Mussel-inspired bioceramics with self-assembled Ca-P/polydopamine composite nanolayer : preparation, formation mechanism, improved cellular bioactivity and osteogenic differentiation of bone marrow stromal cells

The nanostructured surface of biomaterials plays an important role in improving their in vitro cellular bioactivity as well as stimulating in vivo tissue regeneration. Inspired by the mussel’s adhesive versatility, which is thought to be due to the plaque–substrate interface being rich in 3,4-dihydroxy-L-phenylalamine (DOPA) and lysine amino acids, in this study we developed a self-assembly method to prepare a uniform calcium phosphate (Ca-P)/polydopamine composite nanolayer on the surface of b-tricalcium phosphate (b-TCP) bioceramics by soaking b-TCP bioceramics in Tris–dopamine solution. It was found that the addition of dopamine, reaction temperature and reaction time are three key factors inducing the formation of a uniform Ca-P/polydopamine composite nanolayer. The formation mechanism of a Ca-P/polydopamine composite nanolayer involved two important steps: (i) the addition of dopamine to Tris–HCl solution decreases the pH value and accelerates Ca and P ionic dissolution from the crystal boundaries of b-TCP ceramics; (ii) dopamine is polymerized to form self-assembled polydopamine film and, at the same time, nanosized Ca-P particles are mineralized with the assistance of polydopamine, in which the formation of polydopamine occurs simultaneously with Ca-P mineralization (formation of nanosized microparticles composed of calcium phosphate-based materials), and finally a self-assembled Ca-P/polydopamine composite nanolayer forms on the surface of the b-TCP ceramics. Furthermore, the formed self-assembled Ca-P/polydopamine composite nanolayer significantly enhances the surface roughness and hydrophilicity of b-TCP ceramics, and stimulates the attachment, proliferation, alkaline phosphate (ALP) activity and bone-related gene expression (ALP, OCN, COL1 and Runx2) of human bone marrow stromal cells. Our results suggest that the preparation of self-assembled Ca-P/polydopamine composite nanolayers is a viable method to modify the surface of biomaterials by significantly improving their surface physicochemical properties and cellular bioactivity for bone regeneration application.

The effect of hypoxia on the stemness and differentiation capacity of PDLC and DPC

Introduction. Stem cells are regularly cultured under normoxic conditions. However, the physiological oxygen tension in the stem cell niche is known to be as low as 1-2% oxygen, suggesting that hypoxia has a distinct impact on stem cell maintenance. Periodontal ligament cells (PDLCs) and dental pulp cells (DPCs) are attractive candidates in dental tissue regeneration. It is of great interest to know whether hypoxia plays a role in maintaining the stemness and differentiation capacity of PDLCs and DPCs. Methods. PDLCs and DPCs were cultured either in normoxia (20% O2) or hypoxia (2% O2). Cell viability assays were performed and the expressions of pluripotency markers (Oct-4, Sox2, and c-Myc) were detected by qRT-PCR and western blotting. Mineralization, glycosaminoglycan (GAG) deposition, and lipid droplets formation were assessed by Alizarin red S, Safranin O, and Oil red O staining, respectively. Results. Hypoxia did not show negative effects on the proliferation of PDLCs and DPCs. The pluripotency markers and differentiation potentials of PDLCs and DPCs significantly increased in response to hypoxic environment. Conclusions. Our findings suggest that hypoxia plays an important role in maintaining the stemness and differentiation capacity of PDLCs and DPCs.

