Revascularization and bone remodeling of frozen allografts stimulated by intramedullary sustained delivery of FGF‐2 and VEGF

Frozen bone allografts are susceptible to nonunion and fracture due to limited revascularization and incomplete bone remodeling. We aim to revascularize bone allografts by combining angiogenesis from implanted arteriovenous (AV) bundles with delivery of fibroblast growth factor (FGF‐2) and/or vascular endothelial growth factor (VEGF) via biodegradable microspheres. Rat femoral diaphyseal allografts were frozen at ?80°C, and heterotopically transplanted over a major histocompatibility mismatch. A saphenous AV bundle was inserted into the intramedullary canal. Growth factor was encapsulated into microspheres and inserted into the graft, providing localized and sustained drug release. Forty rats were included in four groups: (I) phosphate‐buffered saline, (II) FGF‐2, (III) VEGF, and (IV) FGF‐2 + VEGF. At 4 weeks, angiogenesis was measured by the hydrogen washout method and microangiography. Bone remodeling was evaluated by quantitative histomorphometry and histology. Bone blood flow was significantly higher in groups III and IV compared to control (p < 0.05). Similarly, bone remodeling was higher in VEGF groups. FGF‐2 had little effect on allograft revascularization. No synergistic effect was observed with use of both cytokines. Delivered in microspheres, VEGF proved to be a potent angiogenic cytokine, increasing cortical bone blood flow and new bone formation in frozen allografts revascularized with an implanted AV bundle. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29: 1431–1436, 2011     

Silent information regulator (Sir)T1 inhibits NF‐κB signaling to maintain normal skeletal remodeling

Silent information regulator T1 (SirT1) is linked to longevity and negatively controls NF‐κB signaling, a crucial mediator of survival and regulator of both osteoclasts and osteoblasts. Here we show that NF‐κB repression by SirT1 in both osteoclasts and osteoblasts is necessary for proper bone remodeling and may contribute to the mechanisms linking aging and bone loss. Osteoclast‐ or osteoblast‐specific SirT1 deletion using the Sirt^flox/flox mice crossed to lysozyme M‐cre and the 2.3 kb col1a1‐cre transgenic mice, respectively, resulted in decreased bone mass caused by increased resorption and reduced bone formation. In osteoclasts, lack of SirT1 promoted osteoclastogenesis in vitro and activated NF‐κB by increasing acetylation of Lysine 310. Importantly, this increase in osteoclastogenesis was blocked by pharmacological inhibition of NF‐κB. In osteoblasts, decreased SirT1 reduced osteoblast differentiation, which could also be rescued by inhibition of NF‐κB. In further support of the critical role of NF‐κB signaling in bone remodeling, elevated NF‐κB activity in IκBα^+/? mice uncoupled bone resorption and formation, leading to reduced bone mass. These findings support the notion that SirT1 is a genetic determinant of bone mass, acting in a cell‐autonomous manner in both osteoblasts and osteoclasts, through control of NF‐κB and bone cell differentiation. © 2013 American Society for Bone and Mineral Research.     

Induction of angiogenesis and osteogenesis in surgically revascularized frozen bone allografts by sustained delivery of FGF‐2 and VEGF

Large conventional bone allografts are susceptible to fracture and nonunion due to incomplete revascularization and insufficient bone remodeling. We aim to improve bone blood flow and bone remodeling using surgical angiogenesis combined with delivery of fibroblast growth factor (FGF‐2) and vascular endothelial growth factor (VEGF). Frozen femoral allografts were heterotopically transplanted in a rat model. The saphenous arteriovenous bundle was implanted within the graft medullary canal. Simultaneously, biodegradable microspheres containing phosphate buffered saline (control), FGF‐2, VEGF, or FGF‐2 + VEGF were placed within the graft. Rats were sacrificed at 4 and 18 weeks. Angiogenesis was determined by quantifying bone capillary density and measuring cortical bone blood flow. Bone remodeling was assessed by histology, histomorphometry, and alkaline phosphatase activity. VEGF significantly increased angiogenesis and bone remodeling at 4 and 18 weeks. FGF‐2 did not elicit a strong angiogenic or osteogenic response. No synergistic effect of FGF‐2 + VEGF was observed. VEGF delivered in microspheres had superior long‐term effect on angiogenesis and osteogenesis in surgically revascularized frozen bone structural allografts as compared to FGF‐2 or FGF‐2 + VEGF. Continuous and localized delivery of VEGF by microencapsulation has promising clinical potential by inducing a durable angiogenic and osteogenic response in frozen allografts. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:1556–1562, 2012     

