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Abstract Skeletal development depends on coordinated angiogenesis and osteogenesis. Bone morphogenetic proteins direct bone formation in part by activating SMAD1/5 signaling in osteoblasts. However, the role of SMAD1/5 in skeletal endothelium is unknown. Here, we found that endothelial cell-conditional SMAD1/5 depletion in juvenile mice caused metaphyseal and diaphyseal hypervascularity, resulting in altered trabecular and cortical bone formation. SMAD1/5 depletion induced excessive sprouting and disrupting the morphology of the metaphyseal vessels, with impaired anastomotic loop formation at the chondro-osseous junction. Endothelial SMAD1/5 depletion impaired growth plate resorption and, upon long-term depletion, abrogated osteoprogenitor recruitment to the primary spongiosa. Finally, in the diaphysis, endothelial SMAD1/5 activity was necessary to maintain the sinusoidal phenotype, with SMAD1/5 depletion inducing formation of large vascular loops and elevated vascular permeability. Together, endothelial SMAD1/5 activity sustains skeletal vascular morphogenesis and function and coordinates growth plate remodeling and osteoprogenitor recruitment dynamics in juvenile mouse bone.

Abstract Adequate pain management is essential for ethical and scientific reasons in animal experiments and should completely cover the period of expected pain without the need for frequent re-application. However, current depot formulations of Buprenorphine are only available in the USA and have limited duration of action. Recently, a new microparticulate Buprenorphine formulation (BUP-Depot) for sustained release has been developed as a potential future alternative to standard formulations available in Europe. Pharmacokinetics indicate a possible effectiveness for about 72 h. Here, we investigated whether the administration of the BUP-Depot ensures continuous and sufficient analgesia in two mouse fracture models (femoral osteotomy) and could, therefore, serve as a potent alternative to the application of Tramadol via the drinking water. Both protocols were examined for analgesic effectiveness, side effects on experimental readout, and effects on fracture healing outcomes in male and female C57BL/6N mice. The BUP-Depot provided effective analgesia for 72 h, comparable to the effectiveness of Tramadol in the drinking water. Fracture healing outcome was not different between analgesic regimes. The availability of a Buprenorphine depot formulation for rodents in Europe would be a beneficial addition for extended pain relief in mice, thereby increasing animal welfare.

Osteoarthritis (OA) is the most common form of musculoskeletal disease with significant healthcare costs and unmet needs in terms of early diagnosis and treatment. Many of the drugs that have been developed to treat OA failed in phase 2 and phase 3 clinical trials or produced inconclusive and ambiguous results. High throughput omics technologies are a powerful tool to better understand the mechanisms of the development of OA and other arthritic diseases. In this paper we outline the strategic reasons for increasingly applying deep phenotyping in OA for the benefit of gaining a better understanding of disease mechanisms and developing targeted treatments. This editorial is intended to launch a special themed issue of Osteoarthritis and Cartilage Open addressing the timely topic of “Advances in omics technologies for deep phenotyping in osteoarthritis”. High throughput omics technologies are increasingly being applied in mechanistic studies of OA and other arthritic diseases. Applying multi-omics approaches in OA is a high priority and will allow us to gather new information on disease pathogenesis at the cellular level, and integrate data from diverse omics technology platforms to enable deep phenotyping. We anticipate that new knowledge in this area will allow us to harness the power of Big Data Analytics and resolve the extremely complex and overlapping clinical phenotypes into molecular endotypes, revealing new information about the cellular taxonomy of OA and “druggable pathways”, thus facilitating future drug development.

Bone generally displays a high intrinsic capacity to regenerate. Nonetheless, large osseous defects sometimes fail to heal. The treatment of such large segmental defects still represents a considerable clinical challenge. The regeneration of large bone defects often proves difficult, since it relies on the formation of large amounts of bone within an environment impedimental to osteogenesis, characterized by soft tissue damage and hampered vascularization. Consequently, research efforts have concentrated on tissue engineering and regenerative medical strategies to resolve this multifaceted challenge. In this review, we summarize, critically evaluate, and discuss present approaches in light of their clinical relevance; we also present future advanced techniques for bone tissue engineering, outlining the steps to realize for their translation from bench to bedside. The discussion includes the physiology of bone healing, requirements and properties of natural and synthetic biomaterials for bone reconstruction, their use in conjunction with cellular components and suitable growth factors, and strategies to improve vascularization and the translation of these regenerative concepts to in vivo applications. We conclude that the ideal all-purpose material for scaffold-guided bone regeneration is currently not available. It seems that a variety of different solutions will be employed, according to the clinical treatment necessary.