Sex-specific compromised bone healing in female rats might be associated with a decrease in mesenchymal stem cell quantity

Introduction The clinically known importance of patient sex as a major risk factor for compromised bone healing is poorly reflected in animal models. Consequently, the underlying cellular mechanisms remain elusive. Because mesenchymal stem cells (MSCs) are postulated to regulate tissue regeneration and give rise to essential differentiated cell types, they may contribute to sex-specific differences in bone healing outcomes. Methods We investigated sex-specific variations in bone healing and associated differences in MSC populations. A 1.5 mm osteotomy gap in the femora of 8 male and 8 female 12-month-old Sprague-Dawley rats was stabilized by an external fixator. Healing was analyzed in terms of biomechanical testing, bridging and callus size over time (radiography at 2, 4, and 6 weeks after surgery), and callus volume and geometry by μCT at final follow-up. MSCs were obtained from bone marrow samples of an age-matched group of 12 animals (6 per gender) and analyzed for numbers of colony-forming units (CFUs) and their capacity to differentiate and proliferate. The proportion of senescent cells was determined by β-galactosidase staining. Results Sex-specific differences were indicated by a compromised mechanical competence of the callus in females compared with males (maximum torque at failure, p = 0.028). Throughout the follow-up, the cross-sectional area of callus relative to bone was reduced in females (p ≤ 0.01), and the bridging of callus was delayed (p 2weeks = 0.041). μCT revealed a reduced callus size (p = 0.003), mineralization (p = 0.003) and polar moment of inertia (p = 0.003) in female animals. The female bone marrow contained significantly fewer MSCs, represented by low CFU numbers in both femora and tibiae (p femur = 0.017, p tibia = 0.010). Functional characteristics of male and female MSCs were similar. Conclusion Biomechanically compromised and radiographically delayed bone formation were distinctive in female rats. These differences were concomitant with a reduced number of MSCs, which may be causative for the suboptimal bone healing.

Sulfated glycosaminoglycans mediate the effects of FGF2 on the osteogenic potential of rat calvarial osteoprogenitor cells

Fibroblast growth factor-2 (FGF2) is a powerful promoter of bone growth. We demonstrate here that brief exposure to FGF2 enhances mineralized nodule formation in cultured rat osteoprogenitor cells due to an expansion of cells that subsequently mineralize. This mitogenic effect is mediated via sulfated glycosaminoglycans (GAGs), FGFR1, and the extracellular signal-regulated kinase (ERK) pathway. The GAGs involved in this stimulation are chondroitin sulfates (CS) rather than heparan sulfates (HS). However, continuous FGF2 treatment reduces alkaline phosphatase (ALP) activity, downregulates collagen Ialpha1 (ColIalpha1) and FGFR3 expression, upregulates the expression and secretion of osteopontin (OPN) and inhibits mineralization. The inhibitory effects of FGF2 on FGFR3 expression and ALP activity are also mediated by the ERK pathway, although the effects of FGF2 on ColIalpha1 and OPN expression are mediated by GAGs and PKC activity. Thus short-term activation of FGF2/FGFR1 promotes osteoprogenitor proliferation and subsequent differentiation, while long-term activation of FGF2 signaling disrupts mineralization by modulating osteogenic marker expression. This study thus establishes the central role of sulfated GAGs in the osteogenic progression of osteoprogenitors.

Temporal and functional changes in glycosaminoglycan expression during osteogenesis

Heparan sulfate proteoglycans (HSPGs) are complex and labile macromolecular moieties on the surfaces of cells that control the activities of a range of extracellular proteins, particularly those driving growth and regeneration. Here, we examine the biosynthesis of heparan sulfate (HS) sugars produced by cultured MC3T3-E1 mouse calvarial pre-osteoblast cells in order to explore the idea that changes in HS activity in turn drive phenotypic development during osteogenesis. Cells grown for 5 days under proliferating conditions were compared to cells grown for 20 days under mineralizing conditions with respect to their phenotype, the forms of HS core protein produced, and their HS sulfotransferase biosynthetic enzyme levels. RQ-PCR data was supported by the results from the purification of day 5 and day 20 HS forms by anionic exchange chromatography. The data show that cells in active growth phases produce more complex forms of sugar than cells that have become relatively quiescent during active mineralization, and that these in turn can differentially influence rates of cell growth when added exogenously back to preosteoblasts.