Continuous elevation of PTH increases the number of osteoblasts via both osteoclast‐dependent and ‐independent mechanisms

Sustained parathyroid hormone (PTH) elevation stimulates bone remodeling (ie, both resorption and formation). The former results from increased RANKL synthesis, but the cause of the latter has not been established. Current hypotheses include release of osteoblastogenic factors from osteoclasts or from the bone matrix during resorption, modulation of the production and activity of osteoblastogenic factors from cells of the osteoblast lineage, and increased angiogenesis. To dissect the contribution of these mechanisms, 6‐month‐old Swiss‐Webster mice were infused for 5 days with 470 ng/h PTH(1‐84) or 525 ng/h soluble RANKL (sRANKL). Both agents increased osteoclasts and osteoblasts in vertebral cancellous bone, but the ratio of osteoblasts to osteoclasts and the increase in bone formation was greater in PTH‐treated mice. Cancellous bone mass was maintained in mice receiving PTH but lost in mice receiving sRANKL, indicating that maintenance of balanced remodeling requires osteoblastogenic effects beyond those mediated by osteoclasts. Consistent with this contention, PTH, but not sRANKL, decreased the level of the Wnt antagonist sclerostin and increased the expression of the Wnt target genes Nkd2, Wisp1, and Twist1. Furthermore, PTH, but not sRANKL, increased the number of blood vessels in the bone marrow. Weekly injections of the RANKL antagonist osteoprotegerin at 10 µg/g for 2 weeks prior to PTH infusion eliminated osteoclasts and osteoblasts and prevented the PTH‐induced increase in osteoclasts, osteoblasts, and blood vessels. These results indicate that PTH stimulates osteoclast‐dependent as well as osteoclast‐independent (Wnt signaling) pro‐osteoblastogenic pathways, both of which are required for balanced focal bone remodeling in cancellous bone. © 2010 American Society for Bone and Mineral Research.     

Osteoclastogenesis in the nonadherent cell population of human bone marrow is inhibited by rhBMP-2 alone or together with rhVEGF.

During bone development and repair, angiogenesis, osteogenesis, and bone remodeling are closely associated processes that share some common mediators. In the present study nonadherent human bone marrow mononuclear cells under the induction of sRANKL and M-CSF, differentiated into osteoclasts with TRAP-positive staining, VNR expression, and Ca-P resorptive activity. The effects of various combinations of rhBMP-2 (0, 3, 30, and 300 ng/mL) and rhVEGF (0 and 25 ng/mL) on osteoclastogenesis potentials were examined in this experimental system. The percentages of TRAP-positive multiple nucleated cells represent osteoclast differentiation potential, and the percentages of resorptive areas in the Ca-P coated plates resemble osteoclast resorption capability. The presence of rhBMP-2 at 30 and 300 ng/mL showed inhibitory effects on osteoclast differentiation and their resorptive capability in the human osteoclast culture system. rhVEGF (25 ng/mL) enhanced the resorptive function of osteoclast whenever it was used alone or combined with 3 ng/mL rhBMP-2. However, rhVEGF-induced resorptive function was inhibited by 30 ng/mL and 300 ng/mL rhBMP-2 in a dose-dependent manner. Statistical analysis demonstrated that an interactive effect exists between rhBMP-2 and rhVEGF on human osteoclastogenesis. These findings suggested that an interactive regulation may exist between BMPs and VEGF signaling pathways during osteoclastogenesis; exact mechanisms are yet to be elucidated.     

Distribution of TRAP‐positive cells and expression of HIF‐1α, VEGF,and FGF‐2 in the reparative reaction in patients with osteonecrosis of the femoral head

Osteogenesis and angiogenesis are closely associated with the reparative process in bone. In osteonecrosis of the femoral head (ONFH), although the progression of bone resorption by osteoclasts is considered to be followed by femoral head collapse, the reparative reaction remains unknown. In order to investigate the reparative reaction in patients with ONFH, the distribution of TRAP‐ positive cells and expression of HIF‐1α, VEGF, and FGF‐2 were observed in 51 hips in 42 patients. TRAP‐positive cells were detected around the teres insertion and retinaculum in the early radiologic stage, and increased around the new trabecular bone throughout the reparative interface zone in the late collapsed stage. HIF‐1α expression was detected at the fibrosis area and the transitional area, which included the proximal area of the reparative interface zone adjacent to the necrotic zone. VEGF was expressed at the edematous area of the reparative interface zone, while FGF‐2 was detected widely in the reparative interface zone and the normal zone. In the late radiologic stages, HIF‐1α, VEGF, and FGF‐2 were not detected in the necrotic zone, and they acted in angiogenesis in the reparative interface zone, while TRAP‐positive cells increased around the new bone formation in response to remodeling after the collapse. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27: 694–700, 2009     

