Wnt pathway regulation by demineralized bone is approximated by both BMP‐2 and TGF‐β1 signaling

Allogeneic demineralized bone is used extensively as a clinical graft material because it has osteo/chondroinductive and osteoconductive properties. Demineralized bone powder (DBP) induces chondrogenic differentiation of human dermal fibroblasts (hDFs) in three‐dimensional collagen cultures, but the initiating mechanisms have not been fully characterized nor has it been shown that bone morphogenetic proteins (BMPs) recapitulate DBP's effects on target cells. Among the many signaling pathways regulated in hDFs by DBP prior to in vitro chondrogenesis, there are changes in Wnts and their receptors that may contribute to DBP actions. This study tests the hypothesis that DBP modulation of Wnt signaling entails both BMP and TGF‐β pathways. We compared the effects of DBP, TGF‐β1, or BMP‐2 on Wnt signaling components in hDFs by Wnt signaling macroarray, RT‐PCR, in situ hybridization, and Western immunoblot analyses. Many effects of DBP on Wnt signaling components were not shared by BMP‐2, and likewise DBP effects on Wnt genes and β‐catenin only partially required the TGF‐β pathway, as shown by selective inhibition of TGF‐β/activin receptor‐like kinase. The analyses revealed that 64% (16/25) of the Wnt signaling components regulated by DBP were regulated similarly by the sum of effects by BMP‐2 and by TGF‐β1. In conclusion, signaling mechanisms of inductive DBP in human dermal fibroblasts involve the modulation of multiple Wnt signals through both BMP and TGF‐β pathways. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31: 554–560, 2013     

Influence of estrogen and progesterone on matrix-induced endochondral bone formation

Summary The influence of estradiol and progesterone, alone or in combination, on the discrete phases of matrix-induced endochondral bone formation was investigated. Administration of estradiol and progesterone in combination increased mesenchymal cell proliferation, as indicated by [³H] thymidine incorporaton into acid precipitable material. However, ornithine decarboxylase (ODC) activity was significantly suppressed by the combination of estradiol and progesterone. Also, this treatment did not influence the³⁵SO₄ incorporation into proteoglycans on day 7. Mineralization of newly induced bone was quantitated by alkaline phosphatase,⁴⁵Ca incorporation into bone mineral and calcium content, and was found to be significantly increased by progesterone alone and in combination with estradiol in both matrix-induced plaques and tibial metaphysis. These results demonstrated the stimulatory role of progesterone in combination with estradiol in bone formation and mineralization.     

Influence of testosterone and dihydrotestosterone on bone-matrix induced endochondral bone formation

Summary A bone matrix-induced endochondral bone development model has been used to study the effects of androgens on different stages of bone development in castrated young adult rats. Androgen treatment, especially with dihydrotestosterone (DHT) for 7 days, inhibited³⁵SO₄ incorporation by the developing cartilage in the induced plaques. Castrated control animals maintained for 11 days after implantation of bone matrix showed significantly lower calcium levels in the induced implant than was observed earlier in the unoperated controls. DHT treatment for 11 days caused dramatic increases in levels of calcium in the implants. Testosterone had little effect. When androgen treatment was continued for 21 days, while levels of alkaline phosphatase in the implants were unaffected, levels of calcium in the implants were significantly higher than on day 11 for both castrated control and androgen-treated animals. Peak alkaline phosphatase activity (day 10) is known to precede peak calcium mineralizing activity (day 12) in this model and it is also known that calcium levels remain high thereafter. Evaluation of calcium and alkaline phosphatase levels in the proximal tibial metaphyses of castrated control and androgen-treated groups of animals showed no changes after 11 days treatment. Prolonged treatment (21 days) elevated the levels of alkaline phosphatase whereas no change was observed in calcium levels in the tibial metaphyses. These findings demonstate that androgens stimulate mineralization and that DHT is more active when used for short periods of time and in early stages of bone development in matrix-induced implants.     