Osteogenic differentiation of murine embryonic stem cells is mediated by fibroblast growth factor receptors

The mechanisms involved in the control of embryonic stem (ES) cell differentiation are yet to be fully elucidated. However, it has become clear that the family of fibroblast growth factors (FGFs) are centrally involved. In this study we examined the role of the FGF receptors (FGFRs 1-4) during osteogenesis in murine ES cells. Single cells were obtained after the formation of embryoid bodies, cultured on gelatin-coated plates, and coaxed to differentiate along the osteogenic lineage. Upregulation of genes was analyzed at both the transcript and protein levels using gene array, relative-quantitative PCR (RQ-PCR), and Western blotting. Deposition of a mineralized matrix was evaluated with Alizarin Red staining. An FGFR1-specific antibody was generated and used to block FGFR1 activity in mES cells during osteogenic differentiation. Upon induction of osteogenic differentiation in mES cells, all four FGFRs were clearly upregulated at both the transcript and protein levels with a number of genes known to be involved in osteogenic differentiation including bone morphogenetic proteins (BMPs), collagen I, and Runx2. Cells were also capable of depositing a mineralized matrix, confirming the commitment of these cells to the osteogenic lineage. When FGFR1 activity was blocked, a reduction in cell proliferation and a coincident upregulation of Runx2 with enhanced mineralization of cultures was observed. These results indicate that FGFRs play critical roles in cell recruitment and differentiation during the process of osteogenesis in mES cells. In particular, the data indicate that FGFR1 plays a pivotal role in osteoblast lineage determination.

Stimulation of osteogenic and angiogenic ability of cells on polymers by pulsed laser deposition of uniform akermanite-glass nanolayer

Polymer biomaterials have been widely used for bone replacement/regeneration because of their unique mechanical properties and workability. Their inherent low bioactivity makes them lack osseointegration with host bone tissue. For this reason, bioactive inorganic particles have been always incorporated into the matrix of polymers to improve their bioactivity. However, mixing inorganic particles with polymers always results in inhomogeneity of particle distribution in polymer matrix with limited bioactivity. This study sets out to apply the pulsed laser deposition (PLD) technique to prepare uniform akermanite (Ca2MgSi2O7, AKT) glass nanocoatings on the surface of two polymers (non-degradable polysulfone (PSU) and degradable polylactic acid (PDLLA)) in order to improve their surface osteogenic and angiogenic activity. The results show that a uniform nanolayer composed of amorphous AKT particles (∼30nm) of thickness 130nm forms on the surface of both PSU and PDLLA films with the PLD technique. The prepared AKT-PSU and AKT-PDLLA films significantly improved the surface roughness, hydrophilicity, hardness and apatite mineralization, compared with pure PSU and PDLLA, respectively. The prepared AKT nanocoatings distinctively enhance the alkaline phosphate (ALP) activity and bone-related gene expression (ALP, OCN, OPN and Col I) of bone-forming cells on both PSU and PDLLA films. Furthermore, AKT nanocoatings on two polymers improve the attachment, proliferation, VEGF secretion and expression of proangiogenic factors and their receptors of human umbilical vein endothelial cells (HUVEC). The results suggest that PLD-prepared bioceramic nanocoatings are very useful for enhancing the physicochemical, osteogenic and angiogenic properties of both degradable and non-degradable polymers for application in bone replacement/regeneration.

Multiphasic construct studied in an ectopic osteochondral defect model

In vivo osteochondral defect models predominantly consist of small animals, such as rabbits. Although they have an advantage of low cost and manageability, their joints are smaller and more easily healed compared with larger animals or humans. We hypothesized that osteochondral cores from large animals can be implanted subcutaneously in rats to create an ectopic osteochondral defect model for routine and high-throughput screening of multiphasic scaffold designs and/or tissue-engineered constructs (TECs). Bovine osteochondral plugs with 4 mm diameter osteochondral defect were fitted with novel multiphasic osteochondral grafts composed of chondrocyte-seeded alginate gels and osteoblast-seeded polycaprolactone scaffolds, prior to being implanted in rats subcutaneously with bone morphogenic protein-7. After 12 weeks of in vivo implantation, histological and micro-computed tomography analyses demonstrated that TECs are susceptible to mineralization. Additionally, there was limited bone formation in the scaffold. These results suggest that the current model requires optimization to facilitate robust bone regeneration and vascular infiltration into the defect site. Taken together, this study provides a proof-of-concept for a high-throughput osteochondral defect model. With further optimization, the presented hybrid in vivo model may address the growing need for a cost-effective way to screen osteochondral repair strategies before moving to large animal preclinical trials.

