Janak L. Pathak

Inflammatory factors in the circulation of patients with active rheumatoid arthritis stimulate osteoclastogenesis via endogenous cytokine production by osteoblasts

SummaryThe combination of cytokines present in the circulation of patients with active rheumatoid arthritis might contribute to the generalized bone loss that commonly occurs in these patients, by directly inhibiting osteoblast proliferation and differentiation, but especially by enhancing endogenous cytokine (i.e., receptor activator of nuclear factor-kappa B ligand (RANKL) and interleukin-6 (IL)-6) production by osteoblasts, thereby stimulating osteoclastogenesis.IntroductionGeneralized bone loss, as occurs in patients with rheumatoid arthritis (RA), is related to elevated levels of circulating cytokines. Individual cytokines have deleterious effects on proliferation and differentiation of osteoblast cell lines, but little is known about the effect of the interaction between inflammatory factors in the circulation of patients with active RA on human osteoblast function, including their communication towards other bone cells. We investigated whether serum from patients with active RA enhances cytokine production by osteoblasts, thereby effectively altering osteoblast-stimulated osteoclastogenesis.MethodsSerum was obtained from 20 patients with active RA (active RA sera) and from the same patients in clinical remission (remission RA sera). To determine osteoclastogenesis, RA serum-pretreated primary human osteoblast cultures were established in direct contact with human osteoclast precursors in the presence or absence of osteoprotegerin (OPG) or IL-6 inhibitor.ResultsCompared to remission RA sera, active RA sera inhibited osteoblast proliferation and differentiation in vitro as demonstrated by a reduced DNA content and gene expression of KI-67, collagen type 1, osteopontin, and osteocalcin. Active RA sera inhibited OPG expression and enhanced RANKL and IL-6 expression but did not alter IL-8 expression in osteoblasts. IL-1β, IL-17, and tumor necrosis factor-α (TNF-α) expression were undetectable. In coculture, active RA sera treatment of osteoblasts stimulated while addition of OPG or IL-6 inhibitory antibodies significantly reduced the number of osteoclasts.ConclusionActive RA sera contain circulating factors, likely cytokines and chemokines, that might contribute to bone loss by directly inhibiting osteoblast proliferation and differentiation, but especially, these factors modulate endogenous cytokine production by osteoblasts, thereby affecting osteoclastogenesis.

CXCL8 and CCL20 Enhance Osteoclastogenesis via Modulation of Cytokine Production by Human Primary Osteoblasts

Generalized osteoporosis is common in patients with inflammatory diseases, possibly because of circulating inflammatory factors that affect osteoblast and osteoclast formation and activity. Serum levels of the inflammatory factors CXCL8 and CCL20 are elevated in rheumatoid arthritis, but whether these factors affect bone metabolism is unknown. We hypothesized that CXCL8 and CCL20 decrease osteoblast proliferation and differentiation, and enhance osteoblast-mediated osteoclast formation and activity. Human primary osteoblasts were cultured with or without CXCL8 (2–200 pg/ml) or CCL20 (5–500 pg/ml) for 14 days. Osteoblast proliferation and gene expression of matrix proteins and cytokines were analyzed. Osteoclast precursors were cultured with CXCL8 (200 pg/ml) and CCL20 (500 pg/ml), or with conditioned medium (CM) from CXCL8 and CCL20-treated osteoblasts with or without IL-6 inhibitor. After 3 weeks osteoclast formation and activity were determined. CXCL8 (200 pg/ml) and CCL20 (500 pg/ml) enhanced mRNA expression of KI67 (2.5–2.7-fold), ALP (1.6–1.7-fold), and IL-6 protein production (1.3–1.6-fold) by osteoblasts. CXCL8-CM enhanced the number of osteoclasts with 3–5 nuclei (1.7-fold), and with >5 nuclei (3-fold). CCL20-CM enhanced the number of osteoclasts with 3–5 nuclei (1.3-fold), and with >5 nuclei (2.8-fold). IL-6 inhibition reduced the stimulatory effect of CXCL8-CM and CCL20-CM on formation of osteoclasts. In conclusion, CXCL8 and CCL20 did not decrease osteoblast proliferation or gene expression of matrix proteins. CXCL8 and CCL20 did not directly affect osteoclastogenesis. However, CXCL8 and CCL20 enhanced osteoblast-mediated osteoclastogenesis, partly via IL-6 production, suggesting that CXCL8 and CCL20 may contribute to osteoporosis in rheumatoid arthritis by affecting bone cell communication.