β‐catenin promotes bone formation and suppresses bone resorption in postnatal growing mice

Genetic studies in the mouse have demonstrated multiple roles for β‐catenin in the skeleton. In the embryo, β‐catenin is critical for the early stages of osteoblast differentiation. Postnatally, β‐catenin in mature osteoblasts and osteocytes indirectly suppresses osteoclast differentiation. However, a direct role for β‐catenin in regulating osteoblast number and/or function specifically in the postnatal life has not been demonstrated. Addressing this knowledge gap is important because low‐density lipoprotein receptor‐related protein 5 (LRP5), a coreceptor for WNT signaling proposed to function through β‐catenin, controls osteoblast number and function in postnatal mice or humans. To overcome the neonatal lethality caused by embryonic deletion of β‐catenin in early‐stage osteoblast‐lineage cells, we use the Osx‐CreER^T2 mouse strain to remove β‐catenin in Osterix (Osx)‐expressing cells by administering tamoxifen (TM) temporarily to postnatal mice. Lineage‐tracing experiments in the long bones demonstrate that Osx‐CreER^T2 targets predominantly osteoblast‐lineage cells on the bone surface, but also transient progenitors that contribute to bone marrow stromal cells and adipocytes. Deletion of β‐catenin by this strategy greatly reduces the bone formation activity of the targeted osteoblasts. However, the targeted osteoblasts rapidly turn over and are replaced by an excessive number of non‐targeted osteoblasts, causing an unexpected increase in bone formation, but an even greater increase in osteoclast number and activity produces a net effect of severe osteopenia. With time, the mutant mice also exhibit a marked increase in bone marrow adiposity. Thus, β‐catenin in postnatal Osx‐lineage cells critically regulates bone homeostasis by promoting osteoblast activity and suppressing osteoblast turnover, while restraining osteoclast and marrow fat formation. © 2013 American Society for Bone and Mineral Research.     

Influence of whole body irradiation and local shielding on matrix-induced endochondral bone differentiation

Summary Subcutaneous implantation of demineralized bone matrix into allogeneic rats induces endochondral dochondral bone formation. We have investigated the effects of irradiation on the sequelae of the interaction of collagenous matrix and mesenchymal cells and on cartilage and bone differentiation. Rats were irradiated in a vertical direction with a midline dose of 850 rad. Radiation entered the rats ventrally while a small area of the upper thorax was locally shielded. After irradiation, bone matrix was implanted in shielded and nonshielded sites, and the implants were studied at various stages. On day 3, [³H]thymidine incorporation, an index of cell proliferation, was inhibited by 70% in the nonshielded sites compared to nonirradiated control rats. The degree of inhibition (35%) was less pronounced in shielded sites. Furthermore, there was recovery of cell proliferation in the shielded sites as opposed to the nonshielded contralateral site. A similar pattern was observed on day 7 as assessed by³⁵SO₄ incorporation into proteoglycans during chondrogenesis. Bone formation and mineralization were quantified on day 11 by alkaline phosphatase activity and⁴⁵Ca incorporation. In nonshielded sites, there was a 73% inhibition of alkaline phosphatase activity. In conclusion, radiation impaired progenitor cell proliferation which resulted in decreased cartilage and bone differentiation. These findings imply that local mesenchymal cells proliferate and differentiate into bone in response to implanted collagenous matrix.     

Interferon‐γ plays a role in bone formation in vivo and rescues osteoporosis in ovariectomized mice

Interferon γ (IFN‐γ) is a cytokine produced locally in the bone microenvironment by cells of immune origin as well as mesenchymal stem cells. However, its role in normal bone remodeling is still poorly understood. In this study we first examined the consequences of IFN‐γ ablation in vivo in C57BL/6 mice expressing the IFN‐γ receptor knockout phenotype (IFNγR1^?/?). Compared with their wild‐type littermates (IFNγR1^+/+), IFNγR1^?/? mice exhibit a reduction in bone volume associated with significant changes in cortical and trabecular structural parameters characteristic of an osteoporotic phenotype. Bone histomorphometry of IFNγR1^?/? mice showed a low‐bone‐turnover pattern with a decrease in bone formation, a significant reduction in osteoblast and osteoclast numbers, and a reduction in circulating levels of bone‐formation and bone‐resorption markers. Furthermore, administration of IFN‐γ (2000 and 10,000 units) to wild‐type C57BL/6 sham‐operated (SHAM) and ovariectomized (OVX) female mice significantly improved bone mass and microarchitecture, mechanical properties of bone, and the ratio between bone formation and bone resorption in SHAM mice and rescued osteoporosis in OVX mice. These data therefore support an important physiologic role for IFN‐γ signaling as a potential new anabolic therapeutic target for osteoporosis. © 2011 American Society for Bone and Mineral Research.     

