Primary Osteoblast-like Cells Research Articles

Enhancement of bone regeneration by coadministration of angiogenic and osteogenic factors using messenger RNA

BackgroundBone defects remain a challenge today. In addition to osteogenic activation, the crucial role of angiogenesis has also gained attention. In particular, vascular endothelial growth factor (VEGF) is likely to play a significant role in bone regeneration, not only to restore blood supply but also to be directly involved in the osteogenic differentiation of mesenchymal stem cells. In this study, to produce additive angiogenic-osteogenic effects in the process of bone regeneration, VEGF and Runt-related transcription factor 2 (Runx2), an essential transcription factor for osteogenic differentiation, were coadministered with messenger RNAs (mRNAs) to bone defects in the rat mandible.MethodsThe mRNAs encoding VEGF or Runx2 were prepared via in vitro transcription (IVT). Osteogenic differentiation after mRNA transfection was evaluated using primary osteoblast-like cells, followed by an evaluation of the gene expression levels of osteogenic markers. The mRNAs were then administered to a bone defect prepared in the rat mandible using our original cationic polymer-based carrier, the polyplex nanomicelle. The bone regeneration was evaluated by micro-computerized tomography (μCT) imaging, and histologic analyses.ResultsOsteogenic markers such as osteocalcin (Ocn) and osteopontin (Opn) were significantly upregulated after mRNA transfection. VEGF mRNA was revealed to have a distinct osteoblastic function similar to that of Runx2 mRNA, and the combined use of the two mRNAs resulted in further upregulation of the markers. After in vivo administration into the bone defect, the two mRNAs induced significant enhancement of bone regeneration with increased bone mineralization. Histological analyses using antibodies against the Cluster of Differentiation 31 protein (CD31), alkaline phosphatase (ALP), or OCN revealed that the mRNAs induced the upregulation of osteogenic markers in the defect, together with increased vessel formation, leading to rapid bone formation.ConclusionsThese results demonstrate the feasibility of using mRNA medicines to introduce various therapeutic factors, including transcription factors, into target sites. This study provides valuable information for the development of mRNA therapeutics for tissue engineering.

Impact of Harvesting Method and Donor Age on the Behavior of Human Osteoblast-Like Cells.

Autogenous particulate bone grafts are being utilized in oral implantology for minor grafting procedures. This study aimed to investigate the influence of the bone-harvesting technique, donor age, and donor site on proliferation and differentiation of human primary osteoblast-like cells in the cell culture. Autogenous bone particles (20 samples) were harvested from the maxilla and mandible during surgery using two different protocols, and two types of particulate bone grafts were collected: bone chips and bone sludge. Bone samples were cultured in growth medium and, after 2 to 3 weeks, the cells that grew from bone grafts were cultured in the normal and osteogenic medium for 0, 4, 7, and 20 days. DNA, alkaline-phosphatase (ALP), calcium-content measurements, and Alizarin red/toluidine blue staining were performed. Data were analyzed by repeated-measures analysis of variance with Bonferroni test. The level of statistical significance was set at 5% (P < .05). Total DNA, ALP, and calcium content were significantly higher for the bone chip samples compared to the bone sludge samples. Total DNA and ALP content were significantly higher for the patients in age group 1 (≤ 60 years) compared to age group 2 (> 60 years) and was significantly higher for mandibular samples than maxillary samples on day 20. However, the calcium measurement showed no significant difference concerning donor age and donor site. Data analysis revealed that harvesting technique (bone chips vs bone sludge), donor age (≤ 60 years vs > 60 years), and donor site (maxilla vs mandible) influenced the osteogenic potential of the collected particulate bone graft. The bone chips were superior in terms of osteogenic efficacy and should be considered a suitable option for particulate bone graft collection.

Assessment of Immunological Effects of Low-Level Er: YAG Laser Radiation.