Effect of rhBMP‐2 and VEGF in a vascularized bone allotransplant experimental model based on surgical neoangiogenesis

We have demonstrated survival of living allogeneic bone without long‐term immunosuppression using short‐term immunosuppression and simultaneous creation of an autogenous neoagiogenic circulation. In this study, bone morphogenic protein‐2 (rhBMP‐2), and/or vascular endothelial growth factor (VEGF), were used to augment this process. Femoral diaphyseal bone was transplanted heterotopically from 46 Dark Agouti to 46 Lewis rats. Microvascular repair of the allotransplant nutrient pedicle was combined with intra‐medullary implantation of an autogenous saphenous arteriovenous (AV) bundle and biodegradable microspheres containing buffer (control), rhBMP‐2 or rhBMP‐2 + VEGF. FK‐506 given daily for 14 days maintained nutrient pedicle flow during angiogenesis. After an 18 weeks survival period, we measured angiogenesis (capillary density) from the AV bundle and cortical bone blood flow. Both measures were greater in the combined (rhBMP‐2 + VEGF) group than rhBMP‐2 and control groups (p < 0.05). Osteoblast counts were also higher in the rhBMP‐2 + VEGF group (p < 0.05). A trend towards greater bone formation was seen in both rhBMP2 + VGF and rhBMP2 groups as compared to controls (p = 0.059). Local administration of VEGF and rhBMP‐2 augments angiogenesis, osteoblastic activity and bone blood flow from implanted blood vessels of donor origin in vascularized bone allografts. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31: 561–566, 2013     

Tumor‐derived syndecan‐1 mediates distal cross‐talk with bone that enhances osteoclastogenesis

Tumor‐stimulated bone resorption fuels tumor growth and marks a dramatic decline in the health and prognosis of breast cancer patients. Identifying mechanisms that mediate cross‐talk between tumor and bone remains a key challenge. We previously demonstrated that breast cancer cells expressing high levels of heparanase exhibit enhanced shedding of the syndecan‐1 proteoglycan. Moreover, when these heparanase‐high cells are implanted in the mammary fat pad, they elevate bone resorption. In this study, conditioned medium from breast cancer cells expressing high levels of heparanase was shown to significantly stimulate human osteoclastogenesis in vitro (p < .05). The osteoclastogenic activity in the medium of heparanase‐high cells was traced to the presence of syndecan‐1, intact heparan sulfate chains, and heat‐labile factor(s), including the chemokine interleukin 8 (IL‐8). The enhanced osteoclastogenesis promoted by the heparanase‐high cells results in a dramatic increase in bone resorption in vitro. In addition, the long bones of animals bearing heparanase‐high tumors in the mammary fat pad had significantly higher numbers of osteoclasts compared with animals bearing tumors expressing low levels of heparanase (p < .05). Together these data suggest that syndecan‐1 shed by tumor cells exerts biologic effects distal to the primary tumor and that it participates in driving osteoclastogenesis and the resulting bone destruction. © 2010 American Society for Bone and Mineral Research     

Pasteurella multocida Toxin Stimulates Bone Resorption by Osteoclasts via Interaction with Osteoblasts

In this study we used an in vitro assay system with osteoblast and osteoclast co-cultures to assess the effect of purified recombinant Pasteurella multocida toxin on bone resorption. Resorption was measured by the release of a telopeptide breakdown product of type I collagen. It was found that P. multocida did not stimulate bone resorption by osteoclasts directly and also did not stimulate bone breakdown via the release of collagenase or other proteases from osteoblasts. During co-culture of osteoblasts and osteoclasts, with cell-cell contact prevented, P. multocida toxin produced no significant effect. Osteoblast-conditioned media gave a biphasic effect; low concentrations of P. multocida toxin stimulated bone resorption, whereas 100 ng/ml inhibited resorption by osteoclasts. However, when both cell types were co-cultured with cell-cell contact permitted, P. multocida toxin induced a large concentration-dependent increase in bone resorption over a 7-day period. This suggested that P. multocida toxin causes bone breakdown via an osteoblast-dependent mechanism and that a membrane-bound receptor may be involved. Received: 8 July 1997 / Accepted: 8 April 1998     