Activation of the canonical Wnt signaling pathway induces cementum regeneration

Canonical Wnt signaling is important in tooth development but it is unclear whether it can induce cementogenesis and promote the regeneration of periodontal tissues lost due to disease. Therefore, the aim of this study is to investigate the influence of canonical Wnt signaling enhancers on human periodontal ligament cell (hPDLCs) cementogenic differentiation in vitro and cementum repair in a rat periodontal defect model. Canonical Wnt signaling was induced by (i) local injection of lithium chloride; (ii) local injection of sclerostin antibody; and (iii) local injection of a lentiviral construct overexpressing β-catenin. The results showed that the local activation of canonical Wnt signaling resulted in significant new cellular cementum deposition and the formation of well-organized periodontal ligament fibers, which was absent in the control group. In vitro experiments using hPDLCs showed that the Wnt signaling pathway activators significantly increased mineralization, alkaline phosphatase (ALP) activity, and gene and protein expression of the bone and cementum markers osteocalcin (OCN), osteopontin (OPN), cementum protein 1 (CEMP1), and cementum attachment protein (CAP). Our results show that the activation of the canonical Wnt signaling pathway can induce in vivo cementum regeneration and in vitro cementogenic differentiation of hPDLCs.

FGF-2 induces the proliferation of human periodontal ligament cells and modulates their osteoblastic phenotype by affecting Runx2 expression in the presence and absence of osteogenic inducers

The exact phenotype of human periodontal ligament cells (hPDLCs) remains a controversial area. Basic fibroblast growth factor (FGF‑2) exhibits various functions and its effect on hPDLCs is also controversial. Therefore, the present study examined the effect of FGF‑2 on the growth and osteoblastic phenotype of hPDLCs with or without osteogenic inducers (dexamethasone and β‑glycerophosphate). FGF‑2 was added to defined growth culture medium and osteogenic inductive culture medium. Cell proliferation, osteogenic differentiation and mineralization were measured. The selected differentiation markers, Runx2, collagen type Ⅰ, α1 (Col1a1), osteocalcin (OCN) and epidermal growth factor receptor (EGFR), were investigated by reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR). Runx2 and OCN protein expression was measured by western blotting. FGF‑2 significantly increased the proliferation of hPDLCs, but did not affect alkaline phosphatase activity. RT‑qPCR analysis revealed enhanced mRNA expression of Runx2, OCN and EGFR, but suppressed Col1a1 gene expression in the absence of osteogenic inducers, whereas all these gene levels had no clear trend in their presence. The Runx2 protein expression was clearly increased, but the OCN protein level showed no evident trend. The mineralization assay demonstrated that FGF‑2 inhibited mineralized matrix deposition with osteogenic inducers. These results suggested that FGF‑2 induces the growth of immature hPDLCs, which is a competitive inhibitor of epithelial downgrowth, and suppresses their differentiation into mineralized tissue by affecting Runx2 expression. Therefore, this may lead to the acceleration of periodontal regeneration.