Introduction: Low-level laser radiation has a significant effect on cell proliferation. Various investigations into the effect of Er: YAG laser on the treated cell lines have been published. Determining core targeted proteins is an attractive subject. This research aimed at identifying the critical targeted protein by a low-level Er: YAG laser in primary osteoblast-like cells. Methods: Data were extracted from the literature about proteomic assessment of 3.3 J/cm2 of low-level Er: YAG laser radiation on osteoblast-like cells of rat calvaria. The significant differentially expressed proteins plus 100 first neighbors were analyzed via network analysis and gene ontology enrichment. Results: Nine differentially expressed proteins among the 12 queried proteins were included in the main connected component. Analysis revealed that Cxcl1 was a key targeted protein in response to laser radiation. The presence of Cxcl1 in the significant cellular pathways indicated that cell growth and proliferation were affected. Conclusion: It can be concluded that the immune system is affected by the laser to activate cellular defense against stress.

Baghdadite Ceramics Prevent Senescence in Human Osteoblasts and Promote Bone Regeneration in Aged Rats.

Bone fractures and critical-sized bone defects present significant health threats for the elderly who have limited capacity for regeneration due to the presence of functionally compromised senescent cells. A wide range of synthetic materials has been developed to promote the regeneration of critical-sized bone defects, but it is largely unknown if a synthetic biomaterial (scaffold) can modulate cellular senescence and improve bone regeneration in aged scenarios. The current study investigates the interaction of Baghdadite (Ca3ZrSi2O9) ceramic scaffolds with senescent human primary osteoblast-like cells (HOBs) and its bone regeneration capacity in aged rats. A senescent HOB model was established by repeatedly passaging HOBs till passage 7 (P7). Compared to the clinically used hydroxyapatite/tricalcium phosphate (HA/TCP), Baghdadite prevented senescence induction in P7 HOBs and markedly negated the paracrine effect of P7 HOB secretomes that mediated the up-regulations of cellular senescence-associated gene expression levels in P2 HOBs. We further demonstrated that conditioned media extracted from Baghdadite corrected the dysfunctional mitochondria in P7 HOBs. In vivo, the bone regeneration capacity was enhanced when 3D printed Baghdadite scaffolds were implanted in a calvaria critical-sized bone defect model in both young and aged rats compared to HA/TCP scaffolds, but a better effect was observed in aged rats than in young rats. This study suggests that Baghdadite ceramic represents a novel and promising biomaterial approach to promote bone regeneration capacity in the elderly by providing an anti-senescent microenvironment.

Effects of Low-Level Er:YAG Laser Irradiation on Proliferation and Calcification of Primary Osteoblast-Like Cells Isolated From Rat Calvaria.

Several reports have shown that the photo-bio-modulation of cells by various lasers has favorable biological effects. However, the effects of low-level Er:YAG laser irradiation on osteoblasts remain unclear. The purpose of this study was to evaluate the effects of low-level Er:YAG laser irradiation on proliferation and osteogenic differentiation of primary osteoblast-like cells isolated from the calvariae of 3–5-day-old Wistar rats. Cells were irradiated by Er:YAG laser at energy fluences of 2.2, 3.3, and 4.3 J/cm2, respectively. After irradiation, cell surface temperatures were measured and cell proliferation was evaluated by flow cytometry and CCK-8. Calcification was evaluated by measuring areas of Alizarin red S staining after 7, 14, and 21 days culture in osteoinductive medium. Gene expression in non-irradiated and laser-irradiated cells was evaluated by qPCR at 3, 6, and 12 h, as well as 1, 3, 7, and 14 days after irradiation. Microarray analysis was performed to comprehensively evaluate the gene expression of non-irradiated and irradiated cells at 3.3 J/cm2 at 6 h after irradiation. No pronounced increase of cell surface temperature was induced by irradiation. Irradiation did not affect osteoblast-like cell proliferation. Osteoblast-like cell calcification was significantly increased 7 days after Er:YAG laser irradiation at 3.3 J/cm2. Bglap expression was significantly increased in cells irradiated at 3.3 J/cm2 6 h post-irradiation. Microarray analysis showed that irradiation at 3.3 J/cm2 caused an upregulation of inflammation-related genes and downregulation of Wisp2. Gene set enrichment analysis also clarified enrichment of inflammation-related and Notch signaling gene sets. In conclusion, low-level Er:YAG laser irradiation at 3.3 J/cm2 enhanced calcification of primary osteoblast-like cells via enhanced Bglap expression and enriched Notch signaling.