Regulation of RANKL‐induced osteoclastogenesis by RING finger protein RNF114

Normal bone remodeling is a continuous process orchestrated by bone‐resorbing osteoclasts and bone‐forming osteoblasts, which an imbalance in bone remodeling results in metabolic bone diseases. RANKL, a member of the TNF cytokine family, functions as a key stimulator for osteoclast differentiation and maturation. Here, we report that RNF114, previously identified as a psoriasis susceptibility gene, plays a regulatory role in the RANKL/RANK/TRAF6 signaling pathway that mediates osteoclastogenesis. Our results demonstrated that RNF114 expression was significantly down‐regulated in mouse osteoclast precursor cells undergoing RANKL‐induced osteoclast differentiation. RNF114 knockout did not affect development or viability of the subpopulation of bone marrow macrophages capable of differentiating into osteoclasts in culture. However, in the presence of RANKL, RNF114 knockout bone marrow macrophages exhibited enhanced cell proliferation and augmented osteoclast differentiation, as shown by an increased expression of mature osteoclast markers, increased osteoclastic TRAP activity and bone resorption. Conversely, ectopic expression of RNF114 inhibited CTSK expression, TRAP activity, and bone resorption in RANKL‐treated pre‐osteoclasts. RNF114 also suppressed RANKL‐activated NFATc1 expression and NFAT‐regulated promoter activity. RNF114 suppressed TRAF6‐, but not TAK1/TAB2‐mediated NF‐κB activation downstream of RANKL/RANK. In particular, TRAF6 protein levels were down‐regulated by RNF114, possibly via K48‐mediated proteasome‐dependent degradation. These data suggested that RNF114's inhibitory effect on RANKL‐stimulated osteoclastogenesis was mediated by blocking RANK/TRAF6/NF‐κB signal transduction. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:159–166, 2018.     

Deletion of FGFR3 in Osteoclast Lineage Cells Results in Increased Bone Mass in Mice by Inhibiting Osteoclastic Bone Resorption

Fibroblast growth factor receptor 3 (FGFR3) participates in bone remodeling. Both Fgfr3 global knockout and activated mice showed decreased bone mass with increased osteoclast formation or bone resorption activity. To clarify the direct effect of FGFR3 on osteoclasts, we specifically deleted Fgfr3 in osteoclast lineage cells. Adult mice with Fgfr3 deficiency in osteoclast lineage cells (mutant [MUT]) showed increased bone mass. In a drilled‐hole defect model, the bone remodeling of the holed area in cortical bone was also impaired with delayed resorption of residual woven bone in MUT mice. In vitro assay demonstrated that there was no significant difference between the number of tartrate‐resistant acid phosphatase (TRAP)‐positive osteoclasts derived from wild‐type and Fgfr3‐deficient bone marrow monocytes, suggesting that FGFR3 had no remarkable effect on osteoclast formation. The bone resorption activity of Fgfr3‐deficient osteoclasts was markedly decreased accompanying with downregulated expressions of Trap, Ctsk, and Mmp 9. The upregulated activity of osteoclastic bone resorption by FGF2 in vitro was also impaired in Fgfr3‐deficient osteoclasts, indicating that FGFR3 may participate in the regulation of bone resorption activity of osteoclasts by FGF2. Reduced adhesion but not migration in osteoclasts with Fgfr3 deficiency may be responsible for the impaired bone resorption activity. Our study for the first time genetically shows the direct positive regulation of FGFR3 on osteoclastic bone resorption. © 2016 American Society for Bone and Mineral Research.     

