Autologous blood coagulum is a physiological carrier for BMP6 to induce new bone formation and promote posterolateral lumbar spine fusion in rabbits

In the present study, we describe autologous blood coagulum (ABC) as a physiological carrier for BMP6 to induce new bone formation. Recombinant human BMP6 (rhBMP6), dispersed within ABC and formed as an autologous bone graft substitute (ABGS), was evaluated either with or without allograft bone particles (ALLO) in rat subcutaneous implants and in a posterolateral lumbar fusion (PLF) model in rabbits. ABGS induced endochondral bone differentiation in rat subcutaneous implants. Coating ALLO by ABC significantly decreased the formation of multinucleated foreign body giant cells (FBGCs) in implants, as compared with ALLO alone. However, addition of rhBMP6 to ABC/ALLO induced a robust endochondral bone formation with little or no FBGCs in the implant. In rabbit PLF model, ABGS induced new bone formation uniformly within the implant resulting in a complete fusion when placed between two lumbar transverse processes in the posterolateral gutter with an optimum dose of 100‐μg rhBMP6 per ml of ABC. ABGS containing ALLO also resulted in a fusion where the ALLO was replaced by the newly formed bone via creeping substitution. Our findings demonstrate for the first time that rhBMP6, with ABC as a carrier, induced a robust bone formation with a complete spinal fusion in a rabbit PLF model. RhBMP6 was effective at low doses with ABC serving as a physiological substratum providing a permissive environment by protecting against foreign body reaction elicited by ALLO.     

Neofunction of ACVR1 in fibrodysplasia ossificans progressiva

Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disease characterized by extraskeletal bone formation through endochondral ossification. FOP patients harbor point mutations in ACVR1 (also known as ALK2), a type I receptor for bone morphogenetic protein (BMP). Two mechanisms of mutated ACVR1 (FOP-ACVR1) have been proposed: ligand-independent constitutive activity and ligand-dependent hyperactivity in BMP signaling. Here, by using FOP patient-derived induced pluripotent stem cells (FOP-iPSCs), we report a third mechanism, where FOP-ACVR1 abnormally transduces BMP signaling in response to Activin-A, a molecule that normally transduces TGF-β signaling but not BMP signaling. Activin-A enhanced the chondrogenesis of induced mesenchymal stromal cells derived from FOP-iPSCs (FOP-iMSCs) via aberrant activation of BMP signaling in addition to the normal activation of TGF-β signaling in vitro, and induced endochondral ossification of FOP-iMSCs in vivo. These results uncover a novel mechanism of extraskeletal bone formation in FOP and provide a potential new therapeutic strategy for FOP.Heterotopic ossification (HO) is defined as bone formation in soft tissue where bone normally does not exist. It can be the result of surgical operations, trauma, or genetic conditions, one of which is fibrodysplasia ossificans progressiva (FOP). FOP is a rare genetic disease characterized by extraskeletal bone formation through endochondral ossification (1–6). The responsive mutation for classic FOP is 617G > A (R206H) in the intracellular glycine- and serine-rich (GS) domain (7) of ACVR1 (also known as ALK2), a type I receptor for bone morphogenetic protein (BMP) (8–10). ACVR1 mutations in atypical FOP patients have been found also in other amino acids of the GS domain or protein kinase domain (11, 12). Regardless of the mutation site, mutated ACVR1 (FOP-ACVR1) has been shown to activate BMP signaling without exogenous BMP ligands (constitutive activity) and transmit much stronger BMP signaling after ligand stimulation (hyperactivity) (12–25).To reveal the molecular nature of how FOP-ACVR1 activates BMP signaling, cells overexpressing FOP-ACVR1 (12–20), mouse embryonic fibroblasts derived from Alk2^R206H/+ mice (21, 22), and cells from FOP patients, such as stem cells from human exfoliated deciduous teeth (23), FOP patient-derived induced pluripotent stem cells (FOP-iPSCs) (24, 25) and induced mesenchymal stromal cells (iMSCs) from FOP-iPSCs (FOP-iMSCs) (26) have been used as models. Among these cells, Alk2^R206H/+ mouse embryonic fibroblasts and FOP-iMSCs are preferred because of their accessibility and expression level of FOP-ACVR1 using an endogenous promoter. In these cells, however, the constitutive activity and hyperactivity is not strong (within twofold normal levels) (22, 26). In addition, despite the essential role of BMP signaling in development (27–31), the pre- and postnatal development and growth of FOP patients are almost normal, and HO is induced in FOP patients after physical trauma and inflammatory response postnatally, not at birth (1–6). These observations led us to hypothesize that FOP-ACVR1 abnormally responds to noncanonical BMP ligands induced by trauma or inflammation.Here we show that FOP-ACVR1 transduced BMP signaling in response to Activin-A, a molecule that normally transduces TGF-β signaling (10, 32–34) and contributes to inflammatory responses (35, 36). Our in vitro and in vivo data indicate that activation of TGF-β and aberrant BMP signaling by Activin-A in FOP-cells is one cause of HO in FOP. These results suggest a possible application of anti–Activin-A reagents as a new therapeutic tool for FOP.     

