Nephrocalcinosis in children who received high-dose vitamin D.

BACKGROUND: Vitamin D supplements are readily available as over-the-counter preparations. However, although rare, cases of vitamin D overdose still occur and are associated with nephrocalcinosis and life-threatening hypercalcemia. Errors in manufacturing of nutritional supplements may be a cause of vitamin D intoxication in children. This study aimed to identify factors associated with vitamin D overdose-related nephrocalcinosis in children due to manufacturing errors in supplements. METHODS: This retrospective study reviewed medical charts of pediatric patients with non-registered supplement-related vitamin D overdose at a tertiary referral hospital between 2006 and 2011. Clinical and laboratory characteristics of patients with or without nephrocalcinosis were evaluated. Receiver operating characteristics curve and area under the receiver operating characteristics curve were used to determine the most predictive value of each characteristic. RESULTS: A total of 44 patients (males: 29; age: 7-62 months) were included. Age ≤ 16.5 months, body weight ≤ 10.25 kg, body height ≤ 78.5 cm, body surface area (BSA) ≤ 0.475 m2, 25-hydroxyvitamin D3 ≥ 143 ng/mL, and calcium ≥ 10.65 mg/dL were predictive of developing nephrocalcinosis with a sensitivity and specificity of > 60%. Univariant analysis revealed that BSA was the most significant anthropometric prognostic factor (odds ratio: 12.09; 95% confidence interval: 2.61-55.72; P = 0.001). CONCLUSIONS: Children with smaller BSAs were more vulnerable to high-dose vitamin D3-related nephrocalcinosis. Physicians and parents should be aware of the potential adverse effects of vitamin D overdose in children. A higher resolution version of the Graphical abstract is available as Supplementary information.

Differential androgen deprivation therapies with anti-androgens casodex/bicalutamide or MDV3100/Enzalutamide versus anti-androgen receptor ASC-J9(R) Lead to promotion versus suppression of prostate cancer metastasis.

Despite the fact that androgen deprivation therapy (ADT) can effectively reduce prostate cancer (PCa) size, its effect on PCa metastasis remains unclear. We examined the existing data on PCa patients treated with ADT plus anti-androgens to analyze ADT effects on primary tumor size, prostate-specific antigen (PSA) values, and metastatic incidence. We found that the current ADT with anti-androgens might lead to primary tumor reduction, with PSA decreased yet metastases increased in some PCa patients. Using in vitro and in vivo metastasis models with four human PCa cell lines, we evaluated the effects of the currently used anti-androgens, Casodex/bicalutamide and MDV3100/enzalutamide, and the newly developed anti-AR compounds, ASC-J9® and cryptotanshinone, on PCa cell growth and invasion. In vitro results showed that 10 µm Casodex or MDV3100 treatments suppressed PCa cell growth and reduced PSA level yet significantly enhanced PCa cell invasion. In vivo mice studies using an orthotopic xenograft mouse model also confirmed these results. In contrast, ASC-J9® led to suppressed PCa cell growth and cell invasion in in vitro and in vivo models. Mechanism dissection indicated these Casodex/MDV3100 treatments enhanced the TGF-ß1/Smad3/MMP9 pathway, but ASC-J9® and cryptotanshinone showed promising anti-invasion effects via down-regulation of MMP9 expression. These findings suggest the potential risks of using anti-androgens and provide a potential new therapy using ASC-J9® to battle PCa metastasis at the castration-resistant stage.

Targeting androgen receptor in bone marrow mesenchymal stem cells leads to better transplantation therapy efficacy in liver cirrhosis.