Biodegradable polymer promotes osteogenic differentiation in immortalized and primary osteoblast-like cells

Biodegradable polymers have been broadly used as agents that can complex with and deliver osteoinductive agents, but osteoinductivity of the polymers themselves has been rarely studied. Here we report the osteoinductivity of poly(4-hydroxy-L-proline ester) (PHPE), a biodegradable cationic polymer with cell penetrating properties. Under physiological conditions, PHPE degrades into trans-4-hydroxy-L-proline (trans-Hyp), a non-coded amino acid with essential functions in collagen fibril formation and fibril stability. Treatment of SaOS-2 osteoblast-like cells and hFOB 1.19 primary osteoblast cells with PHPE promoted earlier collagen nodule formation and mineralization of the extracellular matrix compared to untreated cells, even when mineralization activators were absent in the growth medium. Our results indicate that PHPE is a potential osteoinductive agent in vitro that can favor bone regeneration. Moreover, this osteoinductive property could be partly attributed to the degradation product trans-Hyp, which could recapitulate some, but not all of the osteogenic activity. The primary findings of this study can be summarized as follows: treatment of cells with PHPE led to (1) the induction of COL1A1 expression, collagen synthesis and secretion in osteoblast-like cells, (2) mineralization of the ECM in both SaOS-2 and hFOB 1.19 primary osteoblasts, and (3) induction of BMP2 gene and protein expression in osteoblast-like cells, which can promote mineralization of the ECM and regeneration of the bone tissue. Our results suggest that PHPE is a non-cytotoxic polymer and can be potentially used to overcome collagenopathies such as osteogenesis imperfecta.

Ex vivo construction of human primary 3D–networked osteocytes

A human bone tissue model was developed by constructing ex vivo the 3D network of osteocytes via the biomimetic assembly of primary human osteoblastic cells with 20–25μm microbeads and subsequent microfluidic perfusion culture. The biomimetic assembly: (1) enabled 3D–constructed cells to form cellular network via processes with an average cell-to-cell distance of 20–25μm, and (2) inhibited cell proliferation within the interstitial confine between the microbeads while the confined cells produced extracellular matrix (ECM) to form a mechanically integrated structure. The mature osteocytic expressions of SOST and FGF23 genes became significantly higher, especially for SOST by 250 folds during 3D culture. The results validate that the bone tissue model: (1) consists of 3D cellular network of primary human osteocytes, (2) mitigates the osteoblastic differentiation and proliferation of primary osteoblast-like cells encountered in 2D culture, and (3) therefore reproduces ex vivo the phenotype of human 3D–networked osteocytes. The 3D tissue construction approach is expected to provide a clinically relevant and high-throughput means for evaluating drugs and treatments that target bone diseases with in vitro convenience.

Defining the Properties of an Array of \u2013NH2-Modified Substrates for the Induction of a Mature Osteoblast/Osteocyte Phenotype from a Primary Human Osteoblast Population Using Controlled Nanotopography and Surface Chemistry

Accelerating the integration of a joint replacement or the healing of a bone fracture, particularly a complicated non-union fracture, would improve patient welfare and decrease healthcare costs. Currently, an autologous bone graft is the gold standard method for the treatment of complicated non-union fractures, but it is not always possible to harvest such a graft. A proactive highly inductive so-called smart material approach is pertinent in these cases. In this study, the surface chemistry of a previously approved material with desirable bulk material properties was modified to investigate its potential as an economical and effective alternative. The objective was to create stable synthetic chemical coatings that could guide cells along the osteogenic lineage required to generate mineralised tissue that would induce and accelerate bone healing. Primary human osteoblast-like cells were cultured in vitro for 7, 14 and 28 days on amine-terminated (chain length in the range 3–11) silane-modified glass surfaces with controlled nanotopography, to determine how surface chemistry and nanotopography change osteoblast function. The materials were characterised using atomic force microscopy (AFM), scanning electron microscopy (SEM), water contact angle (WCA) and a novel ninhydrin assay. The cells were analysed using qRT-PCR, von Kossa tinctural staining for mineralisation, and visualised using both transmitted white light and electron microscopy. Bone-like nodules, quantified using microscopy, only formed on the short-chain (chain length 3 and 4) amines after 7 days, as did the up-regulation of sclerostin, suggestive of a more mature osteoblast phenotype. In this paper, we report more rapid nodule formation than has previously been observed, without the addition of exogenous factors in the culture medium. This suggests that the coating would improve the integration of implants with bone or be the basis of a smart biomaterial that would accelerate the bone regeneration process.

