Human BMP-2 gene transfer using transcutaneous in vivo electroporation induced both intramembranous and endochondral ossification

It has been generally accepted that bone morphogenetic protein-2 (BMP-2) can induce osteogenesis in skeletal muscles via endochondral ossification. However, it is not clear how the ossification process occurs after the BMP-2 gene transfer to skeletal muscles in rats using in vivo electroporation. In this study, we evaluated the ossification process by BMP-2 gene transfer using in vivo electroporation. The gastrocnemius muscles of Wistar rats were injected with human BMP-2 gene expression vector (pCAGGS-BMP-2), followed by electroporation under the condition of 100 V, 50 msec per 1 sec, x8. Light and electron microscopic and radiographic analyses were performed at 1, 3, 5, 7, and 10 days after treatment. At 7 days, no sign of cartilage and/or bone formation was detected. However, at 10 days after in vivo electroporation, soft X-ray analysis revealed small lucent areas around the plasmid-injected region. Clusters of both cartilage tissues, leading to endochondral ossification and intramembranous bones of various sizes, were observed between muscle fibers. RT-PCR detected osteocalcin mRNA, showing bone formation at 10 days. Our findings strongly suggest that BMP-2 gene transfer using in vivo electroporation induces not only endochondral ossification but also intramembranous ossification.     

Elevated interferon gamma expression in the central nervous system of tumour necrosis factor receptor 1-deficient mice with experimental autoimmune encephalomyelitis

Inflammation in the central nervous system (CNS) can be studied in experimental autoimmune encephalomyelitis (EAE). The proinflammatory cytokines interferon-gamma (IFN-gamma) and tumour necrosis factor (TNF) are implicated in EAE pathogenesis. Signals through the type 1 TNF receptor (TNFR1) are required for severe EAE to develop, whereas deficiency in IFN-gamma or its receptor result in more severe EAE. We investigated IFN-gamma expression in TNFR1-deficient (TNFR1-/-) mice. We describe here that there were more IFN-gamma-secreting T cells present in the CNS of TNFR1-/- mice during EAE compared to wild-type (WT) mice, despite that clinical symptoms were mild, with delayed onset. There was greater expression of IL-12/23p40 by antigen-presenting cells in these mice, and in vitro, TNFR1-/- antigen-presenting cells induced greater secretion of IFN-gamma but not interleukin (IL)-17 when cultured with primed T cells than did WT antigen presenting cells. TNFR1-/- mice with EAE had significantly higher expression of CXCL10 mRNA (but not CCL5 mRNA) in the CNS compared to WT mice with EAE. These data demonstrate that IFN-gamma expression is enhanced in the CNS of TNFR1-/- mice with EAE and suggest that IFN-gamma levels do not necessarily correlate with EAE severity.     

The new bone biology: pathologic, molecular, and clinical correlates

Bone and cartilage and their disorders are addressed under the following headings: functions of bone; normal and abnormal bone remodeling; osteopetrosis and osteoporosis; epithelial-mesenchymal interaction, condensation and differentiation; osteoblasts, markers of bone formation, osteoclasts, components of bone, and pathology of bone; chondroblasts, markers of cartilage formation, secondary cartilage, components of cartilage, and pathology of cartilage; intramembranous and endochondral bone formation; RUNX genes and cleidocranial dysplasia (CCD); osterix; histone deacetylase 4 and Runx2; Ligand to receptor activator of NFkappaB (RANKL), RANK, osteoprotegerin, and osteoimmunology; WNT signaling, LRP5 mutations, and beta-catenin; the role of leptin in bone remodeling; collagens, collagenopathies, and osteogenesis imperfecta; FGFs/FGFRs, FGFR3 skeletal dysplasias, craniosynostosis, and other disorders; short limb chondrodysplasias; molecular control of the growth plate in endochondral bone formation and genetic disorders of IHH and PTHR1; ANKH, craniometaphyseal dysplasia, and chondrocalcinosis; transforming growth factor beta, Camurati-Engelmann disease (CED), and Marfan syndrome, types I and II; an ACVR1 mutation and fibrodysplasia ossificans progressiva; MSX1 and MSX2: biology, mutations, and associated disorders; G protein, activation of adenylyl cyclase, GNAS1 mutations, McCune-Albright syndrome, fibrous dysplasia, and Albright hereditary osteodystrophy; FLNA and associated disorders; and morphological development of teeth and their genetic mutations.     