NDST-1 modulates BMPR and PTHrP signaling during endochondral bone formation in a gene knockout model

GlcNAc N-deacetylase/N-sulfotransferase-1 (NDST-1), a member of the enzyme family catalyzing the first modification step in the biosynthesis of heparan sulfate (HS), was knocked out in mice to investigate its role in embryonic development. NDST-1 null mice exhibited delayed endochondral bone formation including shortened calcified zones in limbs, delayed chondrocyte and osteogenetic differentiation, and increased chondrocyte proliferation. In situ HS binding assay revealed that the binding ability of bone morphogenetic protein (BMP) -2, -4, and -6 to endogenous HS was decreased in mutant phalanges, while that of fibroblast growth factor-1 (FGF-1) was not affected. Up-regulation of BMPR-IA, Phospho-Smad1 (P-Smad1) and parathyroid-hormone related protein (PTHrP), but not the Indian hedgehog, Gli1, Gli3, Patched, and FGFR-3, was observed. Furthermore, block of BMPR signaling with noggin rescued the delayed chondrocyte hypertrophic differentiation in NDST-1 (−/−) mice and recovered the expression of both P-Smad1 and PTHrP proteins. These results suggested that NDST-1-dependent heparan sulfate might negatively modulate BMP and its downstream PTHrP signaling, and thus affect endochondral bone development.     

Phospho‐Smad1 modulation by nedd4 e3 ligase in BMP/TGF‐β signaling

A considerable number of studies have focused on the regulation of mothers against decapentaplegic homologue (Smad)–dependent or –independent pathways in the signaling by each transforming growth factor β (TGF‐β) superfamily member in diverse biologic contexts. The sophisticated regulation of the actions of these molecules and the underlying molecular mechanisms still remain elusive. Here we show new mechanisms of ambilateral R (receptor‐regulated)–Smad regulation of bone morphogenetic protein 2 (BMP‐2)/TGF‐β1 signals. In a specific context, both signals regulate the nonclassic Smads pathway reciprocally, BMP‐2 to Smad2/3 and TGF‐β1 to Smad1/5/8, as well as their own classic linear Smad pathway. Interestingly, in this study, we found that C‐terminal phosphorylated forms of each pathway Smad degraded rapidly 3 hours after stimulation of nonclassic signals but are dramatically restored by treatment with via proteasomal inhibition. Furthermore, an E3 ligase, neural precursor cell expressed, developmentally down‐regulated 4 (Nedd4), also was found as one of the important modulators of the p‐Smad1 in both BMP‐2 and TGF‐β1 action. Overexpressed Nedd4 suppressed the BMP‐induced osteoblast transdifferentiation process of premyoblast C2C12 cells or alkaline phosphatase (ALP) level of human osteosarcoma cells and promoted TGF‐β1‐induced degradation of p‐Smad1 via physical interaction and polyubiquitination. Conversely, siNedd4 potentiated BMP signals through upregulation of p‐Smad1 and ALP activity, the effect of which led to an increased the rate of P_i‐induced calcification of human vascular smooth muscle cells. These new insights about proteasomal degradation–mediated phosphorylated nonclassic Smad regulation of BMP‐2/TGF‐β1 could, in part, help to unravel the complex mechanisms of abnormal nonosseous calcification by the aberrant activity of BMP/TGF‐β/Smads. © 2011 American Society for Bone and Mineral Research.     

