Osteoblast Attachment on Bioactive Glass Air Particle Abrasion-Induced Calcium Phosphate Coating.

Air particle abrasion (APA) using bioactive glass (BG) effectively decontaminates titanium (Ti) surface biofilms and the retained glass particles on the abraded surfaces impart potent antibacterial properties against various clinically significant pathogens. The objective of this study was to investigate the effect of BG APA and simulated body fluid (SBF) immersion of sandblasted and acid-etched (SA) Ti surfaces on osteoblast cell viability. Another goal was to study the antibacterial effect against Streptococcus mutans. Square-shaped 10 mm diameter Ti substrates (n = 136) were SA by grit blasting with aluminum oxide particles, then acid-etching in an HCl-H2SO4 mixture. The SA substrates (n = 68) were used as non-coated controls (NC-SA). The test group (n = 68) was further subjected to APA using experimental zinc-containing BG (Zn4) and then mineralized in SBF for 14 d (Zn4-CaP). Surface roughness, contact angle, and surface free energy (SFE) were calculated on test and control surfaces. In addition, the topography and chemistry of substrate surfaces were also characterized. Osteoblastic cell viability and focal adhesion were also evaluated and compared to glass slides as an additional control. The antibacterial effect of Zn4-CaP was also assessed against S. mutans. After immersion in SBF, a mineralized zinc-containing Ca-P coating was formed on the SA substrates. The Zn4-CaP coating resulted in a significantly lower Ra surface roughness value (2.565 µm; p < 0.001), higher wettability (13.35°; p < 0.001), and higher total SFE (71.13; p < 0.001) compared to 3.695 µm, 77.19° and 40.43 for the NC-SA, respectively. APA using Zn4 can produce a zinc-containing calcium phosphate coating that demonstrates osteoblast cell viability and focal adhesion comparable to that on NC-SA or glass slides. Nevertheless, the coating had no antibacterial effect against S. mutans.

Monocyte Differentiation on Atomic Layer-Deposited (ALD) Hydroxyapatite Coating on Titanium Substrate.

Hydroxyapatite (HA; Ca10(PO4)6(OH)2) coating of bone implants has many beneficial properties as it improves osseointegration and eventually becomes degraded and replaced with new bone. We prepared HA coating on a titanium substrate with atomic layer deposition (ALD) and compared monocyte differentiation and material resorption between ALD-HA and bone. After stimulation with macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-B ligand (RANKL), human peripheral blood monocytes differentiated into resorbing osteoclasts on bovine bone, but non-resorbing foreign body cells were observed on ALD-HA. The analysis of the topography of ALD-HA and bone showed no differences in wettability (water contact angle on ALD-HA 86.2° vs. 86.7° on the bone), but the surface roughness of ALD-HA (Ra 0.713 µm) was significantly lower compared to bone (Ra 2.30 µm). The cellular reaction observed on ALD-HA might be a consequence of the topographical properties of the coating. The absence of resorptive osteoclasts on ALD-HA might indicate inhibition of their differentiation or the need to modify the coating to induce osteoclast differentiation.

Osteoclastogenesis of human peripheral blood, bone marrow, and cord blood monocytes.

Osteoclasts are multinucleated bone resorbing cells that can be differentiated from human monocytes in vitro. There are few studies comparing osteoclastogenesis of different monocyte sources. We compared monocytes from human bone marrow (BM), peripheral blood (PB), and umbilical cord blood (CB) and their osteoclastogenic potential by culturing them with RANKL (20 and 80 ng/ml) and M-CSF (10 ng/ml) for 14 days. We also cultured cells without growth factors, as umbilical cord blood monocytes have been reported to be able to fuse spontaneously into osteoclasts. The data was analysed on d4, d8, d11, and d14. After culture with RANKL and M-CSF, all types of cell cultures developed TRACP -positive multinuclear cells that were able to form resorption pits on human bone slices. Only occasional multinuclear cells and small infrequent resorbed areas could be found in PB and CB-derived cultures without growth factors. BM-derived cells formed greater resorption areas than PB- and CB-derived monocytes. The greatest monocyte population in BM samples were intermediate (CD14++CD16+) and in PB and CB classical monocytes (76.3% and 54.4%, respectively). In conclusion, our data demonstrates that bone resorbing osteoclasts can be differentiated from BM, PB and CB. However, the osteoclast precursor origin can affect the osteoclast properties and function.