UNLABELLED: Transplantation of bone marrow mesenchymal stem cells (BM-MSCs) has been considered as an alternative therapy, replacing liver transplantation in clinical trials, to treat liver cirrhosis, an irreversible disease that may eventually lead to liver cancer development. However, low survival rate of the BM-MSCs leading to unsatisfactory efficacy remains a major concern. Gender differences have been suggested in BM-MSCs therapeutic application, but the effect of the androgen receptor (AR), a key factor in male sexual phenotype, in this application is not clear. Using two liver cirrhosis mouse models induced by CCl4 or thioacetamide, we showed that targeting AR in the BM-MSCs improved their self-renewal and migration potentials and increased paracrine effects to exert anti-inflammatory and anti-fibrotic actions to enhance liver repair. Mechanism dissection studies suggested that knocking out AR in BM-MSCs led to improved self-renewal and migration by alteration of the signaling of epidermal growth factor receptor and matrix metalloproteinase 9 and resulted in suppression of infiltrating macrophages and hepatic stellate cell activation through modulation of interleukin (IL)1R/IL1Ra signaling. Therapeutic approaches using either AR/small interfering RNA or the AR degradation enhancer, ASC-J9, to target AR in BM-MSCs all led to increased efficacy for liver repair. CONCLUSION: Targeting AR, a key factor in male sexual phenotype, in BM-MSCs improves transplantation therapeutic efficacy for treating liver fibrosis.

Effects of erythropoietin in neonates with hypoxic-ischemic encephalopathy receiving therapeutic hypothermia.

BACKGROUND: Minimizing multiple organ dysfunction-related mortality and morbidity is a critical issue for patients with hypoxic-ischemic encephalopathy (HIE) receiving therapeutic hypothermia (TH). Although erythropoietin (EPO) has demonstrated protective effects on various hypoxic-ischemic organs in animal studies and clinical trials in adults, its effects on neonates with HIE require further investigation. METHODS: This study retrospectively analyzed the medical records of neonates with HIE who received TH with or without EPO (TH+EPO vs TH groups) administration in a tertiary referral hospital from January 2016 to January 2021. Data regarding patient characteristics, medical treatment, and clinical (neurological, cardiac, respiratory, gastrointestinal, hepatic, and renal) function assessments were collected. To control for confounding factors and selection bias between the two groups, a 1:1 propensity matching method was applied. RESULTS: A total of 45 neonates with HIE received TH during the study period, with 24 patients (53%) in the TH+EPO group. After matching, each group enrolled 13 cases. No significant difference in mortality or hospital stay between the two groups was noted. During the first 3 days, the patients in the TH+EPO group showed significantly higher blood pressure (BP) than those in the TH group ( p < 0.05 on day 1). The TH+EPO group showed trends of higher blood hemoglobin ( p > 0.05) and creatinine ( p > 0.05) levels and lower estimated glomerular filtration rate ( p > 0.05) and urine output ( p > 0.05) during the first 2 weeks than TH group. CONCLUSION: The use of EPO in addition to TH is safe for neonates with HIE. The neonates with moderate or severe HIE who received EPO may have a lesser risk of hypotension than those who received TH alone. Further clinical studies on renal and cardiac functions and long-term neurological effects of EPO are required.

The selective inhibitory effect of a synthetic tanshinone derivative on prostate cancer cells.