Pharmacological inhibition of PPARγ increases osteoblastogenesis and bone mass in male C57BL/6 mice

Infiltration of bone marrow with fat is a prevalent feature in people with age‐related bone loss and osteoporosis, which correlates inversely with bone formation and positively with high expression levels of peroxisomal proliferator‐activated receptor gamma (PPARγ). Inhibition of PPARγ thus represents a potential therapeutic approach for age‐related bone loss. In this study, we examined the effect of PPARγ inhibition on bone in skeletally mature C57BL/6 male mice. Nine‐month‐old mice were treated with a PPARγ antagonist, bisphenol‐A‐diglycidyl ether (BADGE), alone or in combination with active vitamin D (1,25[OH]₂D₃) for 6 weeks. Micro‐computed tomography and bone histomorphometry indicated that mice treated with either BADGE or BADGE + 1,25(OH)₂D₃ had significantly increased bone volume and improved bone quality compared with vehicle‐treated mice. This phenotype occurred in the absence of alterations in osteoclast number. Furthermore, the BADGE + 1,25(OH)₂D₃‐treated mice exhibited higher levels of unmineralized osteoid. All of the treated groups showed a significant increase in circulating levels of bone formation markers without changes in bone resorption markers, while blood glucose, parathyroid hormone, and Ca⁺ remained normal. Furthermore, treatment with BADGE induced higher levels of expression of vitamin D receptor within the bone marrow. Overall, treated mice showed higher levels of osteoblastogenesis and bone formation concomitant with decreased marrow adiposity and ex vivo adipogenesis. Taken together, these observations demonstrate that pharmacological inhibition of PPARγ may represent an effective anabolic therapy for osteoporosis in the near future. © 2013 American Society for Bone and Mineral Research.     

Circulating γδ T cells and the risk of acute‐phase response after zoledronic acid administration

The use of intravenous nitrogen‐containing bisphosphonates (N‐BPs) is associated with the appearance of an acute phase response (APR) in a proportion of the patients for reasons that are poorly understood. The APR was attributed to the indirect activation of γδ T cells with the release of interferon‐γ and tumor necrosis factor (TNF). Forty patients with postmenopausal or senile osteoporosis (age range = 53–91 years) never previously treated with intravenous (iv) bisphosphonate, received a single 5‐mg zoledronic acid (ZOL) iv infusion over 15 minutes. White blood cells were counted and analyzed with an automated hematology analyzer (ADVIA 2120i Siemens, New York, USA) and by flow cytometer (BD FACSCanto, Becton Dickinson). The occurrence of APR was defined by the occurrence of fever (>37 °C) during the next 2 days. Forty‐two percent of patients (17 of 40) receiving the infusion of ZOL experienced an APR. Compared with the others they were younger (69 ± 7 years versus 74 ± 8 years; p = 0.06), and both the proportion and absolute number of γδ T cells were significant higher (p = 0.02 and p = 0.013, respectively). Nonsignificant differences were found between the two groups for white blood cells and for the other circulating lymphocyte subpopulations. Age was inversely correlated with circulating γδ T cells (p = 0.003) but the difference between the two groups in circulating γδ T cells persisted for age‐adjusted values and vice versa. In conclusion, the results of this study indicate that the number of circulating γδ T cells, together with age, are important determinant of the occurrence of APR after intravenous infusion of ZOL and possibly of any other N‐BPs. © 2012 American Society for Bone and Mineral Research     

Immunohistochemical localization of bone morphogenetic proteins (BMPs) 2, 4, 6, and 7 during induced heterotopic bone formation.

The distribution and staining intensity of bone morphogenetic proteins (BMPs) 2, 4, 6, and 7 were assessed by immunohistochemistry in ectopic bone induced in Nu/Nu mice by Saos-2 cell derived implants. Devitalized Saos-2 cells or their extracts can induce endochondral bone formation when implanted subcutaneously into Nu/Nu mice. BMP staining was mostly cytoplasmic. The most intense BMP staining was seen in hypertrophic and apoptotic chondrocytes, osteoprogenitor cells such as periosteal and perivascular cells, and osteoblasts. BMP staining in osteocytes and osteoclasts was variable, ranging from undetectable to intensely stained, and from minimal to moderately stained in megakaryocytes of the induced bone marrow. BMP-2, 4, 6, and 7 staining in Saos-2 implant-induced bone indicates the following: (1) Saos-2 cell products promote expression of BMPs by host osteoprogenitor cells, which in turn, leads to bone and marrow formation at ectopic sites; (2) strong BMP staining is seen in maturing chondrocytes, and thus may play a role in chondrocyte differentiation and/or apoptosis; (3) BMP expression in perivascular and periosteal cells indicates that osteoprogenitor cells also express BMP; (4) BMP release by osteoclasts may promote osteoblastic differentiation at sites of bone remodeling. These new data can be useful in understanding the role of BMPs in promoting clinical bone repair and in various pathologic conditions.     

