15-30 times greater Ra than PEEK and TAV (15.2µm, 1.0µm, and 0.4µm, respectively). Cell count on day 7 for r3DP was significantly greater than PEEK (p 0.05). ALP activity was significantly greater in the r3DP group (8.8±1.1nmol/µg) compared to both PEEK (5.7±1.4nmol/µg) and TAV (4.4±0.9 nmol/µg) (p 0.05). CONCLUSIONS Optimal cellular activity and osteogenic cell signaling are important for osteoblast differentiation, a process needed to foster a cellular environment favorable for healing. Cells on a roughened 3D-printed TAV substrate exhibited significantly higher proliferation compared to PEEK and significantly higher ALP activity than both TAV and PEEK. Therefore, the r3DP substrate facilitated a cellular environment more favorable for osteoblast differentiation than TAV and PEEK substrates. Future research in an animal model is necessary to evaluate the downstream effects of an implant with a roughened 3D-printed TAV surface on the healing process. FDA DEVICE/DRUG STATUS SABLE (Globus Medical) (Approved for this indication)

Electrospun, synthetic bone void filler promotes human MSC function and BMP-2 mediated spinal fusion.

Synthetic bone grafts are often used to achieve a well-consolidated fusion mass in spinal fusion procedures. These bone grafts function as scaffolds, and ideally support cell function and facilitate protein binding. The aim was to characterize an electrospun, synthetic bone void filler (Reb) for its bone morphogenetic protein (BMP)-2 release properties and support of human mesenchymal stem cell (hMSC) function in vitro, and its efficacy in promoting BMP-2-/bone marrow aspirate-(BMA)-mediated posterolateral spinal fusion (PLF) in vivo. BMP-2 release kinetics from Reb versus standard absorbable collagen sponge (ACS) was determined. hMSC adhesion and proliferation on Reb was tested using cell counting, fluorescence microscopy and MTS. Cell osteogenic differentiation was quantified via cellular alkaline phosphatase (ALP) activity. For in vivo analysis, 18 Lewis rats were treated during PLF surgery with the following groups: (I) Reb + BMA, (II) Reb + BMA + BMP-2 and (III) BMA. A safe, minimally effective dose of BMP-2 was used. Fusion consolidation was followed for 3 months using radiography and micro-CT. After sacrifice, fusion rate and biomechanical stiffness was determined using manual palpation, biomechanical tests and histology. In vitro, BMP-2 release kinetics were similar between Reb versus ACS. MSC proliferation and differentiation were increased in the presence of Reb. At 3 months post-surgery, fusion rates were 29% (group I), 100% (group II), and 0% (group III). Biomechanical stiffness was higher in group II versus I. Micro-CT showed an increased bone volume and connectivity density in group II. Trabecular thickness was increased in group I versus II. H&E staining showed newly formed bone in group II only. Reb possesses a high protein binding affinity and promotes hMSC function. Combination with BMA and minimal dose BMP-2 allowed for 100% bone fusion in vivo. This data suggests that a minimally effective dose of BMP-2 can be used when combined with Reb.

Combinations of growth factors for human mesenchymal stem cell proliferation and osteogenic differentiation.

AimsHere we introduce a wide and complex study comparing effects of growth factors used alone and in combinations on human mesenchymal stem cell (hMSC) proliferation and osteogenic differentiation. Certain ways of cell behaviour can be triggered by specific peptides – growth factors, influencing cell fate through surface cellular receptors.MethodsIn our study transforming growth factor β (TGF-β), basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), insulin-like growth factor 1 (IGF-1), and vascular endothelial growth factor (VEGF) were used in order to induce osteogenesis and proliferation of hMSCs from bone marrow. These cells are naturally able to differentiate into various mesodermal cell lines. Effect of each factor itself is pretty well known. We designed experimental groups where two and more growth factors were combined. We supposed cumulative effect would appear when more growth factors with the same effect were combined. The cellular metabolism was evaluated using MTS assay and double-stranded DNA (dsDNA) amount using PicoGreen assay. Alkaline phosphatase (ALP) activity, as early osteogenesis marker, was observed. Phase contrast microscopy was used for cell morphology evaluation.ResultsTGF-β and bFGF were shown to significantly enhance cell proliferation. VEGF and IGF-1 supported ALP activity. Light microscopy showed initial extracellular matrix mineralization after VEGF/IGF-1 supply.ConclusionA combination of more than two growth factors did not support the cellular metabolism level and ALP activity even though the growth factor itself had a positive effect. This is probably caused by interplay of various messengers shared by more growth factor signalling cascades.Cite this article: Bone Joint Res 2020;9(7):412–420.

Photobiomodulation of human osteoblast-like cells invitro by low-intensity-pulsed LED light.