Prostaglandin D2 Receptors Control Osteoclastogenesis and the Activity of Human Osteoclasts

We recently showed that human osteoblasts synthesize prostaglandin D₂ (PGD₂) and express both DP and CRTH2 receptors. Activation of the DP receptor decreased osteoprotegerin production, whereas activation of the CRTH2 receptor induced osteoblast chemotaxis and decreased RANKL expression. Our objectives in this study were to determine the presence, distribution, and action of these receptors in the functions of human osteoclasts and in osteoclastogenesis. Immunohistochemistry was used to detect the presence of DP and CRTH2 in in vitro–differentiated human osteoclasts in culture and in osteoclasts in situ. The effects of the activation of PGD₂ receptors on the cytoskeleton were determined by fluorescence microscopy. Specific agonists and antagonists allowed the study of the roles of these receptors on bone resorption and osteoclast differentiation. Our results show that in vitro–differentiated human osteoclasts and authentic fetal osteoclasts express both DP and CRTH2 receptors, as shown by immunocytochemistry. Similar results were obtained in osteoclasts from normal, osteoporotic, pagetic, and osteoarthritic adult bone tissues. Stimulation of osteoclasts with PGD₂ induced a robust reorganization of the cytoskeleton with a decrease in the number of cells presenting actin rings and an increase of lamellipodia, effects mediated by the DP and CRTH2 receptors, respectively. PGD₂ showed an inhibitory effect on bone resorption activity acting through the DP receptor. In vitro osteoclastogenesis from peripheral blood mononuclear cells cultured in the presence of RANKL and macrophage‐colony stimulating factor was decreased by activation of either DP or CRTH2 receptors. These results suggest that PGD₂ receptors could be useful targets in certain bone diseases because their specific activation/inhibition leads to a decrease in osteoclastogenesis and to inhibition of bone resorption by osteoclasts.     

Osteoclasts prefer aged bone

Summary We investigated whether the age of the bones endogenously exerts control over the bone resorption ability of the osteoclasts, and found that osteoclasts preferentially develop and resorb bone on aged bone. These findings indicate that the bone matrix itself plays a role in targeted remodeling of aged bones. Introduction Osteoclasts resorb aging bone in order to repair damage and maintain the quality of bone. The mechanism behind the targeting of aged bone for remodeling is not clear. We investigated whether bones endogenously possess the ability to control osteoclastic resorption. Methods To biochemically distinguish aged and young bones; we measured the ratio between the age-isomerized βCTX fragment and the non-isomerized αCTX fragment. By measurement of TRACP activity, CTX release, number of TRACP positive cells and pit area/pit number, we evaluated osteoclastogenesis as well as osteoclast resorption on aged and young bones. Results We found that the αCTX / βCTX ratio is 3:1 in young compared to aged bones, and we found that both α and βCTX are released by osteoclasts during resorption. Osteoclastogenesis was augmented on aged compared to young bones, and the difference was enhanced under low serum conditions. We found that mature osteoclasts resorb more on aged than on young bone, despite unchanged adhesion and morphology. Conclusions These data indicate that the age of the bone plays an important role in controlling osteoclast-mediated resorption, with significantly higher levels of osteoclast differentiation and resorption on aged bones when compared to young bones. Kim Henriksen and Diana J. Leeming contributed equally. Financial disclosure: Morten A. Karsdal, Per Qvist and Claus Christiansen own stock options in Nordic Bioscience A/S     

Enhanced Osteoclastogenesis Causes Osteopenia in Twisted Gastrulation‐Deficient Mice Through Increased BMP Signaling

The uncoupling of osteoblastic and osteoclastic activity is central to disorders such as osteoporosis, osteolytic malignancies, and periodontitis. Numerous studies have shown explicit functions for bone morphogenetic proteins (BMPs) in skeletogenesis. Their signaling activity has been shown in various contexts to be regulated by extracellular proteins, including Twisted gastrulation (TWSG1). However, experimental paradigms determining the effects of BMP regulators on bone remodeling are limited. In this study, we assessed the role of TWSG1 in postnatal bone homeostasis. Twsg1‐deficient (Twsg1^?/?) mice developed osteopenia that could not be explained by defective osteoblast function, because mineral apposition rate and differentiation markers were not significantly different compared with wildtype (WT) mice. Instead, we discovered a striking enhancement of osteoclastogenesis in Twsg1^?/? mice, leading to increased bone resorption with resultant osteopenia. Enhanced osteoclastogenesis in Twsg1^?/? mice was caused by increased cell fusion, differentiation, and function of osteoclasts. Furthermore, RANKL‐mediated osteoclastogenesis and phosphorylated Smad1/5/8 levels were enhanced when WT osteoclasts were treated with recombinant BMP2, suggesting direct regulation of osteoclast differentiation by BMPs. Increase in detectable levels of phosphorylated Smad 1/5/8 was noted in osteoclasts from Twsg1^?/? mice compared with WT mice. Furthermore, the enhanced osteoclastogenesis in Twsg1^?/? mice was reversed in vitro in a dose‐dependent manner with exposure to Noggin, a BMP antagonist, strongly suggesting that the enhanced osteoclastogenesis in Twsg1 mutants is attributable to increased BMP signaling. Thus, we present a novel and previously uncharacterized role for TWSG1 in inhibiting osteoclastogenesis through regulation of BMP activity.     