Different bone remodeling levels of trabecular and cortical bone in response to changes in Wnt/β‐catenin signaling in mice

Trabecular bone and cortical bone have different bone remodeling levels, and the underlying mechanisms are not fully understood. In the present study, the expression of Wnt/β‐catenin signaling and its downstream molecules along with bone mass in trabecular and cortical bone were compared in wild‐type mice, constitutive activation of β‐catenin (CA‐β‐catenin) mice and β‐catenin deletion mice. It was found that the expression level of most of the examined genes such as Wnt3a, β‐catenin, osteocalcin and RANKL/OPG ratio were significantly higher in trabecular bone than in cortical bone in wild‐type mice. CA‐β‐catenin resulted in up‐regulated expression of the above‐mentioned genes except for RANKL/OPG ratio, which were down‐regulated. Also, CA‐β‐catenin led to increased number of osteoblasts, decreased number of osteoclasts and increased bone mass in both the trabecular bone and cortical bone compared with wild‐type mice; however, the extent of changes was much greater in the trabecular bone than in the cortical bone. By contrast, null β‐catenin led to down‐regulated expression of the above‐mentioned genes except for RANKL/OPG ratio. Furthermore, β‐catenin deletion led to decreased number of osteoblasts, increased number of osteoclasts and decreased bone mass when compared with wild‐type mice. Again, the extent of these changes was more significant in trabecular bone than cortical bone. Taken together, we found that the expression level of Wnt/β‐catenin signaling and bone remodeling‐related molecules were different in cortical bone and trabecular bone, and the trabecular bone was more readily affected by changes in the Wnt/β‐catenin signaling pathway. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:812–819, 2017.

     

Characterization of γδ T cell subsets in organ transplantation

γδ T cells are innate‐type lymphocytes that preferentially act as regulators of local effector immune responses. Recent reports found an altered distribution of the two main subpopulations of blood γδ T cells (Vδ1 and Vδ2) in operationally tolerant liver transplant recipients. Based on this, γδ T cells subset quantification was proposed as a biomarker of immunologic risk in liver transplantation. The specific characteristics of γδ T cell subsets in transplantation remain however unknown. We have investigated here the phenotype, repertoire and functional properties of γδ T cell subsets in a large population of allograft recipients. Our results indicate that alterations in the γδ T cell compartment are not restricted to tolerant liver recipients. In fact, most immunosuppressed liver and kidney recipients also display an enlarged peripheral blood γδ T cell pool mainly resulting from an expansion of Vδ1 T cells exhibiting an oligoclonal repertoire and different phenotypic and cytokine production traits than Vδ2 T cells. We propose that persistent viral infections are likely to contribute to these alterations. Our data provide novel insight in the biology of γδ T cells and a rationale for exploring these lymphocytes in more depth into the pathogenesis of viral infections in transplantation.     

Additive effects of hyperbaric oxygen and platelet‐derived growth factor‐BB in chondrocyte transplantation via up‐regulation expression of platelet‐derived growth factor‐β receptor