BACKGROUND: Androgen receptor (AR) is the main therapeutic target for the treatment of prostate cancer (PCa). Anti-androgens to reduce or prevent androgens binding to AR are widely used to suppress AR-mediated PCa growth; however, the androgen depletion therapy (ADT) is only effective for a short period of time. Here we tested PTS33, a new sodium derivative of cryptotanshinone, which can effectively inhibit the DHT-induced AR transactivation and PCa cell growth, and then explored the effects of PTS33 on inhibiting the expressions of AR target genes and proteins. METHODS: PCa cells, LNCaP, CWR22Rv1, C4-2, PC-3, and DU145, were treated with PTS33 and luciferase assay was used to evaluate the ability of each to regulate AR transactivation. RT-PCR was used to evaluate the mRNA levels of AR target genes such as PSA, TMPRSS2, and TMEPA1. Western blot was used to determine AR, PSA, estrogen receptor alpha (ERα), glucocorticoid receptor (GR), and progesterone receptor (PR) protein expression. Cell growth and IC50 were determined by MTT assay after 48 hr treatment. RESULTS: Our data showed that PTS33 selectively inhibits AR activities, but PTS33 does not repress the activities of other nuclear receptors, including ERα, GR, and PR. At a low concentration, 2 µM of PTS33 effectively suppresses the growth of AR-positive PCa cells, and has little effect on AR-negative PCa cells. Furthermore, our data indicated that PTS33 could modulate AR transactivation and suppress the AR target genes (PSA, TMPRSS2, and TMEPA1) expression in both androgen responsive PCa LNCaP cells and castration-resistant C4-2 cells. In addition, PTS33 can also inhibit estrogen/Δ5-androstenediol induced AR activities. The mechanistic studies indicate that PTS33 can inhibit AR function by suppression of AR protein expression, the AR N-C interaction, and AR-coregulator interaction. CONCLUSIONS: PTS33 has shown a good efficacy to inhibit AR transactivation, block AR regulated gene expression, and reduce cell growth in AR positive PCa cells. The structure of PTS33 could be used as a base for development of novel AR signaling inhibitors to treat PCa.

Effect of Aging on the Macrophage Response to Titanium Particles.

Macrophage-mediated inflammatory reaction to implant wear particles drives bone loss around total joint replacements (TJR). Although most TJR recipients are elderly, studies linking wear particle-activated macrophages and peri-implant osteolysis have not taken into account the multiple effects that aging has on the innate immune system and, in particular, on macrophages. To address this, we compared the wear particle responses of bone marrow macrophages obtained from young (2-month) and aged (18-month) mice. Macrophages were polarized to M0, M1, or M2 phenotypes in vitro, challenged with titanium particles, and their inflammatory response was characterized at multiple time points by quantitative reverse-transcription polymerase chain reaction and enzyme-linked immunosorbent assay. In addition, age-dependent changes in activation of transcription factor nuclear factor-κB were analyzed by a lentiviral vector-based luciferase reporter system. The particle stimulation experiment was further repeated using human primary macrophages isolated from blood donors of different ages. We found that the pro-inflammatory responses were generally higher in macrophages obtained from young mice, but differences between the age groups remained small and of uncertain biological significance. Noteworthily, M2 polarization effectively suppressed the particle-induced inflammation in both young and aged macrophages. These results suggest that aging of the innate immune system per se plays no significant role in the response of macrophages to titanium particles, whereas induction of M2 polarization appears a promising strategy to limit macrophage-mediated inflammation regardless of age. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:405-416, 2020.

Macrophage Effects on Mesenchymal Stem Cell Osteogenesis in a Three-Dimensional In Vitro Bone Model.

As musculoskeletal (MSK) disorders continue to increase globally, there is an increased need for novel, in vitro models to efficiently study human bone physiology in the context of both healthy and diseased conditions. For these models, the inclusion of innate immune cells is critical. Specifically, signaling factors generated from macrophages play key roles in the pathogenesis of many MSK processes and diseases, including fracture, osteoarthritis, infection etc. In this study, we aim to engineer three-dimensional (3D) and macrophage-encapsulated bone tissues in vitro, to model cell behavior, signaling, and other biological activities in vivo, in comparison to current two-dimensional models. We first investigated and optimized 3D culture conditions for macrophages, and then co-cultured macrophages with mesenchymal stem cells (MSCs), which were induced to undergo osteogenic differentiation to examine the effect of macrophage on new bone formation. Seeded within a 3D hydrogel scaffold fabricated from photocrosslinked methacrylated gelatin, macrophages maintained high viability and were polarized toward an M1 or M2 phenotype. In co-cultures of macrophages and human MSCs, MSCs displayed immunomodulatory activities by suppressing M1 and enhancing M2 macrophage phenotypes. Lastly, addition of macrophages, regardless of polarization state, increased MSC osteogenic differentiation, compared with MSCs alone, with proinflammatory M1 macrophages enhancing new bone formation most effectively. In summary, this study illustrates the important roles that macrophage signaling and inflammation play in bone tissue formation.