Mechanosensor polycystin-1 potentiates differentiation of human osteoblastic cells by upregulating Runx2 expression via induction of JAK2/STAT3 signaling axis.

Polycystin-1 (PC1) has been proposed as a chief mechanosensing molecule implicated in skeletogenesis and bone remodeling. Mechanotransduction via PC1 involves proteolytic cleavage of its cytoplasmic tail (CT) and interaction with intracellular pathways and transcription factors to regulate cell function. Here we demonstrate the interaction of PC1-CT with JAK2/STAT3 signaling axis in mechanically stimulated human osteoblastic cells, leading to transcriptional induction of Runx2 gene, a master regulator of osteoblastic differentiation. Primary osteoblast-like PC1-expressing cells subjected to mechanical-stretching exhibited a PC1-dependent increase of the phosphorylated(p)/active form of JAK2. Specific interaction of PC1-CT with pJAK2 was observed after stretching while pre-treatment of cells with PC1 (anti-IgPKD1) and JAK2 inhibitors abolished JAK2 activation. Consistently, mechanostimulation triggered PC1-mediated phosphorylation and nuclear translocation of STAT3. The nuclear phosphorylated(p)/DNA-binding competent pSTAT3 levels were augmented after stretching followed by elevated DNA-binding activity. Pre-treatment with a STAT3 inhibitor either alone or in combination with anti-IgPKD1 abrogated this effect. Moreover, PC1-mediated mechanostimulation induced elevation of Runx2 mRNA levels. ChIP assays revealed direct regulation of Runx2 promoter activity by STAT3/Runx2 after mechanical-stretching that was PC1-dependent. Our findings show that mechanical load upregulates expression of Runx2 gene via potentiation of PC1-JAK2/STAT3 signaling axis, culminating to possibly control osteoblastic differentiation and ultimately bone formation.

Testing of antibiotic releasing implant coatings to fight bacteria in combat-associated osteomyelitis - an in-vitro study.

Surgical procedures to prevent osteomyelitis after trauma can be supported by local application of antibiotics. This in-vitro study investigated the release and impact of antibiotics from implant coatings against bacteria associated with combat-related osteomyelitis. K-wires were coated with poly(D,L-lactide) and ciprofloxacin, gentamicin, colistin, daptomycin or cefoxitin in different concentrations. The release was quantified and antimicrobial activity tested for different gram-positive or gram-negative bacteria, alone and in combination. To exclude toxic effects, primary osteoblast-like cells were exposed to antibiotic coating concentrations. All antibiotics alone and in combination showed an initial burst release with dose dependent antimicrobial activity and no negative effects on osteoblast-like cells, except for cefoxitin. Implant coatings can be customized with single or double antibiotic coatings to effectively fight different bacteria and also mixed infections in the treatment of a combat-acquired osteomyelitis. However, optimal drug load and degradation behaviour of individual antibiotics have to be considered.