Immune function of the decoy receptor osteoprotegerin

Osteoprotegerin (OPG) is a member of the tumor necrosis factor receptor (TNFR) superfamily. OPG has an important function as a protector of bone, demonstrated by the fact that OPG(-/-) mice have severe osteoporosis. OPG acts as a decoy receptor, binding to RANK ligand (RANKL), thus preventing the interaction between receptor activator of NF-kappaB (RANK) and RANKL. This interaction is required for the development of functionally active osteoclasts. Osteoclasts are cells of the monocytic lineage that are responsible for bone resportion. Furthermore, as well as being an important player in the regulation of bone metabolism, OPG also has a role in the regulation of the immune response. Dendritic cells (DCs) express RANK and T cells express RANKL. The ligation of RANK by RANKL can activate both T cells and DCs. Furthermore, both B cells and DCs secrete OPG, and this secretion is regulated by the CD40 receptor. OPG(-/-) mice have B-cell developmental defects. Also, DCs isolated from OPG(-/-) mice more efficiently present antigen in vitro and secrete elevated amounts of inflammatory cytokines when stimulated with bacterial products, or soluble RANKL in vitro. Taken together, this suggests that OPG plays an important role in the immune response regulating the interactions between T cells and DCs.     

Osteogenesis and growth of the nasal ventral conchae of the piglet.

The present study investigated the osteogenesis and growth of the nasal ventral conchae of piglets aged from 1 to 28 days. Serial transverse sections of paraffin wax-embedded noses were stained and examined by light microscopy. Bone formation occurred in a rostrocaudal direction in the ventral scroll, dorsal scroll, connecting zone, transverse lamina and articular lamina, successively, and occurred by two ossification processes: endochondral ossification and intramembranous bone apposition. Endochondral ossification was responsible for the longitudinally, rostrally directed growth. Rapid transverse bone growth and modelling were centrifugally directed and occurred by intramembranous bone apposition at the eccentric side of the scrolls and resorption at the concentric scroll side. Elongation of the distal scroll extremities took place by intramembranous bone apposition.     

The use of ASCs engineered to express BMP2 or TGF-β3 within scaffold constructs to promote calvarial bone repair

Calvarial bone healing is difficult and grafts comprising adipose-derived stem cells (ASCs) and PLGA (poly(lactic-co-glycolic acid)) scaffolds barely heal rabbit calvarial defects. Although calvarial bone forms via intramembranous ossification without cartilage templates, it was suggested that chondrocytes/cartilages promote calvarial healing, thus we hypothesized that inducing ASCs chondrogenesis and endochondral ossification involving cartilage formation can improve calvarial healing. To evaluate this hypothesis and selectively induce osteogenesis/chondrogenesis, rabbit ASCs were engineered to express the potent osteogenic (BMP2) or chondrogenic (TGF-β3) factor, seeded into either apatite-coated PLGA or gelatin sponge scaffolds, and allotransplanted into critical-size calvarial defects. Among the 4 ASCs/scaffold constructs, gelatin constructs elicited in vitro chondrogenesis, in vivo osteogenic metabolism and calvarial healing more effectively than apatite-coated PLGA, regardless of BMP2 or TGF-β3 expression. The BMP2-expressing ASCs/gelatin triggered better bone healing than TGF-β3-expressing ASCs/gelatin, filling ≈86% of the defect area and ≈61% of the volume at week 12. The healing proceeded via endochondral ossification, instead of intramembranous pathway, as evidenced by the formation of cartilage that underwent osteogenesis and hypertrophy. These data demonstrated ossification pathway switching and significantly augmented calvarial healing by the BMP2-expressing ASCs/gelatin constructs, and underscored the importance of growth factor/scaffold combinations on the healing efficacy and pathway.     