Production of GFP and Luciferase-Expressing Reporter Macrophages for In Vivo Bioluminescence Imaging.

Macrophages have emerged as crucial regulators of tissue homeostasis, inflammation, and tissue regeneration. In vivo bioluminescence imaging could offer a powerful tool to study many poorly understood aspects of macrophage biology. Thus, we recently developed a straightforward method for the production of large numbers of green fluorescent protein (GFP) and firefly luciferase (fLUC)-expressing reporter macrophages for various in vivo bioluminescence imaging applications. Lentivirus vector containing the GFP/fLUC reporter gene is produced and mouse bone marrow macrophages are isolated following established protocols. Macrophages are then exposed to the lentivirus in the presence of 10 µM cyclosporine for 24 h. After a 24-h recovery period, the transduction is repeated. Three days after the second infection the cells are ready to be used in vivo. Following this cyclosporine-mediated double infection strategy up to 60% of the macrophages express GFP in flow cytometry. The macrophages maintain their ability to polarize to M1 and M2 phenotypes and, when injected to the systemic circulation of a mouse model, reporter cells are both easily detectable with BLI and migrate to a local site of inflammation. These GFP/fLUC-expressing reporter macrophages could prove to be useful tools to study the role of macrophages in health and disease.

Mesenchymal stem cells in the aseptic loosening of total joint replacements.

Peri-prosthetic osteolysis remains as the main long-term complication of total joint replacement surgery. Research over four decades has established implant wear as the main culprit for chronic inflammation in the peri-implant tissues and macrophages as the key cells mediating the host reaction to implant-derived wear particles. Wear debris activated macrophages secrete inflammatory mediators that stimulate bone resorbing osteoclasts; thus bone loss in the peri-implant tissues is increased. However, the balance of bone turnover is not only dictated by osteoclast-mediated bone resorption but also by the formation of new bone by osteoblasts; under physiological conditions these two processes are tightly coupled. Increasing interest has been placed on the effects of wear debris on the cells of the bone-forming lineage. These cells are derived primarily from multipotent mesenchymal stem cells (MSCs) residing in bone marrow and the walls of the microvasculature. Accumulating evidence indicates that wear debris significantly impairs MSC-to-osteoblast differentiation and subsequent bone formation. In this review, we summarize the current understanding of the effects of biomaterial implant wear debris on MSCs. Emerging treatment options to improve initial implant integration and treat developing osteolytic lesions by utilizing or targeting MSCs are also discussed. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1195-1207, 2017.

The biological response to orthopedic implants for joint replacement. II: Polyethylene, ceramics, PMMA, and the foreign body reaction.

Novel evidence-based prosthetic designs and biomaterials facilitate the performance of highly successful joint replacement (JR) procedures. To achieve this goal, constructs must be durable, biomechanically sound, and avoid adverse local tissue reactions. Different biomaterials such as metals and their alloys, polymers, ceramics, and composites are currently used for JR implants. This review focuses on (1) the biological response to the different biomaterials used for TJR and (2) the chronic inflammatory and foreign-body response induced by byproducts of these biomaterials. A homeostatic state of bone and surrounding soft tissue with current biomaterials for JR can be achieved with mechanically stable, infection free and intact (as opposed to the release of particulate or ionic byproducts) implants. Adverse local tissue reactions (an acute/chronic inflammatory reaction, periprosthetic osteolysis, loosening and subsequent mechanical failure) may evolve when the latter conditions are not met. This article (Part 2 of 2) summarizes the biological response to the non-metallic materials commonly used for joint replacement including polyethylene, ceramics, and polymethylmethacrylate (PMMA), as well as the foreign body reaction to byproducts of these materials. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1685-1691, 2017.