Regulation of bone mass and osteoclast function depend on the F-actin modulator SWAP-70

Bone remodeling involves tightly regulated bone‐resorbing osteoclasts and bone‐forming osteoblasts. Determining osteoclast function is central to understanding bone diseases such as osteoporosis and osteopetrosis. Here, we report a novel function of the F‐actin binding and regulatory protein SWAP‐70 in osteoclast biology. F‐actin ring formation, cell morphology, and bone resorption are impaired in Swap‐70^?/? osteoclasts, whereas the expression of osteoclast differentiation markers induced in vitro by macrophage colony‐stimulating factor (M‐CSF) and receptor activator of NF‐κB ligand (RANKL) remains unaffected. Swap‐70^?/? mice develop osteopetrosis with increased bone mass, abnormally dense bone, and impaired osteoclast function. Ectopic expression of SWAP‐70 in Swap‐70^?/? osteoclasts in vitro rescues their deficiencies in bone resorption and F‐actin ring formation. Rescue requires a functional pleckstrin homology (PH) domain, known to support membrane localization of SWAP‐70, and the F‐actin binding domain. Transplantation of SWAP‐70–proficient bone marrow into Swap‐70^?/? mice restores osteoclast resorption capacity in vivo. The identification of the role of SWAP‐70 in promoting osteoclast function through modulating membrane‐proximal F‐actin rearrangements reveals a new pathway to control osteoclasts and bone homeostasis. © 2012 American Society for Bone and Mineral Research.     

Insulin-like growth factor-binding protein-2 is required for osteoclast differentiation

Global deletion of the Igfbp2 gene results in the suppression of bone turnover. To investigate the role of insulin‐like growth factor‐binding protein‐2 (IGFBP‐2) in regulating osteoclast differentiation, we cultured Igfbp2^?/? bone marrow cells and found a reduction in the number of osteoclasts and impaired resorption. Addition of full‐length IGFBP‐2 restored osteoclast differentiation, fusion, and resorption. To determine the molecular domains of IGFBP‐2 that were required for this effect to be manifest, Igfbp2^?/? bone marrow cells were transfected with constructs in which the heparin‐binding (HBD) or the IGF‐binding domains of IGFBP‐2 were mutated. We found that both domains were necessary for osteoclastogenesis because expression of the mutated forms of either domain failed to support the formation of functionally mature osteoclasts. To discern the mechanism by which IGFBP‐2 regulates osteoclast formation, PTEN abundance and phosphorylation status as well as AKT responsiveness to IGF‐I were analyzed. Igfbp2^?/? cells had elevated levels of PTEN and phospho‐PTEN compared with controls. Expression of wild‐type IGFBP‐2 reduced the level of PTEN to that of wild‐type cells. Cells expressing the IGF‐binding mutant showed suppression of PTEN and phospho‐PTEN equivalent to the wild‐type protein, whereas those expressing the IGFBP‐2 HBD mutant showed no PTEN suppression. When the ability of IGF‐I to stimulate AKT activation, measured by Thr³⁰⁸ and Ser⁴⁷³ phosphorylation, was analyzed, stimulation of Ser⁴⁷³ in response to IGF‐I in preosteoclasts required the presence of intact IGFBP‐2. This effect was duplicated by the addition of a CK2 inhibitor that prevents the phosphorylation of PTEN. In contrast, in fully differentiated osteoclasts, stimulation of Thr³⁰⁸ phosphorylation required the presence of intact IGFBP‐2. We conclude that IGFBP‐2 is an important regulator of osteoclastogenesis and that both the heparin‐ and the IGF‐binding domains of IGFBP‐2 are essential for the formation of fully differentiated and functional osteoclasts. © 2012 American Society for Bone and Mineral Research     