Bone de novo deposition in ASC protein-dependent

Event Abstract Back to Event Bone de novo deposition in ASC protein-dependent Monica Calasans-Maia1, Suelen C. Sartoretto1, Sara Gemini-Piperni2, Thais P. Santos3, Inaya C. Lima3, Alexandre M. Rossi4, Gutemberg G. Alves5, Jose M. Granjeiro1, 2, Willian F. Zambuzzi6, Anna M. Teti7 and Nadia Rucci7 1 Fluminense Federal University, Dentistry School, Brazil 2 INMETRO, Bioengineering Programme, Brazil 3 COPPE - UFRJ, Nuclear Instrumentation Department, Brazil 4 Brazilian Center for Physics Research, LABIOMAT, Brazil 5 Fluminense Federal University, Clinical Research Unit, Italy 6 UNESP - Botucatu, Chemistry and Biochemistry, Brazil 7 University of L'Aquila, Department of Biotechnological and Applied Clinical Sciences, Italy One of the most important strategies in regenerative medicine is to fill bone defects with biocompatible materials in order to support functional bone healing. In particular, properties such as osteoconductivity and bio-absorption are improved by partial substitution of CO3 and PO3- in Hydroxyapatite (HA). However, biomaterial implantation also has adverse effects, mainly inflammatory reactions at the early stages. Recent studies demonstrate the role of ASC protein as a mediator of the inflammatory response due to the recruitment and formation of the inflammasome complex. Thus, it has been shown that ASC KO mice present resistance to inflammatory diseases, such as rheumatoid arthritis and multiple sclerosis, but the contributions of the inflammasome component of ASC in the biological response to inorganic biomaterials for implanting have not been yet investigated. In order to address the function of ASC in the bone de novo deposition, we subjected wild type/C57BL/6 and ASC KO mice to three inorganic biomaterials, taking particular note of tibia defect healing. First, primary osteoprogenitor cells from both genotypes were collected. Positive staining for the activity of the osteoblast marker alkaline phosphatase (ALP) and the osteoclast marker tartrate resistant acid phosphatase (TRAcP) were detected histochemically. Thereafter, in order to validate our hypothesis that the ASC protein regulated osteoblast differentiation and bone de novo deposition, we evaluate the healing process of tibia defects on both wild type and ASC KO mice genotypes. Histological and histomorfometric analysis was performed. X-ray micro-computed tomography (μCT) analysis was used in order to confirm the data. Our results showed that primary osteoblast-like cells from ASC KO mice presented a lower level of ALP staining when compared to the same condition osteoblast-like cells from wild type mice, regardless of indirect exposure to the different inorganic biomaterials, suggesting ASC as a novel dependent protein involved in osteoblast differentiation in vitro, independent of inorganic biomaterial influence. Furthermore, our set of in vivo results showed that ASC KO mice were not able to fully heal tibia defects until after 28 days, independent of the inorganic biomaterial used as a scaffold, while wild type tibia defects presented a higher bone de novo volume. Altogether, our results strongly suggest that bone de novo deposition is a biological ASC-dependent event, which should be explored within the field of bone bioengineering. FAPESP; FAPERJ; CNPq; Marie Curie International Research Staff Exchange Scheme Fellowship to SGP within the 7th European Community Framework Programme.

Primary osteoblast-like cells from patients with end-stage kidney disease reflect gene expression, proliferation, and mineralization characteristics ex vivo

Osteocytes regulate bone turnover and mineralization in chronic kidney disease (CKD). Since osteocytes are derived from osteoblasts, alterations in osteoblast function may regulate osteoblast maturation, osteocytic transition, bone turnover and skeletal mineralization. Thus, primary osteoblasts-like cells were cultured from bone chips obtained from 24 pediatric ESKD patients. RNA expression in cultured cells was compared to RNA expression in cells from healthy individuals, to RNA expression in the bone core itself, and to parameters of bone histomorphometry. Proliferation and mineralization rates of patient cells were compared to rates in healthy control cells. Associations were observed between bone osteoid accumulation, as assessed by bone histomorphometry, and bone core RNA expression of osterix, matrix gla protein, PTH receptor 1, and RANKL. Gene expression of osteoblast markers was increased in cells from ESKD patients and signaling genes including Cyp24A1, Cyp27B1, VDR, and NHERF1 correlated between cells and bone cores. Cells from patients with high turnover renal osteodystrophy proliferated more rapidly and mineralized more slowly than did cells from healthy controls. Thus, primary osteoblasts obtained from patients with ESKD retain changes in gene expression ex vivo that are also observed in bone cores specimens. Evaluation of these cells in vitro may provide further insights into the abnormal bone biology that persists, despite current therapies, in patients with ESKD.