CCL2, CCL5, and IGF-1 participate in the immunomodulation of osteogenesis during M1/M2 transition in vitro.

The modulation of macrophage phenotype from pro-inflammatory (M1) to tissue healing (M2) via exogenous addition of interleukin-4 (IL-4) facilitates osteogenesis; however, the molecular mediators underlying this phenomenon remain unknown. This study characterizes the IL-4-dependent paracrine crosstalk between macrophages and osteoprogenitors and its effect on osteogenesis in vitro. Primary murine M1 were co-cultured with MC3T3 cells (M1-MC3T3) in both transwell plates and direct co-cultures. To modulate M1 to M2, M1-MC3T3 were treated with IL-4 (20 ng/mL) at day 3 after seeding (M1 + IL-4-MC3T3). Selected molecular targets were assessed at days 3 and 6 after seeding at protein and mRNA levels. Mineralization was assessed at day 21. Transwell M1 + IL-4-MC3T3 significantly enhanced the secretion of CCL2/MCP-1, IGF-1 and to a lesser degree, CCL5/RANTES at day 6. At day 3, alkaline phosphatase (Alpl) was upregulated in direct M1-MC3T3. At day 6, Smurf2 and Insulin growth factor-1 (IGF-1) were downregulated and upregulated, respectively, in direct M1 + IL-4-MC3T3. Finally, M1 + IL-4-MC3T3 increased bone matrix mineralization compared with MC3T3 cells in transwell, but this was significantly less than M1-MC3T3. Taken together, macrophage subtypes enhanced the osteogenesis in transwell setting and the transition from M1 to M2 was associated with an increase in bone anabolic factors CCL2/MCP-1, CCL5/RANTES and IGF-1 in vitro. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 3069-3076, 2017.

Pro-inflammatory M1 macrophages promote Osteogenesis by mesenchymal stem cells via the COX-2-prostaglandin E2 pathway.

Bone fractures are among the most common orthopaedic problems that affect individuals of all ages. Immediately after injury, activated macrophages dynamically contribute to and regulate an acute inflammatory response that involves other cells at the injury site, including mesenchymal stem cells (MSCs). These macrophages and MSCs work in concert to modulate bone healing. In this study, we co-cultured undifferentiated M0, pro-inflammatory M1, and anti-inflammatory M2 macrophages with primary murine MSCs in vitro to determine the cross-talk between polarized macrophages and MSCs and their effects on osteogenesis. After 4 weeks of co-culture, MSCs grown with macrophages, especially M1 macrophages, had enhanced bone mineralization compared to MSCs grown alone. The level of bone formation after 4 weeks of culture was closely associated with prostaglandin E2 (PGE2) secretion early in osteogenesis. Treatment with celecoxib, a cyclooxygenase-2 (COX-2) selective inhibitor, significantly reduced bone mineralization in all co-cultures but most dramatically in the M1-MSC co-culture. We also found that the presence of macrophages reduced the secretion of osteoprotegerin (OPG), the decoy RANKL receptor, suggesting that macrophages may indirectly modulate osteoclast activity in addition to enhancing bone formation. Taken together, these findings suggest that an initial pro-inflammatory phase modulated by M1 macrophages promotes osteogenesis in MSCs via the COX-2-PGE2 pathway. Understanding the complex interactions between macrophages and MSCs provide opportunities to optimize bone healing and other regenerative processes via modulation of the inflammatory response. This study provides one possible biological mechanism for the adverse effects of non-steroidal anti-inflammatory drugs on fracture healing and bone regeneration. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2378-2385, 2017.

* Murine Model of Progressive Orthopedic Wear Particle-Induced Chronic Inflammation and Osteolysis.