Osteoblast Attachment on Titanium Coated with Hydroxyapatite by Atomic Layer Deposition.

BACKGROUND: The increasing demand for bone implants with improved osseointegration properties has prompted researchers to develop various coating types for metal implants. Atomic layer deposition (ALD) is a method for producing nanoscale coatings conformally on complex three-dimensional surfaces. We have prepared hydroxyapatite (HA) coating on titanium (Ti) substrate with the ALD method and analyzed the biocompatibility of this coating in terms of cell adhesion and viability. METHODS: HA coatings were prepared on Ti substrates by depositing CaCO3 films by ALD and converting them to HA by wet treatment in dilute phosphate solution. MC3T3-E1 preosteoblasts were cultured on ALD-HA, glass slides and bovine bone slices. ALD-HA and glass slides were either coated or non-coated with fibronectin. After 48h culture, cells were imaged with scanning electron microscopy (SEM) and analyzed by vinculin antibody staining for focal adhesion localization. An 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) test was performed to study cell viability. RESULTS: Vinculin staining revealed similar focal adhesion-like structures on ALD-HA as on glass slides and bone, albeit on ALD-HA and bone the structures were thinner compared to glass slides. This might be due to thin and broad focal adhesions on complex three-dimensional surfaces of ALD-HA and bone. The MTT test showed comparable cell viability on ALD-HA, glass slides and bone. CONCLUSION: ALD-HA coating was shown to be biocompatible in regard to cell adhesion and viability. This leads to new opportunities in developing improved implant coatings for better osseointegration and implant survival.

Collagen XIII-derived ectodomain regulates bone angiogenesis and intracortical remodeling.

Osteoporosis is the most common degenerative bone disease that occurs when the balance of bone production and resorption is perturbed. Loss of bone mass or alteration in its quality leads to significant weakening of the bones and subsequently to higher fracture risk. Collagen XIII (ColXIII) is a conserved transmembrane protein expressed in many mesenchymal tissues. Here we show that ColXIII is a regulator of bone remodeling niche. In this study, we found that ColXIII expression is significantly upregulated in osteoporotic patients. In view of that, we studied bone homeostasis in ColXIII-overexpressing mice (Col13a1oe) up to 72 weeks of age and observed a cortical bone overgrowth followed by a drastic bone loss, together with increased bone vascularization. Moreover, our results demonstrate that the ColXIII-derived ectodomain enhances angiogenesis through ß1-integrins and the JNK pathway. Consequently, these data suggest that ColXIII has a role in age-dependent cortical bone deterioration with possible implications for osteoporosis and fracture risk.

Peripheral blood monocytes show increased osteoclast differentiation potential compared to bone marrow monocytes.

Bone marrow (BM) and peripheral blood (PB) derived mononuclear cells are precursors of in vitro osteoclast differentiation. However, few studies have compared the phenotypic and functional properties of osteoclasts generated from these sources and the effects of different growth factors on osteoclastogenesis. Both cell types differentiated into functional osteoclasts, but culturing the cells with or without transforming growth factor beta (TGF-ß) and dexamethasone revealed differences in their osteoclastogenic capacity. When receptor activator for nuclear factor κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) were used for differentiation, we did not observe differences in bone resorption activity or expression of osteoclastogenic genes calcitonin receptor (CR) and nuclear factor of activated T-cells (NFATc1) between the osteoclasts formed from the two sources. Addition of TGF-ß and dexamethasone led to higher number of nuclei in multinuclear cells and increased expression of tartrate resistant acid phosphatase (TRACP) 5a and 5b, CR and NFATc1 in PB- derived osteoclasts depicting the higher osteoclastogenic potential and responsiveness to TGF-ß and dexamethasone in PB monocytes. These results conclude that the choice of the osteoclast precursor source as well as the choice of osteoclastogenic growth factors are essential matters in determining the phenotypic characteristics of heterogeneous osteoclast populations.

Corrigendum to "Gap junctional communication is involved in differentiation of osteoclasts from bone marrow and peripheral blood monocytes".

[This corrects the article DOI: 10.1016/j.heliyon.2018.e00621.].