Spectroscopic characterization and solubility investigation on the effects of As(V) on mineral structure tooeleite (Fe6(AsO3)4SO4(OH)4·H2O)

Tooeleite is an unique ferric arsenite sulfate mineral, which has the potential significance of directly fixing As(III) as mineral trap. The tooeleite and various precipitates were hydrothermally synthesized under the different of initial As(III)/As(V) molar ratios and characterized by XRD, FTIR, XPS and SEM. The crystallinity of tooeleite decreases with the amount of As(V). The precipitate is free of any crystalline tooeleite at the level of that XRD could detect when the ratio of As(III)/As(V) of 7:3 and more. The characteristic bands of tooeleite are observed in 772, 340, 696 and 304 cm−1, which are assigned to the ν1, ν2, ν3 and ν4 vibrations of AsO33−. These intensities of bands gradually decreases with the presence of As(V) and its increasing. An obviously wide band is observed in 830 cm−1, which is the ν1 vibration of AsO4. The result of XPS reveals that the binding energies of As3d increase from 44.0 eV to 45.5 eV, which indicates that the amount of As(V) in the precipitates increases. The concentrations of arsenic released of these precipitates are 350–650 mg/L. The stability of tooeleite decreases by comparison when the presence of coexisting As(V) ions.

The ultrastructural relationship between osteocytes and dental implants following osseointegration

Background Osteocytes, the most abundant cells in bone, havemultiple functions, including acting as mechanosensors and regulating mineralization. It is clear that osteocytes influence bone remodeling by controlling the differentiation and activity of osteoblasts and osteoclasts. Determining the relationship between titanium implants and osteocytes may therefore benefit our understanding of the process of osseointegration. Purpose The aim of this study was to visualize the ultrastructural relationship between osteocytes and the titanium implant surface following osseointegration in vivo. Materials and Methods Titanium implants were placed in the maxillary molar regions of eight female Sprague Dawley rats, 3 months old. The animals were sacrificed 8 weeks after implantation, and undecalcified tissue sections were prepared. Resin-cast samples were subsequently acid-etched with 37% phosphoric acid prior to examination using scanning electron microscopy. Results Compared with mature bone, where the osteocytes were arranged in an ordered fashion, the osteocytes appeared less organized in the newly formed bone around the titanium implant. Further, a layer of mineralization with few organic components was observed on the implant surface. This study shows for the first time that osteocytes and their dendrites are directly connected with the implant surface. Conclusions: This study shows the direct anchorage of osteocytes via dendritic processes to a titanium implant surface in vivo. This suggests an important regulatory role for osteocytes and their lacunar-canalicular network in maintaining long-term osseointegration.

Multiple sulfur isotope analyses support a magmatic model for the volcanogenic massive sulfide deposits of the Teutonic Bore volcanic complex, Yilgarn Craton, Western Australia

We report sensitive high mass resolution ion microprobe, stable isotopes (SHRIMP SI) multiple sulfur isotope analyses (32S, 33S, 34S) to constrain the sources of sulfur in three Archean VMS deposits—Teutonic Bore, Bentley, and Jaguar—from the Teutonic Bore volcanic complex of the Yilgarn Craton, Western Australia, together with sedimentary pyrites from associated black shales and interpillow pyrites. The pyrites from VMS mineralization are dominated by mantle sulfur but include a small amount of slightly negative mass-independent fractionation (MIF) anomalies, whereas sulfur from the pyrites in the sedimentary rocks has pronounced positive MIF, with ∆33S values that lie between 0.19 and 6.20‰ (with one outlier at −1.62‰). The wall rocks to the mineralization include sedimentary rocks that have contributed no detectable positive MIF sulfur to the VMS deposits, which is difficult to reconcile with the leaching model for the formation of these deposits. The sulfur isotope data are best explained by mixing between sulfur derived from a magmatic-hydrothermal fluid and seawater sulfur as represented by the interpillow pyrites. The massive sulfide lens pyrites have a weighted mean ∆33S value of −0.27 ± 0.05‰ (MSWD = 1.6) nearly identical with −0.31 ± 0.08‰ (MSWD = 2.4) for pyrites from the stringer zone, which requires mixing to have occurred below the sea floor. We employed a two-component mixing model to estimate the contribution of seawater sulfur to the total sulfur budget of the two Teutonic Bore volcanic complex VMS deposits. The results are 15 to 18% for both Teutonic Bore and Bentley, much higher than the 3% obtained by Jamieson et al. (2013) for the giant Kidd Creek deposit. Similar calculations, carried out for other Neoarchean VMS deposits give value between 2% and 30%, which are similar to modern hydrothermal VMS deposits. We suggest that multiple sulfur isotope analyses may be used to predict the size of Archean VMS deposits and to provide a vector to ore deposit but further studies are needed to test these suggestions.