Periprosthetic osteolysis and subsequent aseptic loosening of total joint replacements are driven by byproducts of wear released from the implant. Wear particles cause macrophage-mediated inflammation that culminates with periprosthetic bone loss. Most current animal models of particle-induced osteolysis are based on the acute inflammatory reaction induced by wear debris, which is distinct from the slowly progressive clinical scenario. To address this limitation, we previously developed a murine model of periprosthetic osteolysis that is based on slow continuous delivery of wear particles into the murine distal femur over a period of 4 weeks. The particle delivery was accomplished by using subcutaneously implanted osmotic pumps and tubing, and a hollow titanium rod press-fit into the distal femur. In this study, we report a modification of our prior model in which particle delivery is extended to 8 weeks to better mimic the progressive development of periprosthetic osteolysis and allow the assessment of interventions in a setting where the chronic particle-induced osteolysis is already present at the initiation of the treatment. Compared to 4-week samples, extending the particle delivery to 8 weeks significantly exacerbated the local bone loss observed with µCT and the amount of both peri-implant F4/80+ macrophages and tartrate-resistant acid phosphatase-positive osteoclasts detected with immunohistochemical and histochemical staining. Furthermore, systemic recruitment of reporter macrophages to peri-implant tissues observed with bioluminescence imaging continued even at the later stages of particle-induced inflammation. This modified model system could provide new insights into the mechanisms of chronic inflammatory bone loss and be particularly useful in assessing the efficacy of treatments in a setting that resembles the clinical scenario of developing periprosthetic osteolysis more closely than currently existing model systems.

Decreased osteogenesis in mesenchymal stem cells derived from the aged mouse is associated with enhanced NF-κB activity.

Aging is associated with significant bone loss and delayed fracture healing. NF-κB activation is highly correlated with inflammatory-associated bone diseases including infection, wear particle exposure, and chronic inflammation during natural aging processes. The critical roles of NF-κB in both the pro-inflammatory response and osteoclast-mediated bone resorption have been well defined. However, the biological effects of NF-κB activation in mesenchymal stem cell (MSC)-mediated bone formation remain largely unknown. In the current study, bone marrow-MSCs were isolated from young (8 weeks old) and aged (72 weeks old) mice. NF-κB activity in MSCs at basal levels and under different biological conditions were determined by our recently established lentiviral vector-based luciferase reporter assay. We found that NF-κB activity was increased in aged MSCs at basal levels or when exposed to low dose (10 or 100 ng/ml) lipopolysaccharide (LPS); this effect was not seen when the cells were exposed to higher dose (1 µg/ml) LPS. During osteogenesis, NF-κB activity was increased in aged MSCs at weeks 1 and 2, but showed no significant difference at week 3. Both Smurf2 and TAZ, the NF-κB target genes that regulate osteogenic differentiation, were increased in aged MSCs. In addition, the expression of RANKL was dramatically increased, and OPG was decreased in aged MSCs. Our findings suggest that targeting NF-κB activity in MSCs has the potential to modulate aging-associated bone loss, or enhance bone-healing in aged patients. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:281-288, 2017.

Mutant CCL2 protein coating mitigates wear particle-induced bone loss in a murine continuous polyethylene infusion model.

Wear particle-induced osteolysis limits the long-term survivorship of total joint replacement (TJR). Monocyte/macrophages are the key cells of this adverse reaction. Monocyte Chemoattractant Protein-1 (MCP-1/CCL2) is the most important chemokine regulating trafficking of monocyte/macrophages in particle-induced inflammation. 7ND recombinant protein is a mutant of CCL2 that inhibits CCL2 signaling. We have recently developed a layer-by-layer (LBL) coating platform on implant surfaces that can release biologically active 7ND. In this study, we investigated the effect of 7ND on wear particle-induced bone loss using the murine continuous polyethylene (PE) particle infusion model with 7ND coating of a titanium rod as a local drug delivery device. PE particles were infused into hollow titanium rods with or without 7ND coating implanted in the distal femur for 4 weeks. Specific groups were also injected with RAW 264.7 as the reporter macrophages. Wear particle-induced bone loss and the effects of 7ND were evaluated by microCT, immunohistochemical staining, and bioluminescence imaging. Local delivery of 7ND using the LBL coating decreased systemic macrophage recruitment, the number of osteoclasts and wear particle-induced bone loss. The development of a novel orthopaedic implant coating with anti-CCL2 protein may be a promising strategy to mitigate peri-prosthetic osteolysis.