Gap junctional communication is involved in differentiation of osteoclasts from bone marrow and peripheral blood monocytes.

AIMS: The aim of the study was to compare the influence of gap junctional communication (GJC) in osteoclastogenesis from bone marrow (BM) and peripheral blood (PB) monocytes. These widely used sources differ in purity, since BM cultures contain a significant number of stromal cells. We studied whether stimulation of GJC in BM monocyte/stromal cell cultures differs from the effect in pure PB monocyte cultures. We compared the differentiation also in acidosis, which is a known inducer of bone resorption. MAIN METHODS: Human BM and PB monocytes were isolated from BM aspirates or whole blood samples. The cells were cultured on human bone slices with osteoclastogenic growth factors and a GJC modulator, antiarrhythmic peptide AAP10, at physiological and acidic pH. KEY FINDINGS: Both BM and PB monocytes differentiated into osteoclasts. Acidosis increased resorption in both cultures but stimulated cell fusion only in BM cultures, which demonstrates the role of stromal cells in osteoclastogenesis. At physiological pH, AAP10 increased the number of multinuclear cells and bone resorption in both BM and PB cultures indicating that GJC is involved in differentiation in both of these osteoclastogenesis assays. Interestingly, in PB cultures at pH 6.5 the stimulation of GJC with AAP10 inhibited both osteoclastogenesis and bone resorption suggesting a different role of GJC in BM and PB monocytes at stressed environment. SIGNIFICANCE: The study is conducted with primary human tissue samples and adds new knowledge on factors affecting osteoclastogenesis from different monocyte sources.

Osteoclasts and Remodeling Based Bone Formation.

Osteoclasts are multinuclear cells of the monocyte macrophage lineage. They are responsible for bone remodeling by first resorbing packets of bone, which are subsequently replaced by new bone produced by osteoblasts. Osteoblasts are derived from mesenchymal stem cells, and thus osteogenesis can also be induced in various tissues at extra skeletal sites. Fifty years ago it was discovered that demineralized bone matrix is able to induce ectopic bone formation. Since that time the differentiation of bone cells has been studied intensively. The aim was to produce bone for the repair of bone defects. The molecular basis of bone remodeling has been established in great detail and the mechanism of how bone resorption and bone formation are coupled in bone remodeling sites has been delineated. Osteoclasts resorb bone, but they also secrete anabolic signals that induce mesenchymal stem cells and osteoblasts to initiate osteogenesis in resorption lacuna (remodeling) or another nonresorbed site (modeling). It is this osteoclast derived influence on mesenchymal stem cells and osteoblasts that could be utilized in tissue engineering. So far investigators have tried to find ways to induce bone formation by activating mesenchymal stem cells, but a better understanding of the remodeling paradigm of bone, the intrinsic regulation of bone formation through osteoclastic resorption, could be utilized for tissue engineering. Scaffold materials like decellularized natural tissue extracellular matrices or bone type resorbable mineral matrices induce resorption and simultaneously induce bone formation.

Osteoclastogenesis is influenced by modulation of gap junctional communication with antiarrhythmic peptides.

Osteoclasts are formed by the fusion of mononuclear precursor cells of the monocyte-macrophage lineage. Among several putative mechanisms, gap-junctional intercellular communication (GJC) has been proposed to have a role in osteoclast fusion and bone resorption. We examined the role of GJC in osteoclastogenesis and in vitro bone resorption with mouse bone marrow hematopoietic stem cells and RAW 264.7 cells. Blocking of gap junctions with 18-α-glycyrrhetinic acid (18GA) led to inhibition of osteoclastogenesis and in vitro bone resorption. Similarly, the GJC inhibitor GAP27 inhibited osteoclast formation. GJC modulation with the antiarrhythmic peptides (AAPs) led to increased amounts of multinuclear RAW 264.7 osteoclasts as well as increased number of nuclei per multinuclear cell. In the culture of bone marrow hematopoietic stem cells in the presence of bone marrow stromal cells AAP reduced the number of osteoclasts, and coculture of MC3T3-E1 preosteoblasts with RAW 264.7 macrophages prevented the action of AAPs to promote osteoclastogenesis. The present data indicate that AAPs modulate the fusion of the pure culture of cells of the monocyte-macrophage lineage. However, the fusion is influenced by GJC in cells of the osteoblast lineage.