Age-related impairment of bones' adaptive response to loading in mice is associated with sex-related deficiencies in osteoblasts but no change in osteocytes.

Bones adjust their mass and architecture to be sufficiently robust to withstand functional loading by adapting to their strain environment. This mechanism appears less effective with age, resulting in low bone mass. In male and female young adult (17-week-old) and old (19-month-old) mice, we investigated the effect of age in vivo on bones' adaptive response to loading and in vitro in primary cultures of osteoblast-like cells derived from bone cortices. Right tibias were axially loaded on alternate days for 2 weeks. Left tibias were non-loaded controls. In a separate group, the number of sclerostin-positive osteocytes and the number of periosteal osteoblasts were analyzed 24 hours after a single loading episode. The responses to strain of the primary osteoblast-like cells derived from these mice were assessed by EGR2 expression, change in cell number and Ki67 immunofluorescence. In young male and female mice, loading increased trabecular thickness and the number of trabecular connections. Increase in the number of trabecular connections was impaired with age but trabecular thickness was not. In old mice, the loading-related increase in periosteal apposition of the cortex was less than in young ones. Age was associated with a lesser loading-related increase in osteoblast number on the periosteal surface but had no effect on loading-related reduction in the number of sclerostin-positive osteocytes. In vitro, strain-related proliferation of osteoblast-like cells was lower in cells from old than young mice. Cells from aged female mice demonstrated normal entry into the cell cycle but subsequently arrested in G2 phase, reducing strain-related increases in cell number. Thus, in both male and female mice, loading-related adaptive responses are impaired with age. This impairment is different in females and males. The deficit appears to occur in osteoblasts' proliferative responses to strain rather than earlier strain-related responses in the osteocytes. © 2014 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals, Inc. on behalf of the American Society for Bone and Mineral Research.

In vitro effect of FGIN-1-27, a ligand to 18 kDa mitochondrial translocator protein, in human osteoblast-like cells

Ligands of 18 kDa mitochondrial translocator protein (TSPO) differ in their cellular effects. We hypothesize that different TSPO ligands might exert different cellular responses. Therefore, following previous studies that showed different cellular responses to two specific TSPO ligands, PK 11195 and protoporphyrin IX, in human osteoblast-like cells in vitro, we now report the cellular response to another specific TSPO ligand, FGIN-1-27 (10(-5) M) (MW 436 kDa), in order to characterize the effects of each TSPO ligand. We found in primary culture of the human osteoblast-like cells that cell numbers were decreased by an average of 30% (p < 0.001) following exposure to 10(-5) M of FGIN-1-27 in comparison to vehicle controls. Cellular [(18)F]-FDG incorporation and ATP content were suppressed, by an average of 43% (p < 0.001) and 83% (p < 0.001), respectively. Mitochondrial mass and ΔΨm increased by an average of 26% (p < 0.01) and 425% (p < 0.0001) respectively. Lactate dehydrogenase activity was enhanced in culture media by 60% (p < 0.05), indicating overall cell death, while no increase in apoptotic levels was observed. Cellular proliferation, as determined by BrdU assay, was not affected. Synthesis of mRNA of TSPO, VDAC 1, and hexokinase 2 decreased in 0.3, 0.3 and 0.5 fold respectively, with accompanying decreases in protein expression of TSPO and Voltage Dependent Anion Channel 1 by 23% (p < 0.001) and 98% (p < 0.001), respectively, but without changes in hexokinase 2 protein expression. Thus it appears that 10(-5) M FGIN-1-27 reduces cell viability, cell metabolism, and mitochondrial function. Previously we found similar effects of PK 11195 on mitochondrial function and cell metabolism and of protoporphyrin IX on cell death in primary osteoblast-like cells.