Excessive bone formation in a mouse model of ankylosing spondylitis is associated with decreases in Wnt pathway inhibitors

Introduction: Ankylosing spondylitis (AS) is unique in its pathology where inflammation commences at the entheses before progressing to an osteoproliferative phenotype generating excessive bone formation that can result in joint fusion. The underlying mechanisms of this progression are poorly understood. Recent work has suggested that changes in Wnt signalling, a key bone regulatory pathway, may contribute to joint ankylosis in AS. Using the proteoglycan-induced spondylitis (PGISp) mouse model which displays spondylitis and eventual joint fusion following an initial inflammatory stimulus, we have characterised the structural and molecular changes that underlie disease progression. Methods: PGISp mice were characterised 12 weeks after initiation of inflammation using histology, immunohistochemistry (IHC) and expression profiling. Results: Inflammation initiated at the periphery of the intervertebral discs progressing to disc destruction followed by massively excessive cartilage and bone matrix formation, as demonstrated by toluidine blue staining and IHC for collagen type I and osteocalcin, leading to syndesmophyte formation. Expression levels of DKK1 and SOST, Wnt signalling inhibitors highly expressed in joints, were reduced by 49% and 63% respectively in the spine PGISp compared with control mice (P < 0.05) with SOST inhibition confirmed by IHC. Microarray profiling showed genes involved in inflammation and immune-regulation were altered. Further, a number of genes specifically involved in bone regulation including other members of the Wnt pathway were also dysregulated. Conclusions: This study implicates the Wnt pathway as a likely mediator of the mechanism by which inflammation induces bony ankylosis in spondyloarthritis, raising the potential that therapies targeting this pathway may be effective in preventing this process.

The effect of LRP5 polymorphisms on bone mineral density is apparent in childhood

Bone mass acquired during childhood is the primary determinant of adult bone mineral density (BMD) and osteoporosis risk. Bone accrual is subject to genetic influences. Activating and inactivating LRP5 gene mutations elicit extreme bone phenotypes, while more common LRP5 polymorphisms are associated with normal variation of BMD. Our aim was to test the hypothesis that LRP5 gene polymorphisms influence bone mass acquisition during childhood. The association between LRP5 gene polymorphisms and bone size and mineralization was examined in 819 unrelated British Caucasian children (n = 429 boys) aged 9 years. Height, weight, pubertal status (where available), total-body and spinal bone area, bone mineral content (BMC), BMD, and area-adjusted BMC (aBMC) were assessed. Dual-energy X-ray absorptiometry (DXA)-gene associations were assessed by linear regression, with adjustment for age, gender, pubertal status, and body size parameters. There were 140, 79, 12, and 2 girls who achieved Tanner stages I-IV, respectively, and 179 and 32 boys who achieved Tanner stages I and II, respectively. The rs2306862 (N740N) coding polymorphism in exon 10 of the LRP5 gene was associated with spinal BMD and aBMC (each P = 0.01) and total-body BMD and aBMC (P = 0.04 and 0.03, respectively). Adjusting for pubertal stage strengthened associations between this polymorphism and spinal BMD and aBMC (P = 0.01 and 0.002, respectively). Individuals homozygous for the T allele had greater spinal BMD and aBMC scores than those homozygous for the C allele. A dose effect was apparent as the mean spinal BMD and aBMC of heterozygous TC individuals were intermediate between those of their TT and CC counterparts. The N740N polymorphism in exon 10 of LRP5 was associated with spinal BMD and aBMC in pre- and early pubertal children. These results indicate that LRP5 influences volumetric bone density in childhood, possibly through effects on trabecular bone formation.