The 4-1BB ligand and 4-1BB expressed on osteoclast precursors enhance RANKL-induced osteoclastogenesis via bi-directional signaling

The 4-1BB is a costimulatory molecule similar to the receptor activator of NF-kappaB ligand (RANKL), both of which are key factors for the differentiation of osteoclasts and are expressed mainly by activated T cells. The 4-1BB shares common signaling pathways with RANK, suggesting a potential role in osteoclastogenesis. In this study, the role of 4-1BB and 4-1BB ligand (4-1BBL) in osteoclastogenesis was investigated using 4-1BB(-/-) and 4-1BB(+/+) mice. Osteoclast precursors normally express 4-1BB and 4-1BBL after exposure to RANKL, which was confirmed by semi-quantitative RT-PCR and flow cytometry. The 4-1BB(-/- )mice had a slightly increased bone mass accompanied by a reduced osteoclastogenic ability of 4-1BB(-/-) bone marrow-derived macrophages (BMM) ex vivo. In addition, 4-1BB(-/-) BMM demonstrated hypophosphorylation of JNK and p38 and decreased induction of c-Fos in response to RANKL stimulation. Retroviral transduction of wild-type as well as partial-length 4-1BB, which lacks TNF receptor-associated factor 2-binding sites for signaling, restored the osteoclastogenic ability of 4-1BB(-/-) BMM. Furthermore, both recombinant 4-1BB and 4-1BBL enhanced RANKL-induced osteoclastogenesis by 4-1BB(+/+) BMM and the induction of c-Fos and NFATc1.Together, these results indicate that 4-1BBL and 4-1BB expressed on osteoclast precursors enhance RANKL-induced osteoclastogenesis via bi-directional signaling, findings that may delineate the complex nature of the 4-1BBL and 4-1BB interaction.     

调节破骨细胞分化的RANK特异性基序的再确认

目的： 进一步确认核转录因子NF-κB受体激动剂（RANK）蛋白中调节破骨细胞(OC)分化的特异性基序(motif),为阐明OC分化机制提供理论依据。方法: 分别突变RANK蛋白膜内部分第533-540之间的8个氨基酸（突变前氨基酸序列为DIIVVYVS,突变后氨基酸序列为ELLAAFAA）,用定点突变方法构建8个由肿瘤坏死因子受体1(TNFR1)和RANK跨膜部分和膜内部分共同组成的TNFR1/RANK突变嵌合体(TNFR1/RANK-533-TNFR1/RANK-540),每1个突变体在其RANK膜内部分含1个突变氨基酸。用包装细胞plat E分别将各突变体包装成逆转录病毒,通过逆转录病毒感染分别将这些突变体转染到TNFR1/TNFR2基因敲除小鼠的骨髓巨噬细胞(BMM)中。用TNF-ɑ和单核细胞集落刺激因子（M-CSF）刺激、观察转染哪种突变体的BMM不能诱导OC形成,不能诱导OC形成的突变体所包含的突变前氨基酸就是RANK调节OC分化的关键氨基酸,多个关键氨基酸组成的片段就是RANK特异性motif。结果: 转染TNFR1/RANK-533、TNFR1/RANK-539和TNFR1/RANK-540的BMM均能分化成OC,表明氨基酸D533、V539、S540突变后对OC分化无影响;转染TNFR1/RANK-534的BMM有极少数能分化成OC,表明氨基酸I534突变后对OC分化有部分影响;而转染TNFR1/RANK-535、TNFR1/RANK-536、TNFR1/RANK-537和TNFR1/RANK-538的BMM均不能分化成OC,表明氨基酸I535、V536 、V537和Y538突变后对OC分化起关键影响。结论: I534、I535、V536 、V537和Y538这5个氨基酸组成的片段（534-IIVVY-538）可能就是RANK调节OC分化的特异性motif。     

OPG/RANKL/RANK系统及其临床应用

人OPG，RANK和RANKL是3个肿瘤坏死因子家族新成员，主要存在于人的骨髓及其他少数组织中。2000年美国骨与矿物质研究协会对其给予了标准化命名，并统称为OPG/RANKL/RANK系统。作为破骨细胞形成、分化和骨吸收调节的关键调节物，OPG/RANKL/RANK系统在骨质疏松、骨硬化病、类风湿性关节炎、骨肿瘤、Paget’s病、牙周炎及正畸牙移动等临床疾病的发病机制及治疗方面均取得了较大进展。     