Analysis of parameters influencing the release of antibiotics mixed with bone grafting material using a reliable mixing procedure

Local infections arising from fracture fixation, defect reconstruction or joint replacement can cause extreme pain and impaired healing, lead to revision operations, prolong hospital stay and increase costs. Treatment options including prophylaxis are afforded by the use of grafts and biomaterials loaded with antibiotics. These can produce local therapeutic concentrations with a reduced systemic concentration and reduced systemic side-effects. Patient-specific loading of osteogenic graft materials with antibiotic could be an important option for orthopaedic surgeons. A local therapeutic concentration must be available for the desired duration and cytotoxic effects must be kept within an acceptable range. The present study investigates a simple and reliable mixing procedure that could be used for the perioperative combination of antibiotic powders and solutions with bone grafting materials. The potential influence of concentration and sampling regime on the release kinetics of gentamicin, tobramycin and vancomycin was studied over a period of 56days and potency and cytotoxicity were evaluated. In all treatment groups, gentamicin and tobramycin were completely released within 3days whilst vancomycin was released over a period of 14days. The results clearly show that the main parameter influencing release is the molecular weight of the drug. Growth of Staphylococcus aureus was inhibited in all 3 treatment groups for at least 3days. Cell viability and alkaline phosphatase activity of primary osteoblast-like cells were not significantly affected by the antibiotic concentrations obtained from the elution experiments.Bone grafting is an established component of surgery for bone defect filling and for biological stimulation of healing. Patient-specific enhancement of such procedures by incorporation of antibiotics for infection prevention or by addition of cytokines for promotion of impaired healing or for treatment of critical size defects will be a relevant issue in the future.

In vitro response to alkaline phosphatase coatings immobilized onto titanium implants using electrospray deposition or polydopamine‐assisted deposition

Immobilization of biomolecules onto implant surfaces is one of the most straightforward strategies to control the interaction between an implant and its biological environment. Recently, it was shown that the enzyme alkaline phosphatase (ALP) could be efficiently immobilized onto titanium implants in a single step using polydopamine. We hypothesized that such polydopamine-ALP coatings can enhance the early attachment of cells and increase mineralization. Therefore, the current study aimed at immobilization of ALP onto titanium by means of either one- or two-step polydopamine-assisted immobilization or electrospray deposition, the comparative characterization of these experimental substrates and subsequent cell behavioral analysis using primary osteoblast-like cells. Uncoated titanium and ALP-free polydopamine coatings served as controls. Despite significant ALP surface activity and lower water contact for angles ALP-containing surface modifications, only marginal effects on early cell behavior (i.e., cell spreading) and osteogenic differentiation (i.e., proliferation, differentiation and mineralization) were observed in comparison to uncoated titanium.

Effects and differentiation activity of IGF-I, IGF-II, insulin and preptin on human primary bone cells

The importance of the complex interrelated regulatory pathways involving IGF factors and pancreatic hormones can be observed in several metabolic diseases, where the deregulation of these factors has a wide impact on bone health. These findings have stimulated us to compare the effect of IGF-I, IGF-II, insulin and preptin on human bone cells. The effect on cell differentiation and cell activity of osteoblasts and osteoclasts has been analysed. We have observed a significant effect by IGF-I, a modest effect by IGF-II and preptin and no effect after insulin administration on human primary osteoblast-like cells. All studied factors have shown an induction on human primary osteoclast differentiation and bone resorption activity, with IGF-I being the most potent factor. We hypothesize that these findings may be on the basis of decreased bone mass density observed in several diseases.

Lrp5 Is Not Required for the Proliferative Response of Osteoblasts to Strain but Regulates Proliferation and Apoptosis in a Cell Autonomous Manner