Macrophages are essential players in the process of fracture healing, acting by remodeling of the extracellular matrix and enabling vascularization. Whilst activated macrophages of M1-like phenotype are present in the initial pro-inflammatory phase of hours to days of fracture healing, an anti-inflammatory M2-like macrophage phenotype is supposed to be crucial for the induction of downstream cascades of healing, especially the initiation of vascularization. In a mouse-osteotomy model, we provide a comprehensive characterization of vessel (CD31+, Emcn+) and macrophage phenotypes (F4/80, CD206, CD80, Mac-2) during the process of fracture healing. To this end, we phenotype the phases of vascular regeneration—the expansion phase (d1–d7 after injury) and the remodeling phase of the endothelial network, until tissue integrity is restored (d14–d21 after injury). Vessels which appear during the bone formation process resemble type H endothelium (CD31hiEmcnhi), and are closely connected to osteoprogenitors (Runx2+, Osx+) and F4/80+ macrophages. M1-like macrophages are present in the initial phase of vascularization until day 3 post osteotomy, but they are rare during later regeneration phases. M2-like macrophages localize mainly extramedullary, and CD206+ macrophages are found to express Mac-2+ during the expansion phase. VEGFA expression is initiated by CD80+ cells, including F4/80+ macrophages, until day 3, while subsequently osteoblasts and chondrocytes are main contributors to VEGFA production at the fracture site. Using Longitudinal Intravital Microendoscopy of the Bone (LIMB) we observe changes in the motility and organization of CX3CR1+ cells, which infiltrate the injury site after an osteotomy. A transient accumulation, resulting in spatial polarization of both, endothelial cells and macrophages, in regions distal to the fracture site, is evident. Immunofluorescence histology followed by histocytometric analysis reveals that F4/80+CX3CR1+ myeloid cells precede vascularization.

Fractures are one of the most frequently occurring traumatic events worldwide. Approximately 10% of fractures lead to bone healing disorders, resulting in strain for affected patients and enormous costs for society. In order to shed light into underlying mechanisms of bone regeneration (habitual or disturbed), and to develop new therapeutic strategies, various in vivo, ex vivo and in vitro models can be applied. Undeniably, in vivo models include the systemic and biological situation. However, transferability towards the human patient along with ethical concerns regarding in vivo models have to be considered. Fostered by enormous technical improvements, such as bioreactors, on-a-chip-technologies and bone tissue engineering, sophisticated in vitro models are of rising interest. These models offer the possibility to use human cells from individual donors, complex cell systems and 3D models, therefore bridging the transferability gap, providing a platform for the introduction of personalized precision medicine and finally sparing animals. Facing diverse processes during fracture healing and thus various scientific opportunities, the reliability of results oftentimes depends on the choice of an appropriate model. Hence, we here focus on categorizing available models with respect to the requirements of the scientific approach.

Fractures in horses-whether simple fractures with just one clean break, or incomplete greenstick with stress fractures, or complications such as shattered bones can all be either minimal or even catastrophic. Thus, improvement in fracture healing is a hallmark in equine orthopedics. The fracture healing process implements a complex sequence of events including the initial inflammatory phase removing damaged tissue, re-establishment of vessels and mesenchymal stromal cells, a soft and hard callus phase closing the fracture gap as well as the remodeling phase shaping the bone to a scar-free tissue. Detailed knowledge on processes in equine fracture healing in general and on the initial phase in particular is apparently very limited. Therefore, we generated equine in vitro fracture hematoma models (FH models) to study time-dependent changes in cell composition and RNA-expression for the most prominent cells in the FH model (immune cells, mesenchymal stromal cells) under conditions most closely adapted to the in vivo situation (hypoxia) by using flow cytometry and qPCR. In order to analyze the impact of mesenchymal stromal cells in greater detail, we also incubated blood clots without the addition of mesenchymal stromal cells under the same conditions as a control. We observed a superior survival capacity of mesenchymal stromal cells over immune cells within our FH model maintained under hypoxia. Furthermore, we demonstrate an upregulation of relevant angiogenic, osteogenic and hypoxia-induced markers within 48 h, a time well-known to be crucial for proper fracture healing.

The posterior lateral line system (pLLS) of aquatic animals comprises small clustered mechanosensory organs along the side of the animal. They develop from proneuromasts, which are deposited from a migratory primordium on its way to the tip of the tail. We here show, that the Neural Cell Adhesion Molecule Ncam1b is an integral part of the pathways initiating and regulating the development of the pLLS in zebrafish. We find that morpholino-knockdowns of ncam1b (i) reduce cell proliferation within the primordium, (ii) reduce the expression of Fgf target gene erm, (iii) severely affect proneuromast formation, and (iv) affect primordium migration. Ncam1b directly interacts with Fgf receptor Fgfr1a, and a knockdown of fgfr1a causes similar phenotypic changes as observed in ncam1b-morphants. We conclude that Ncam1b is involved in activating proliferation by triggering the expression of erm. In addition, we demonstrate that Ncam1b is required for the expression of chemokine receptor Cxcr7b, which is crucial for directed primordial migration. Finally, we show that the knockdown of ncam1b destabilizes proneuromasts, suggesting a further function of Ncam1b in strengthening the cohesion of proneuromast cells.