兔胫骨截骨延长区愈合过程的病理学观察

目的 :探讨截骨延长区骨愈合的病理学特点和新骨形成规律。方法 :取健康兔 2 7只建立胫骨骨延长动物模型 ,分别于延长的第 0、1、3、5、10、15、2 0、30、40天处死动物 ,在延长区取材进行病理组织学观察 ,同时用抗骨形成蛋白 (BMP)单抗作免疫组化染色。结果 :延长前即出现血肿 ,延长第 1天血肿机化 ,第 3天出现新的血肿 ,第 5天软骨形成和新血肿机化 ,第 15天出现膜内成骨、纤维性成骨和软骨内成骨 ,第 2 0天延长区大部分被骨性骨痂取代 ,第 30天开始改建。免疫组化染色显示延长区纤维母细胞、骨母细胞和软骨细胞均不同程度表达BMP。结论 :延长区存在膜内成骨、纤维性成骨和软骨内成骨 3种方式 ,以软骨内成骨为主 ;内源性BMP参与了延长区愈合过程。     

Does static precede dynamic osteogenesis in endochondral ossification as occurs in intramembranous ossification?

Endochondral ossification takes place with calcified cartilage cores providing a rigid scaffold for new bone formation. Intramembranous ossification begins in connective tissue and new bone formed by a process of static ossification (SO) followed by dynamic ossification (DO) as previously described. The aim of the present study was to determine if the process of endochondral ossification is similar to that of intramembranous ossification with both a static and a dynamic phase of osteogenesis. Endochondral ossification centers of the tibiae and humeri of newborn and young growing rabbits were studied by light and transmission electron microscopy. The observations clearly showed that in endochondral ossification, the calcified trabeculae appeared to be lined first by osteoclasts. The osteoclasts were then replaced by flattened cells (likely cells of the reversal phase) and finally by irregularly arranged osteoblastic laminae, typical of DO. This cellular sequence did not include osteoblasts seen in the phase of SO. These findings clearly support our working hypothesis that SO only forms in soft tissues to provide a rigid framework for DO, and that DO requires a rigid mineralized surface. The presence of osteocytes in contact with the calcified cartilage also suggests the existence of stationary osteoblasts in endochondral ossification. Stationary osteoblasts did not appear to be a unique feature of SO. The presence of stationary osteoblasts may appear to provide the initial osteocytes during osteogenesis that may function as mechanosensors throughout the bone tissue. If this is the case, then bone would be capable of sensing mechanical strains from its inception.     

Proinflammatory cytokine (TNFalpha/IL-1alpha) induction of human osteoclast formation

TNFalpha and IL-1alpha are potent stimulators of bone resorption in vivo and in vitro. Recently, it has been demonstrated that these two cytokines directly induce osteoclastogenesis in mouse marrow cultures. This study determined whether TNFalpha (+/- IL-1alpha) is also capable of inducing human osteoclastogenesis. The CD14(+) monocyte fraction of human peripheral mononuclear cells was cultured with TNFalpha +/- IL-1alpha in the presence of M-CSF. TNFalpha induced the formation of multinucleated cells (MNCs) which were positive for TRAP, VNR and cathepsin K and showed evidence of resorption pit formation. IL-1alpha stimulated TNFalpha-induced lacunar resorption two- to four-fold. Osteoprotegerin, the decoy receptor for RANKL, did not inhibit this process. Anti-human IL-1alpha neutralizing antibodies significantly inhibited resorption without inhibiting the formation of TRAP(+)/VNR(+) MNCs. These results suggest that, in the presence of M-CSF, TNFalpha is sufficient for inducing human osteoclast differentiation from circulating precursors by a process which is distinct from the RANK/RANKL signalling pathway.     