BMP2-coprecipitated calcium phosphate granules enhance osteoinductivity of deproteinized bovine bone, and bone formation during critical-sized bone defect healing

Most materials used clinically for filling critical-sized bone defects (CSBD), such as deproteinized bovine bone (DBB), lack osteoinductivity so that their therapeutic effects are far from satisfactory. The effect of bone morphogenic protein 2 (BMP2)-coprecipitated biomimetic calcium phosphate granules (BMP2-cop.BioCaP) on osteoinduction of DBB graft(s) during CSBD healing is still unknown. We investigated whether BMP2-cop.BioCaP affects the osteoinductivity of DBB, bone formation, and foreign body reaction during CSBD healing. DBB + BMP2-cop.BioCaP, DBB, DBB + BMP2, DBB + BioCaP, and autologous bone grafts were implanted in the CSBD of sheep. Bone formation, DBB/BioCaP degradability, foreign body reaction, and osteoinductivity of DBB were analyzed histologically and histomorphometrically at week 4 and 8. Combination of BMP2-cop.BioCaP and DBB healed CSBD as effectively as autologous bone grafts. About 95% of the BMP2-cop.BioCaP had been degraded and replaced by new bone at week 8 in the DBB + BMP2-cop.BioCaP-group. Foreign body reaction was reduced in the DBB + BMP2-cop.BioCaP-group compared to the other groups. The independent use of the BMP2-cop.BioCaP did not achieve a satisfactory bone repair. In conclusion, the BMP2-cop.BioCaP showed good degradability and biocompatibility, and enhanced osteoinductivity of DBB during CSBD healing in sheep, suggesting BMP2-cop.BioCaP as a potential osteoinducer to enhance the therapeutic effects of the graft materials in clinic.

Antibacterial and wound healing properties of chitosan/poly(vinyl alcohol)/zinc oxide beads (CS/PVA/ZnO)

Treatment against bacterial infection is crucial for wound healing. Development of cost-effective antibacterial agent with wound healing properties is still in high demand. In this study we aimed to design chitosan/poly(vinyl alcohol)/zinc oxide (CS/PVA/ZnO) beads as novel antibacterial agent with wound healing properties. CS/PVA/ZnO beads were synthesized, and characterized by using XRD, FTIR, SEM, and TEM analysis. Pure chitosan exhibits two peaks at 2θ=10 and 20 and the CS/PVA polymer matrix exhibit the peaks at 2θ=19.7° and another of low intensity at 2θ=11.5°. Pure ZnO shows the characteristic peaks at (100), (002), (101), (102), (110), (103), (200), and (112) that were in good agreement with wurtzite ore having hexagonal lattice structure. The antibacterial activity of CS/PVA/ZnO against Escherichia coli, and Staphylococcus aureus were evaluated with the zone of inhibition method. Antibacterial activity of CS/PVA/ZnO was higher than that of chitosan (CS) and poly(vinyl alcohol (PVA). Hemocompatibility and biocompatibility of CS/PVA/ZnO were tested in in vitro. Wound healing properties of CS/PVA/ZnO were tested in mice skin wound. CS/PVA/ZnO showed strong antimicrobial, wound healing effect, hemocompatibility and biocompatibility. Hence the results strongly support the possibility of using this novel CS/PVA/ZnO material for the anti bacterial and wound healing application.

Osteocyte isolation and culture methods

The aim of this paper is to present several popular methods for in vitro culture of osteocytes and osteocyte cell lines. Osteocytes are located extremely suitably within the calcified bone matrix to sense mechanical signals, and are equipped with a multitude of molecular features that allow mechanosensing. However, osteocytes are more than specialized mechanosensing cells. Several signaling molecules are preferentially produced by osteocytes, and osteocytes hold a tight reign over osteoblast and osteoclast formation and activity, but also have a role as endocrine cell, communicating with muscles or organs as remote as the kidneys. In order to facilitate further research into this fascinating cell type, three protocols will be provided in this paper. The first protocol will be on the culture of mouse (early) osteocyte cell lines, the second on the isolation and culture of primary mouse bone cells, and the third on the culture of fully embedded human osteocytes within their own three-dimensional bone matrix.