Inflammation, fracture and bone repair.

The reconstitution of lost bone is a subject that is germane to many orthopedic conditions including fractures and non-unions, infection, inflammatory arthritis, osteoporosis, osteonecrosis, metabolic bone disease, tumors, and periprosthetic particle-associated osteolysis. In this regard, the processes of acute and chronic inflammation play an integral role. Acute inflammation is initiated by endogenous or exogenous adverse stimuli, and can become chronic in nature if not resolved by normal homeostatic mechanisms. Dysregulated inflammation leads to increased bone resorption and suppressed bone formation. Crosstalk among inflammatory cells (polymorphonuclear leukocytes and cells of the monocyte-macrophage-osteoclast lineage) and cells related to bone healing (cells of the mesenchymal stem cell-osteoblast lineage and vascular lineage) is essential to the formation, repair and remodeling of bone. In this review, the authors provide a comprehensive summary of the literature related to inflammation and bone repair. Special emphasis is placed on the underlying cellular and molecular mechanisms, and potential interventions that can favorably modulate the outcome of clinical conditions that involve bone repair.

The effects of immunomodulation by macrophage subsets on osteogenesis in vitro.

BACKGROUND: Bone formation and remodeling are influenced by the inflammatory state of the local microenvironment. In this regard, macrophages are postulated to play a crucial role in modulating osteogenesis. However, the differential effects of macrophage subsets and their plasticity on bone formation are currently unknown. METHODS: Polarized primary murine macrophages and preosteoblastic MC3T3 cells were co-cultured to investigate the effect of non-activated M0, pro-inflammatory M1, and tissue-regenerative M2 macrophages on the osteogenic ability of MC3T3-E1 cells in vitro. Furthermore, to model the physiological transition from inflammation to tissue regeneration, M1-MC3T3 co-cultures were treated with interleukin-4 (IL-4) at different time points to modulate the M1 phenotype towards M2. Macrophage phenotypic markers were assessed by flow cytometry and enzyme-linked immunosorbent assay. A time course study of osteogenic markers at different time points was conducted: alkaline phosphatase (ALP) mRNA levels were evaluated at week 1, ALP activity and osteocalcin and osteopontin mRNA levels at week 2, and matrix mineralization and osteocalcin and osteopontin protein concentrations at week 3. Supernatant collected 72 hours after seeding or IL-4 treatment, whichever was later, was analyzed for oncostatin M, a cytokine released by macrophages that has been recognized to enhance osteogenesis. Unpaired t test or one-way ANOVA with Tukey's or Dunnett's post hoc tests were used for statistical comparison of the groups. RESULTS: Co-culture with any of the macrophage subtypes increased the osteogenic ability of MC3T3 cells as indicated by increases in ALP activity and matrix mineralization. Increased ALP activity, osteocalcin concentration, and matrix mineralization demonstrated that osteogenesis by M1-MC3T3 co-cultures was further enhanced by macrophage phenotype modulation to M2 via IL-4 treatment 72 hours after seeding. Increased oncostatin M protein concentration in untreated M1-MC3T3 co-cultures and M1-MC3T3 co-cultures treated with IL-4 at 72 hours correlated with greater ALP activity and matrix mineralization. CONCLUSIONS: These results suggest that a transient inflammatory phase is crucial for enhanced bone formation. Macrophage plasticity may offer new strategies for modulating the local inflammatory microenvironment with the aim of potentially enhancing bone repair.