IFN-gamma-producing human T cells directly induce osteoclastogenesis from human monocytes via the expression of RANKL

The current study explored our hypothesis that IFN-gamma-producing human T cells inhibit human osteoclast formation. Activated T cells derived from human PBMC were divided into IFN-gamma-producing T cells (IFN-gamma(+) T cells) and IFN-gamma-non-producing T cells (IFN-gamma(-) T cells). IFN-gamma(+) T cells were cultured with human monocytes in the presence of macrophage-CSF alone. The concentration of soluble receptor activator of NF-kappaB ligand (RANKL) and IFN-gamma, and the amount of membrane type RANKL expressed on T cells, were measured by ELISA. In the patients with early rheumatoid arthritis (RA) treated with non-steroidal anti-inflammatory drugs alone, CD4+ T cells expressing both IFN-gamma and RANKL were detected by flow cytometry. Surprisingly, IFN-gamma(+) T cells, but not IFN-gamma(-) T cells, induced osteoclastogenesis from monocytes, which was completely inhibited by adding osteoprotegerin and increased by adding anti-IFN-gamma antibodies. The levels of both soluble and membrane type RANKL were elevated in IFN-gamma(+) T cells. The ratio of CD4+ T cells expressing both IFN-gamma and RANKL in total CD4+ T cells from PBMC was elevated in RA patients. Contrary to our hypothesis, IFN-gamma(+) human T cells induced osteoclastogenesis through the expression of RANKL, suggesting that Th1 cells play a direct role in bone resorption in Th1 dominant diseases such as RA.     

Angiogenesis and intramembranous osteogenesis

Background: Angiogenesis is likely critical for the process of intramembranous osteogenesis; however, the developmental relationship between blood vessels and bone mineralization is not well studied within intramembranous bones. Given its importance, changes in angiogenesis regulation are likely to contribute to evolutionarily and medically relevant craniofacial variation. Results: We summarize what is known about the association between angiogenesis and intramembranous osteogenesis, supplementing with information from the better‐studied processes of endochondral ossification and distraction osteogenesis. Based on this review, we introduce a model of angiogenesis during early intramembranous osteogenesis as well as a series of null hypotheses to be tested. Conclusions: This model can serve as a basis of future research on the spatio‐temporal association and regulatory interactions of mesenchymal, vascular, and bone cells, which will be required to illuminate the potential effects of angiogenesis dysregulation on craniofacial skeletal phenotypes. Developmental Dynamics 242:909–922, 2013. © 2013 Wiley Periodicals, Inc.     

Leptin increases growth of primary ossification centers in fetal mice

The effect of peripheral leptin on fetal primary ossification centers during the early phases of bone histogenesis was investigated by administration of leptin to pregnant mice. Fourteen pregnant mice were divided into two groups. The treated pregnant group was subcutaneously injected in the intrascapular region with supraphysiologic doses (2 mg kg⁻¹) of leptin (Vinci Biochem, Firenze, Italy) in a volume of 0.1 mL per 10 g body weight, at the 7th, 9th and 11th day of gestation. The control group was treated with physiological solution in the same manner and same times as the treated group. The new-born mice were killed 1 day after birth and the primary ossification centers were stained with Alizarin Red S after diaphanizing the soft tissues in 1% potassium hydroxide. The development of both endochondral and intramembranous ossification centers was morphometrically analysed in long bones. The results showed that the ossification centers of mice born by mothers treated with leptin grow more rapidly in both length and cross-sectional area compared with mice born by the untreated mothers. As the development of long bones depends on endochondral ossification occurring at proximal and distal epiphyseal plates as well as on intramembranous ossification along the periosteal surface, it appears that leptin activates the differentiation and proliferation of both chondrocytes and osteoblasts. The role of leptin as a growth factor of cartilage and bone is discussed in the light of the data reported in the literature.     