Bisphenol A (BPA) the mighty and the mutagenic

Graphical abstract

Traditional Chinese medicine formula Bi-Qi capsule alleviates rheumatoid arthritis-induced inflammation, synovial hyperplasia, and cartilage destruction in rats

BackgroundTraditional Chinese medicine (TCM) formula Bi-Qi capsule (Bi-Qi) is a commonly prescribed drug to treat rheumatoid arthritis (RA). However, the mechanism of Bi-Qi-mediated amelioration of RA pathogenesis is still a mystery. Collagen induced arthritis (CIA) in rats is an established model that shares many similarities with RA in humans. In this study we investigated the effect of Bi-Qi on the pathogenesis of CIA in rats.MethodsCIA was developed in Sprague-Dawley (S.D) rats (n = 60, female) and used as a model resembling RA in humans. Rats were treated with a high or moderate dose of Bi-Qi, or methotrexate (MTX). Effects of the treatment on local joint and systemic inflammation, synovial hyperplasia, cartilage destruction, and other main features in the pathogenesis of CIA were analyzed.ResultsInflamed and swollen ankles and joints were observed in arthritic rats, while Bi-Qi or MTX treatment alleviated these symptoms. Only the Bi-Qi moderate dose decreased RA-induced serum levels of tumor necrosis factor-alpha (TNF-α). Both Bi-Qi and MTX reduced the interleukin (IL)-18 serum level. Protein levels of cartilage oligomeric matrix protein and osteopontin in serum, synovium, and cartilage were elevated in arthritic rats, while Bi-Qi alleviated these effects. Synovial hyperplasia, inflammatory cell infiltration in synovium and a high degree of cartilage degradation was observed in RA, and Bi-Qi or MTX alleviated this effect. Bi-Qi at the moderate dose was the most effective in mitigating CIA-related clinical complications.ConclusionsOur findings showed that Bi-Qi alleviates CIA-induced inflammation, synovial hyperplasia, cartilage destruction, and the other main features in the pathogenesis of CIA. This provides fundamental evidence for the anti-arthritic properties of Bi-Qi and corroborates the use of Bi-Qi TCM formula for the treatment of RA.

Design of polyaspartic acid peptide-poly (ethylene glycol)-poly (ε-caprolactone) nanoparticles as a carrier of hydrophobic drugs targeting cancer metastasized to bone

Treatment of cancer metastasized to bone is still a challenge due to hydrophobicity, instability, and lack of target specificity of anticancer drugs. Poly (ethylene glycol)-poly (ε-caprolactone) polymer (PEG-PCL) is an effective, biodegradable, and biocompatible hydrophobic drug carrier, but lacks bone specificity. Polyaspartic acid with eight peptide sequences, that is, (Asp)8, has a strong affinity to bone surface. The aim of this study was to synthesize (Asp)8-PEG-PCL nanoparticles as a bone-specific carrier of hydrophobic drugs to treat cancer metastasized to bone. 1H nuclear magnetic resonance, Fourier transform infrared spectroscopy, and transmission electron microscopy data showed that (Asp)8-PEG-PCL nanoparticles (size 100 nm) were synthesized successfully. (Asp)8-PEG-PCL nanoparticles did not promote erythrocyte aggregation. Fluorescence microscopy showed clear uptake of Nile red-loaded (Asp)8-PEG-PCL nanoparticles by cancer cells. (Asp)8-PEG-PCL nanoparticles did not show cytotoxic effect on MG63 and human umbilical vein endothelial cells at the concentration of 10–800 μg/mL. (Asp)8-PEG-PCL nanoparticles bound with hydroxyapatite 2-fold more than PEG-PCL. Intravenously injected (Asp)8-PEG-PCL nanoparticles accumulated 2.7-fold more on mice tibial bone, in comparison to PEG-PCL. Curcumin is a hydrophobic anticancer drug with bone anabolic properties. Curcumin was loaded in the (Asp)8-PEG-PCL. (Asp)8-PEG-PCL showed 11.07% loading capacity and 95.91% encapsulation efficiency of curcumin. The curcumin-loaded (Asp)8-PEG-PCL nanoparticles gave sustained release of curcumin in high dose for >8 days. The curcumin-loaded (Asp)8-PEG-PCL nanoparticles showed strong antitumorigenic effect on MG63, MCF7, and HeLa cancer cells. In conclusion, (Asp)8-PEG-PCL nanoparticles were biocompatible, permeable in cells, a potent carrier, and an efficient releaser of hydrophobic anticancer drug and were bone specific. The curcumin-loaded (Asp)8-PEG-PCL nanoparticles showed strong antitumorigenic ability in vitro. Therefore, (Asp)8-PEG-PCL nanoparticles could be a potent carrier of hydrophobic anticancer drugs to treat the cancer metastasized to bone.