NF-κB decoy oligodeoxynucleotide mitigates wear particle-associated bone loss in the murine continuous infusion model.

UNLABELLED: Total joint replacement is a cost-effective surgical procedure for patients with end-stage arthritis. Wear particle-induced chronic inflammation is associated with the development of periprosthetic osteolysis. Modulation of NF-κB signaling in macrophages, osteoclasts, and mesenchymal stem cells could potentially mitigate this disease. In the current study, we examined the effects of local delivery of decoy NF-κB oligo-deoxynucleotide (ODN) on wear particle-induced bone loss in a murine continuous femoral particle infusion model. Ultra-high molecular weight polyethylene particles (UHMWPE) with or without lipopolysaccharide (LPS) were infused via osmotic pumps into hollow titanium rods placed in the distal femur of mice for 4weeks. Particle-induced bone loss was evaluated by µCT, and immunohistochemical analysis of sections from the femur. Particle infusion alone resulted in reduced bone mineral density and trabecular bone volume fraction in the distal femur. The decoy ODN reversed the particle-associated bone volume fraction loss around the implant, irrespective of the presence of LPS. Particle-infusion with LPS increased bone mineral density in the distal femur compared with particle-infusion alone. NF-κB decoy ODN reversed or further increased the bone mineral density in the femur (3-6mm from the distal end) exposed to particles alone or particles plus LPS. NF-κB decoy ODN also inhibited macrophage infiltration and osteoclast number, but had no significant effects on osteoblast numbers in femurs exposed to wear particles and LPS. Our study suggests that targeting NF-κB activity via local delivery of decoy ODN has great potential to mitigate wear particle-induced osteolysis. STATEMENT OF SIGNIFICANCE: Total joint replacement is a cost-effective surgical procedure for patients with end-stage arthritis. Chronic inflammation is crucial for the development of wear particle-associated bone loss. Modulation of NF-κB signaling in macrophages (pro-inflammatory cells), osteoclasts (bone-resorbing cells), and osteoblasts (bone-forming cells) could potentially mitigate this disease. Here we demonstrated that local delivery of decoy NF-κB oligo-deoxynucleotide (ODN) mitigated ultra-high molecular weight polyethylene (UHMWPE) wear particle induced bone loss in a clinically relevant murine model. The protective effects of decoy ODN was associated with reduced macrophage infiltration and osteoclast activation, but had no significant effects on osteoblast numbers. Our study suggests that targeting NF-κB activity via local delivery of decoy ODN has great potential to mitigate wear particle-induced bone loss.

The effect of local IL-4 delivery or CCL2 blockade on implant fixation and bone structural properties in a mouse model of wear particle induced osteolysis.

Modulation of macrophage polarization and prevention of CCL2-induced macrophage chemotaxis are emerging strategies to reduce wear particle induced osteolysis and aseptic total joint replacement loosening. In this study, the effect of continuous IL-4 delivery or bioactive implant coating that constitutively releases a protein inhibitor of CCL2 signaling (7ND) on particle induced osteolysis were studied in the murine continuous femoral intramedullary particle infusion model. Polyethylene particles with or without IL-4 were infused into mouse distal femurs implanted with hollow titanium rods using subcutaneous infusion pumps. In another experimental group, particles were infused into the femur through a 7ND coated rod. After 4 weeks, fixation of the implant was assessed using a pullout test. The volume of trabecular bone and the geometry of the local cortical bone were assessed by µCT and the corresponding structural properties of the cortical bone determined by torsional testing. Continuous IL-4 delivery led to increased trabecular bone volume as well as enhanced local bone geometry and structural properties, while 7ND implant coating did not have effect on these parameters. The results suggest that local IL-4 treatment is a promising strategy to mitigate wear particle induced osteolysis. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2255-2262, 2016.