Osteoinductive effect of bone bank allografts on human osteoblasts in culture

Incorporation of a human bone allograft requires osteoclast activity and growth of recipient osteoblasts. The aim of this work was to study the effects produced by autoclavated and ?80°C frozen bone allografts on osteoblast proliferation and synthesis of interleukin 6 (IL6), activator of bone resorption, aminoterminal propeptide of procollagen I (PINP), marker of bone matrix formation, and osteoprotegerin (OPG), inhibitor of osteoclast activity and differentiation. Allografts were obtained from human femoral heads. Human osteoblasts were cultured in the presence (problem group) or in the absence (control group) of allografts during 15 days. Allografts produced a decrease in osteoblast proliferation in the first week of the experiment, and an increase in IL6 mRNA, both at 3 h and 2 days, and an increase in the IL6 released to the culture medium the second day of the experiment. We found a decrease in OPG released to the culture on the 2nd and fourth days. These results suggest an increase in bone resorption and a decrease in bone formation in the first week of the experiment. In the second week, allografts produced an increase in osteoblast proliferation and PINP release to the culture medium, indicating an increase in bone formation; an increase in OPG released to the culture medium, which would indicate a decrease in bone resorption; and a decrease in IL6, indicating a decrease in bone resorption stimulation. These results demonstrate that autoclavated and ?80°C frozen bone allografts produce in bone environmemt changes that regulate their own incorporation to the recipient bone. © 2007 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:200–207, 2008     

Effects of Surgical Angiogenesis on Segmental Bone Reconstruction With Cryopreserved Massive‐Structural Allografts in a Porcine Tibia Model

Cryopreserved bone allografts (CBA) used to reconstruct segmental bone defects provide immediate structural stability, but are vulnerable to infection, non‐union and late stress fracture as the majority of the allograft remains largely avascular. We sought to improve the bone vascularity and bone formation of CBAs by surgical angiogenesis with an implanted arteriovenous (AV) bundle, using a porcine tibial defect model. Cryopreserved tibial bone allografts were transplanted in swine leukocyte antigen (SLA) mismatched Yucatan minipigs to reconstruct a 3.5 cm segmental tibial defect. A cranial tibial AV‐bundle was placed within its intramedullary canal to induce angiogenesis. The AV bundle was patent in eight pigs and ligated in a control group of eight pigs. At 20 weeks neo‐angiogenesis was evaluated by micro‐angiography. Bone formation was measured by quantitative histomorphometry and micro‐computed tomography. Seven of eight AV‐bundles in the revascularized group were patent. One had thrombosed due to allograft displacement. Total vascular volume was higher in the revascularized allografts compared to the ligated group (p = 0.015). Revascularized allografts had increased levels of bone formation on the allograft endosteal surface compared to the ligated control group (p = 0.05). Surgical angiogenesis of porcine tibial CBAs by intramedullary implantation of an AV‐bundle creates an enhanced autogenous neoangiogenic circulation and accelerates active bone formation on allograft endosteal surfaces. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1698–1708, 2019     

Monocytes mediate osteoclastic bone resorption by prostaglandin production

Summary Monocytes are frequently found adjacent to active bone resorption surfaces in both physiological and pathological situations and may play a key role in bone resorption. There is strong circumstantial evidence that monocytes are precursors for osteoclasts in vivo, and recently they have been shown to resorb devitalized bone directly. The present study shows that monocytes can also resorb bone by stimulation of osteoclasts. Live fetal rodent bones prelabeled with⁴⁵Ca and cultured for 48–96 h in the presence of human monocytes or monocyte-conditioned medium released 80% more mineral than bones cultured in control medium. Bone matrix sustained comparable resorption as demonstrated by a 2-fold decrement in the extracted dry weights of the bones cultured in monocyte-conditioned medium. Histological examination of the bones cultured with monocytes or monocyte-conditioned medium showed increased osteoclast number and activity when compared with bones cultured in control medium. Known inhibitors of osteoclastic activity (phosphate 6 × 10⁻³M, cortisol 10⁻⁶M, and calcitonin 50 mU/ml) inhibited monocyte-conditioned medium-mediated bone resorption. The monocyte-conditioned medium contained sufficient prostaglandin E to account for the bone resorption. Indomethacin 10⁻⁵M added to the monocyte cultures blocked monocyte-conditioned media-induced bone resorption and prostaglandin release. These experiments suggest that monocytes stimulate osteoclastic bone resorption by prostaglandin production. Monocyte-induced bone resorption is partly reversed by inhibitors of osteoclast function. Monocyte-induced osteoclastic bone resorption may play an important role in physiologic bone remodeling and in bone destruction that occurs in chronic inflammatory diseases such as rheumatoid arthritis and periodontal disease.