Visible light irradiation is an emerging area in regenerative medicine research. We hypothesized that low‐intensity‐pulsed LED light irradiance may exert photobiomodulatory effects on cultured osteoblast‐like cells. To test this hypothesis, we investigated cell proliferation and markers of cell maturation and metabolic activity following pulsed LED irradiance. Monolayer explant cultures of human osteoblast‐like cells were exposed four times in 24‐h intervals to 2 min of pulsed white LED irradiance of 2.4–2.5 mW·cm−2 and its different spectra of 0.2–0.5 mW·cm−2 (frequency range of 10–40 Hz). Cell proliferation was estimated from microscopic cell counting and cell death by lactate dehydrogenase activity in culture media (measured by a colorimetric method). The early markers of osteoblast maturation and metabolic activity, that is, cellular alkaline phosphatase activity and osteocalcin content, were measured using a colorimetric method and ELISA, respectively. Irradiance of 40 Hz caused the highest increase in cell number (P < 0.01). Osteocalcin content in cells decreased following 40 Hz and 10 Hz irradiance (P < 0.05). The 40 Hz blue range irradiance (diffuse transmittance 420–580 nm, maximal cell irradiance 0.5 mW·cm−2) caused a decrease in alkaline phosphatase cellular activity (P < 0.001) and an increase in media osteocalcin content (P < 0.05). The 40 Hz green range (diffuse transmittance 560–650 nm, maximal cell irradiance 0.4 mW·cm−2) irradiance caused an increase in the number of cells and in cell death. In summary, pulsed (40 Hz) white light irradiance has photomodulatory effects, with its green range spectrum affecting cell proliferation and cell death, and its blue range spectrum affecting cellular maturation and metabolism. The results indicate a low‐intensity threshold of photobiomodulation of osteoblast‐like cells in vitro.

Smart Surface-Enhanced Resonance Raman Scattering Nanoprobe for Monitoring Cellular Alkaline Phosphatase Activity during Osteogenic Differentiation.

High-efficiency induction of bone marrow mesenchymal stem cells (BMSCs) to osteogenic differentiation in vitro can help solve a series of bone diseases such as bone injury, fracture repair, and osteoporosis. In order to explore the optimal conditions for different chemical inducers to promote BMSCs differentiation and the possible differentiation mechanisms, we developed a smart nanoprobe that can achieve in situ alkaline phosphatase (ALP) activity detection during osteogenic differentiation in cells. The smart nanoprobe (Au@BCIP) was designed as the surface decoration of gold nanoparticles (AuNPs) with 5-bromo-4-chloro-3-indolyl phosphate (BCIP). The nanoprobe was co-cultured with differentiated BMSCs at different stages to monitor ALP activity based on an ALP-catalyzed hydrolysis reaction with BCIP as a substrate. The product can be quickly oxidized by dissolved oxygen to obtaina Raman-active species (5,5'-dibromo-4,4'-dichloro-1H,1H-[2,2'] biindolylidene-3,3'-dione). The SERS sensitivity was greatly improved by resonating the excitation wavelength of 632.8 nm. It is a new strategy for tracing bone disease-related ALP activity in an in vivo model with high sensitivity and selectivity and non-invasion. By using this nanoprobe, osteogenic differentiation of cells under osteogenic supplements was assessed and the p38 MAPK signaling pathway for osteogenic differentiation was experimentally evidenced, which are of significance for understanding BMSCs and regulating their osteogenic differentiation process.

Burst, Short, and Sustained Vitamin D3 Applications Differentially Affect Osteogenic Differentiation of Human Adipose Stem Cells.

Incorporation of 1,25(OH)2 vitamin D3 (vitD3) into tissue-engineered scaffolds could aid the healing of critical-sized bone defects. We hypothesize that shorter applications of vitD3 lead to more osteogenic differentiation of mesenchymal stem cells (MSCs) than a sustained application. To test this, release from a scaffold was mimicked by exposing MSCs to exactly controlled vitD3 regimens. Human adipose stem cells (hASCs) were seeded onto calcium phosphate particles, cultured for 20 days, and treated with 124 ng vitD3, either provided during 30 min before seeding ([200 nM]), during the first two days ([100 nM]), or during 20 days ([10 nM]). Alternatively, hASCs were treated for two days with 6.2 ng vitD3 ([10 nM]). hASCs attached to the calcium phosphate particles and were viable (~75%). Cell number was not affected by the various vitD3 applications. VitD3 (124 ng) applied over 20 days increased cellular alkaline phosphatase activity at Days 7 and 20, reduced expression of the early osteogenic marker RUNX2 at Day 20, and strongly upregulated expression of the vitD3 inactivating enzyme CYP24. VitD3 (124 ng) also reduced RUNX2 and increased CYP24 applied at [100 nM] for two days, but not at [200 nM] for 30 min. These results show that 20-day application of vitD3 has more effect on hASCs than the same total amount applied in a shorter time span.