Although Lrp5 is known to be an important contributor to the mechanisms regulating bone mass, its precise role remains unclear. The aim of this study was to establish whether mutations in Lrp5 are associated with differences in the growth and/or apoptosis of osteoblast-like cells and their proliferative response to mechanical strain in vitro. Primary osteoblast-like cells were derived from cortical bone of adult mice lacking functional Lrp5 (Lrp5−/−), those heterozygous for the human G171V High Bone Mass (HBM) mutation (LRP5 G171V) and their WT littermates (WTLrp5, WTHBM). Osteoblast proliferation over time was significantly higher in cultures of cells from LRP5 G171V mice compared to their WTHBM littermates, and lower in Lrp5−/− cells. Cells from female LRP5 G171V mice grew more rapidly than those from males, whereas cells from female Lrp5−/− mice grew more slowly than those from males. Apoptosis induced by serum withdrawal was significantly higher in cultures from Lrp5−/− mice than in those from WTHBM or LRP5 G171V mice. Exposure to a single short period of dynamic mechanical strain was associated with a significant increase in cell number but this response was unaffected by genotype which also did not change the ‘threshold’ at which cells responded to strain. In conclusion, the data presented here suggest that Lrp5 loss and gain of function mutations result in cell-autonomous alterations in osteoblast proliferation and apoptosis but do not alter the proliferative response of osteoblasts to mechanical strain in vitro.

Myocyte Enhancer Factor 2C, an Osteoblast Transcription Factor Identified by Dimethyl Sulfoxide (DMSO)-enhanced Mineralization

Rapid mineralization of cultured osteoblasts could be a useful characteristic in stem cell-mediated therapies for fracture and other orthopedic problems. Dimethyl sulfoxide (DMSO) is a small amphipathic solvent molecule capable of stimulating cell differentiation. We report that, in primary human osteoblasts, DMSO dose-dependently enhanced the expression of osteoblast differentiation markers alkaline phosphatase activity and extracellular matrix mineralization. Furthermore, similar DMSO-mediated mineralization enhancement was observed in primary osteoblast-like cells differentiated from mouse mesenchymal cells derived from fat, a promising source of starter cells for cell-based therapy. Using a convenient mouse pre-osteoblast model cell line MC3T3-E1, we further investigated this phenomenon showing that numerous osteoblast-expressed genes were elevated in response to DMSO treatment and correlated with enhanced mineralization. Myocyte enhancer factor 2c (Mef2c) was identified as the transcription factor most induced by DMSO, among the numerous DMSO-induced genes, suggesting a role for Mef2c in osteoblast gene regulation. Immunohistochemistry confirmed expression of Mef2c in osteoblast-like cells in mouse mandible, cortical, and trabecular bone. shRNAi-mediated Mef2c gene silencing resulted in defective osteoblast differentiation, decreased alkaline phosphatase activity, and matrix mineralization and knockdown of osteoblast specific gene expression, including osteocalcin and bone sialoprotein. A flow on knockdown of bone-specific transcription factors, Runx2 and osterix by shRNAi knockdown of Mef2c, suggests that Mef2c lies upstream of these two important factors in the cascade of gene expression in osteoblasts.

Effect of Laser Micromachining of Titanium on Viability and Responsiveness of Osteoblast-Like Cells

Laser engineering may create hemispherical porosities on titanium surfaces obtaining regular and predetermined rough titanium surfaces. The aim of this study was to assess the viability and the proliferation of primary osteoblast-like cells (OB) to growth factors on titanium surfaces with a different roughness in vitro. OB were obtained from volunteers undergoing wisdom tooth removal following a standardized protocol. OB were allowed to attach on 4 different titanium surfaces: sandblasted titanium (SBT) disks, 5-, 10-, and 20-μm regular laser-engineered micropore titanium disks. A well with no disk was used as control. Cell morphology was evaluated with scanning electron microscopy. Viability was measured with MTT (3[4,5 dimethylthiazol 2yl]2,5 diphenyltetrazolium bromide) assay. Proliferation rate of attached cells was evaluated with Cell Counting Kit-8 48 hours after platelet-released supernatant (PRS) application. Statistical analysiswas performed with analysis of variance test. All surfaces showed OB attachment on scanning electron microscopy. OB appeared more numerous on 20T surfaces. Laser-engineered surfaces showed higher OB viability than SBT (P < 0.01). In terms of proliferation, viability increase was noted for all groups after platelet-released supernatant application. 20T and SBT disks seemed to trigger the higher cellular proliferation (20T vs 10T, P < 0.05). Laser-engineered porous titanium surfaces promote viability and proliferation of OB. In particular, hemispherical porosity of 20 μm seems to trigger the higher OB response. Further research is needed to confirm these data.