The present study investigated the effects of hyperbaric oxygen (HBO) and platelet‐derived growth factor‐BB (PDGF‐BB) in chondrocyte transplantation. In vitro, chondrocytes were treated with HBO, PDGF‐BB, and HBO combined with PDGF‐BB (H+P). Cell growth was analyzed using cell counting, MTT assay, and FACS analysis. mRNA expression of the PDGF‐α receptor (PDGFR‐α) and β receptor (PDGFR‐β) was detected by RT‐PCR. Protein expression of PDGFR‐β was detected by Western blotting. In vivo, chondrocytes and PDGF‐BB were suspended in alginate as a transplantation system. Cartilage defects were grafted with this system and with or without HBO treatment. Released PDGF‐BB concentration was quantified by ELISA. After 8 weeks, animals were sacrificed and the repaired tissues were examined. In vitro data suggested that each treatment increased cell growth via the up‐regulated mRNA expression of PDGFR‐α and increased cell accumulation in the S‐phase. The H+P treatment was more additive in cell growth and in mRNA and protein expression of PDGFR‐β than HBO or PDGF‐BB. In vivo results suggested that PDGF‐BB delivery lasted for more than 5 weeks. Scoring results showed that each treatment significantly increased the cartilage repair. Safranin‐O and type II collagen staining confirmed the hyaline‐like cartilage regeneration in the repaired tissues. In situ up‐regulation of PDGFR‐β expression partially explains the additive effect of H+P treatment in cartilage repair. Accordingly, H+P offers a potential treatment method for cartilage repair. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:1439–1446, 2009     

Reconstitution of T and NK cells after haploidentical hematopoietic cell transplantation using αβ T cell–depleted grafts and the clinical implication of γδ T cells

To investigate reconstitution of T and NK cells after αβ T lymphocyte–depleted haploidentical hematopoietic cell transplantation (HHCT) and the clinical implications of γδ T cells, we analyzed 50 pediatric patients who received 55 HHCTs using αβ T cell–depleted grafts. The number of CD3+ T cells and CD8+ T cells recovered rapidly and reached donor levels at days 180 and 60, respectively. Recovery of NK cells was rapid, and the median of NK cells at day 14 was comparable to the donor level. At day 14, median percentage of γδ T lymphocytes was 70.5%. After day 14, the percentage of γδ T cells gradually decreased, while the percentage of αβ T cells gradually increased. Patients with a low percentage (≤21%) of γδ T cells at day 30 had significantly higher incidence of cytomegalovirus (CMV) reactivation compared to patients with a high percentage (>70%) of γδ T cells (P < .01). In patients with acute leukemia, patients with high percentage of γδ T cells at day 30 showed significantly higher relapse‐free survival compared to those with low percentage of γδ T cells (P = .02). Data suggest that early recovery of γδ T cells decreases the risk of CMV reactivation and leukemia relapse.     

Overexpression of Smurf2 Stimulates Endochondral Ossification Through Upregulation of β‐Catenin

Ectopic expression of Smurf2 in chondrocytes and perichondrial cells accelerated endochondral ossification by stimulating chondrocyte maturation and osteoblast development through upregulation of β‐catenin in Col2a1‐Smurf2 embryos. The mechanism underlying Smurf2‐mediated morphological changes during embryonic development may provide new mechanistic insights and potential targets for prevention and treatment of human osteoarthritis. Introduction : Our recent finding that adult Col2a1‐Smurf2 mice have an osteoarthritis‐like phenotype in knee joints prompted us to examine the role of Smurf2 in the regulation of chondrocyte maturation and osteoblast differentiation during embryonic endochondral ossification. Materials and Methods : We analyzed gene expression and morphological changes in developing limbs by immunofluorescence, immunohistochemistry, Western blot, skeletal preparation, and histology. A series of markers for chondrocyte maturation and osteoblast differentiation in developing limbs were examined by in situ hybridization. Results : Ectopic overexpression of Smurf2 driven by the Col2a1 promoter was detected in chondrocytes and in the perichondrium/periosteum of 16.5 dpc transgenic limbs. Ectopic Smurf2 expression in cells of the chondrogenic lineage inhibited chondrocyte differentiation and stimulated maturation; ectopic Smurf2 in cells of the osteoblastic lineage stimulated osteoblast differentiation. Mechanistically, this could be caused by a dramatic increase in the expression of β‐catenin protein levels in the chondrocytes and perichondrial/periosteal cells of the Col2a1‐Smurf2 limbs. Conclusions : Ectopic expression of Smurf2 driven by the Col2a1 promoter accelerated the process of endochondral ossification including chondrocyte maturation and osteoblast differentiation through upregulation of β‐catenin, suggesting a possible mechanism for development of osteoarthritis seen in these mice.