pH dependent silver nanoparticles releasing titanium implant: A novel therapeutic approach to control peri-implant infection

Peri-implant infection control is crucial for implant fixation and durability. Antimicrobial administration approaches to control peri-implant infection are far from satisfactory. During bacterial infection, pH level around the peri-implant surface decreases as low as pH 5.5. This change of pH can be used as a switch to control antimicrobial drug release from the implant surface. Silver nanoparticles (AgNPs) have broad-spectrum antimicrobial properties. In this study, we aimed to design a pH-dependent AgNPs releasing titania nanotube arrays (TNT) implant for peri-implant infection control. The nanotube arrays were fabricated on the surface of titanium implant as containers; AgNPs were grafted on TNT implant surface via a low pH-sensitive acetal linker (TNT-AL-AgNPs). SEM, TEM, AFM, FTIR as well as XPS data showed that AgNPs have been successfully linked to TNT via acetal linker without affecting the physicochemical characteristics of TNT. The pH 5.5 enhanced AgNPs release from TNT-AL-AgNPs implant compared with pH 7.4. AgNPs released at pH 5.5 robustly increased antimicrobial activities against gram-positive and gram-negative bacteria compared with AgNPs released at pH 7.4. TNT-AL-AgNPs implant enhanced osteoblast proliferation, differentiation, and did not affect osteoblast morphology in vitro. In conclusion, incorporation of AgNPs in TNT via acetal linker maintained the surface characteristics of TNT. TNT-AL-AgNPs implant was biocompatible to osteoblasts and showed osteoinductive properties. AgNPs were released from TNT-AL-AgNPs implant in high dose at pH 5.5, and this release showed strong antimicrobial properties in vitro. Therefore, this novel design of low pH-triggered AgNPs releasing TNT-AL-AgNPs could be an infection-triggered antimicrobial releasing implant model to control peri-implant infection.

Sustained Release of Zoledronic Acid from Mesoporous TiO2-Layered Implant Enhances Implant Osseointegration in Osteoporotic Condition

Implant surface modification that provides local sustained release of osteoinductive therapeutic agents enhances implant stability. We designed a mesoporous TiO2-layered titanium implant (MLT) by modified anodization technique that allowed local sustained release of zoledronic acid up to 21 days. Mesoporous layer has pore size 15 nm, depth ∼30 μm, volume 0.32 cm3/g, surface area 112.3 m2/g, surface roughness 20 nm and water contact angle 18.3°. Zoledronic acid-loaded MLT (MLT-Z) was biocompatible, showed anabolic effect on bone forming osteoblasts and catabolic effect on bone resorbing osteoclasts. MLT or MLT-Z implants were implanted in osteoporotic rat-tail vertebrae. Smooth implant in healthy rats were used as a positive control. Histomorphometric analysis showed that bone implant contact of smooth implant in osteoporotic rats was reduced by 4.1-fold compared to healthy rats and MLT-Z rescued the effect by 53%. Similar effect was observed in implant fixation, mechanical stability, BV/TV ratio, Tb.N, Tb.Th and OI% among the groups. Histological and μ-CT images strongly supported the above-mentioned findings. In conclusion, a novel surface-fabricated MLT-Z gives local sustained drug release, robustly enhances implant osseointegration and stability in osteoporotic condition, suggesting it as a promising implant model for patients with compromised bone quality.