Mechanisms involved in bone resorption

Osteoclasts, which are present only in bone, are multinucleated giant cells with the capacity to resorb mineralized tissues. These osteoclasts are derived from hemopoietic progenitors of the monocyte-macrophage lineage. Osteoblasts or bone marrow-derived stromal cells are involved in osteoclastogenesis through a mechanism involving cell-to-cell contact with osteoclast progenitors. Experiments on the osteopetrotic op/op mouse model have established that a product ofosteo blasts, macrophage colony-stimulating factor (M-CSF), regulates differentiation of osteoclast progenitors into osteoclasts. Recent discovery of osteoclast differentiation factor (ODF)/receptor activator of NF-κ Bligand (RANKL) allowed elucidation of the precise mechanism by which osteoblasts regulate osteoclastic bone resorption. Treatment of osteoblasts with bone-resorbing factors up-regulated expression of RANKL mRNA. In contrast, TNF α stimulates osteoclast differentiation in the presence of M-CSF through a mechanism independent of the RANKL system. IL-1 also directly acts on mature osteoclasts as a potentiator of osteoclast activation. In addition, TGF-β super family members, such as bone morphogenetic proteins(BMPs) strikingly enhanced osteoclast differentiation from their progenitors and survival of mature osteoclasts induced by RANKL. These results suggest that BMP-mediated signals cross-communicate with RANKL-mediated ones in inducing osteoclast differentiation and function. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genetic and pharmacological inhibition of retinoic acid receptor γ function promotes endochondral bone formation

The nuclear retinoic acid receptors (RARs) play key roles in skeletal development and endochondral ossification. Previously, we showed that RARγ regulates chondrogenesis and that pharmacological activation of RARγ blocked heterotopic ossification (HO), pathology in which endochondral bone forms in soft tissues. Thus, we reasoned that pharmacological inhibition of RARγ should enhance endochondral ossification, leading to a potential therapeutic strategy for bone deficiencies. We created surgical bone defects in wild type and RARγ‐null mice and monitored bone healing. Fibrous, cartilaginous, and osseous tissues formed in both groups by day 7, but more cartilaginous tissue formed in mutants within and around the defects compared to controls. Next, we implanted a mixture of Matrigel and rhBMP2 subdermally to induce ectopic endochondral ossification. Administration of RARγ antagonists significantly stimulated ectopic bone formation in wild type but not in RARγ‐null mice. The antagonist‐induced increases in bone formation were preceded by increases in cartilage formation and were accompanied by higher levels of phosphorylated Smad1/5/8 (pSmad1/5/8) compared to vehicle‐treated control. Higher pSmad1/5/8 levels were also observed in cartilaginous tissues forming in healing bone defects in RARγ‐null mice, and increases in pSmad1/5/8 levels and Id1‐luc activity were observed in RARγ antagonist‐treated chondrogenic cells in culture. Our data show that genetic or pharmacological interference with RARγ stimulates endochondral bone formation and does so at least in part by stimulating canonical BMP signaling. This pharmacologic strategy could represent a new tool to enhance endochondral bone formation in the setting of various orthopedic surgical interventions and other skeletal deficiencies. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1096–1105, 2017.

     

纳米级羟基磷灰石/骨形态发生蛋白复合物修复兔桡骨大段缺损及局部血管内皮细胞生长因子的表达

摘要 背景：研究发现,在小段骨缺损中植入骨或仿生骨组织,坏死组织逐渐被替换,移植骨中会长入有活性的血管肉芽组织,移植骨被吸收,新骨主动形成。但在大段骨缺损,这一过程发生较慢且不完全。 目的：观察纳米级羟基磷灰石材料复合骨形态发生蛋白后大段骨缺损修复能力及诱导生成血管能力。 方法：制作兔桡骨大段骨缺损模型,抽签随机分2组,选择一侧分别植入纳米级羟基磷灰石/骨形态发生蛋白、纳米级羟基磷灰石修复,均以另一侧为空白对照。植入后6个月行大体观察、X射线、组织形态学检查、组织切片碱性磷酸酶染色、成骨量分析、血管内皮细胞生长因子阳性细胞率及阳性血管数检测。 结果与结论：空白对照组基本无骨组织生成。纳米级羟基磷灰石植入后被新生骨组织分割成小块,材料原有结构破坏。纳米级羟基磷灰石/骨形态发生蛋白组较纳米级羟基磷灰石组残存材料更少,材料降解更为彻底。纳米级羟基磷灰石/骨形态发生蛋白组成骨量、血管内皮细胞生长因子表达及血管内皮细胞生长因子阳性血管数目均明显高于纳米级羟基磷灰石组(P < 0.001),且血管内皮细胞生长因子表达与血管内皮细胞生长因子阳性血管数目成正比关系。说明纳米级羟基磷灰石与骨形态发生蛋白复合后,骨修复能力进一步增强,诱导血管生成能力明显提高。 关键词：纳米级羟基磷灰石;血管内皮细胞生长因子;骨形态发生蛋白;血管生成;纳米生物材料 doi:10.3969/j.issn.1673-8225.2011.12.006