Chondrogenic pre-induction of human mesenchymal stem cells on β-TCP: Enhanced bone quality by endochondral heterotopic bone formation

New techniques to heal bone defects include the combination of bone substitute materials with mesenchymal stem cells (MSC). To find solutions not hampered by low material resorbability or high donor variability of human MSC, the potency of such composites is usually evaluated by heterotopic bone formation assays in immunocompromised animals. The aim of this study was to investigate whether resorbable phase-pure β-tricalcium-phosphate (β-TCP) could support heterotopic bone formation by MSC comparable to partially resorbable hydroxyapatite/tricalcium-phosphate (HA/TCP). Furthermore, in light of disappointing results with osteogenic in vitro priming of MSC, we tested whether chondrogenic pre-induction of constructs may allow for enhanced bone formation by triggering the endochondral pathway. β-TCP granules of three different sizes and HA/TCP were seeded with MSC and transplanted subcutaneously into immunocompromised mice either immediately or after a chondrogenic pre-induction for 6 weeks. After 8 weeks, explants were analysed by histology. β-TCP seeded with unprimed MSC revealed intramembranous bone formation without haematopoietic marrow with 3.8-fold more bone formed with granules smaller than 0.7 mm than with 0.7–1.4 mm particles (p ? 0.018). Chondrogenic pre-induction of β-TCP/MSC composites resulted in collagen type II and proteoglycan-rich cartilage-like tissue which, after transplantation, underwent endochondral ossification, yielding ectopic bone produced by human cells while haematopoietic marrow was derived from the mouse. Transdifferentiation of MSC-derived chondrocytes to osteoblasts or direct osteogenesis of cartilage-resident MSC is postulated to explain the human origin of new bone. In conclusion, β-TCP was significantly more osteo-permissive (p = 0.004) than HA/TCP for human MSC, and chondrogenic priming of β-TCP/MSC represented a superior approach capable of supporting full bone formation, including marrow organization.     

Absence of mechanical loading in utero influences bone mass and architecture but not innervation in Myod-Myf5-deficient mice

Although the responses of bone to increased loading or exercise have been studied in detail, our understanding of the effects of decreased usage of the skeleton has been limited by the scarcity of suitable models. Such models should ideally not affect bone innervation, which appears to be a mediator of physiological responses of bone to unloading. MyoD-/-/Myf5-/- (dd/ff) mice lack skeletal muscle, so the fetuses develop without any active movement in utero and die soon after birth. We used micro-compter tomography and histology to analyse their bone development and structure during endochondral ossification in parallel with the establishment of bone innervation. Long bones from mutant mice were found to be profoundly different from controls, with shorter mineralized zones and less mineralization. They lacked many characteristics of adult bones - curvatures, changes in shaft diameter and traction epiphyses where muscles originate or insert - that were evident in the controls. Histologically, dd/ff mice showed the same degree of endochondral development as wild-type animals, but presented many more osteoclasts in the newly layed bone. Innervation and the expression pattern of semaphorin-3A signalling molecules were not disturbed in the mutants. Overall, we have found no evidence for a major defect of development in dd/ff mice, and specifically no alteration or delay in endochondral ossification and bone innervation. The altered morphological features of dd/ff mice and the increased bone resorption show the role of muscle activity in bone shaping and the consequences of bone unloading.     

Advances in bone biology and new treatments for bone loss

Recent advances in bone biology have led to a more detailed understanding of bone remodeling which is a process that leads to resorption of old bone and replacement by formation of new bone. The most important discoveries in this process of bone remodeling were those of the RANK Ligand/RANK/OPG system which is now recognized the dominant pathway regulating bone resorption. RANK Ligand (RANKL) is a cytokine belonging to the tumor necrosis factor family and is expressed by osteoblasts; it binds to membrane bound receptor RANK on osteoclasts and promotes differentiation of marrow cells through various stages to multinucleated osteoclasts which resorb bone. Several hormones such as parathyroid hormone, calcitriol and prostaglandins stimulate RANK Ligand expression by osteoblasts. Osteoblasts also secrete osteoprotegerin (OPG) which is a soluble receptor that is a potent antagonist of osteoclast formation by binding and inactivating RANKL and OPG is therefore an important regulator of bone resorption. OPG is stimulated by estrogen. OPG has been genetically engineered and in human subjects is a potent inhibitor of bone resorption. Another method for preventing bone resorption is to develope antibodies against RANKL and this has been shown to be a successful strategy. A single subcutaneous injection of this antibody (Denosumab) every 6 months proved to be a potent inhibitor of bone resorption and clinical fracture trials using this agent are now underway. These are novel developments that have risen from basic research in bone biology and other discoveries in the bone remodeling process can be expected to lead to further treatment options for various bone diseases.