A human 3D bone-sensory nerve co-culture model to investigate bone-derived pain

背景骨源性疼痛的条件，如癌症转移，不愈合的骨折，骨质疏松性骨折，或骨关节炎（OA）是众所周知的难以管理和普遍的，估计有150万骨癌转移，2200万骨不连骨折，890万骨质疏松性骨折和5.3亿OA患者目前在世界各地。复杂的信号传导存在于骨骼和神经之间，其耦合健康和疾病中的病理和疼痛。感觉神经侵袭和敏化与骨疾病相关，并且是骨关节炎、骨折和骨转移的动物模型和人类患者中疼痛反应的内在原因。该项目旨在（i）开发一个人源化的3D骨-感觉神经共培养模型，（ii）使用该模型研究骨结构变化与局部疼痛感觉之间的联系机制，（iii）测试该模型是否可以筛选2种已知的预防疼痛和影响骨结构的药物。由于这些神经在矿化骨中的位置，研究骨-神经相互作用具有挑战性。目前，不存在感觉神经-骨界面的人类衍生模型。已经产生了一些使用来自脊柱的神经的骨痛模型，这些神经被称为来自动物和人类的背根神经节（DRG），但是在技术上具有挑战性并且不被普遍采用。动物模型广泛用于骨源性疼痛的研究。2019年，发表了650多篇使用动物模型研究骨源性疼痛病理学的原创研究论文，其中4%是大型动物，包括犬，平均每次研究通常使用23只动物，总计超过14000只动物。一家制药公司测试骨痛药物可能每年使用1000多只动物。最近一项关于骨科动物试验的综述15显示，在9年的时间里，40%的骨科学术动物研究从未发表，其中近一半从未被引用，总计近10，000只动物的生命对骨科知识没有合理的影响。骨关节炎的动物模型每组使用>10只动物以揭示对骨结构和疼痛的影响。例如，在骨关节炎研究中，将使用132-198只小鼠或360只大鼠来测试1种药物。我们新的骨感觉神经模型将促进低成本研究，以确定新的分子靶点和预先筛选疼痛药物。它将减少早期临床前研究动物的使用，并取代目前用于识别和测试缓解骨源性疼痛的新疗法的模型。我们将通过在国家和国际会议和研讨会（例如骨科研究学会临床前模型部分，英国骨科研究学会，骨关节炎研究学会国际）上的传播，以及通过与我们目前在制药行业的合作者的药物进行验证，来促进该模型在动物试验中的使用。

Background Bone-derived pain from conditions such as cancer metastasis, non-healing fracture, osteoporotic fracture, or osteoarthritis (OA) are notoriously hard to manage and prevalent, with an estimated 1.5 million bone cancer metastases, 22 million non-union factures, 8.9 million osteoporotic fractures and 530 million OA sufferers currently worldwide. Complex signalling exists between bone and nerves which couple pathology and pain in health and disease. Sensory nerve invasion and sensitisation are associated with bone diseases, and intrinsic to pain responses in animal models and human patients with osteoarthritis, fracture and bone metastasis. This project aims to (i) develop a humanised 3D bone-sensory nerve co-culture model, (ii) use the model to investigate mechanisms linking changes in bone structure to local pain sensation and (iii) test whether the model can screen 2 drugs known to prevent pain and influence bone structure.Investigating bone-nerve interactions is challenging due to the position of these nerves within mineralised bone. Currently, no human derived models of the sensory nerve-bone interface exist. Some models of bone pain have been produced that use nerves from the spine, called dorsal root ganglia (DRG) from animals and humans, but are technically challenging and not commonly adopted. Animal models are extensively used for the investigation of bone derived pain. In 2019, over 650 original research papers were published investigating bone derived pain in pathology using animal models, 4% of which were large animals including canines, with an average of 23 animals typically used per study, totalling over 14000 animals. A pharmaceutical company testing drugs for bone pain may use more than 1000 animals p.a. In a recent review on orthopaedic animal testing15, it was revealed that over a 9-year period, 40% of academic orthopaedic animal studies were never published and almost half of those published were never cited totalling nearly 10,000 animal lives with no justifiable impact on orthopaedic knowledge. Animal models of osteoarthritis use >10 animals per group to reveal effects on bone structure and pain. For example, in osteoarthritis research 132-198 mice or 360 rats would be used to test 1 drug. Our new bone-sensory nerve model will facilitate low-cost research to identify new molecular targets and pre-screening of drugs for pain. It will reduce early-stage preclinical research animal usage, and replace models currently used to identify and test new treatments to ease bone derived pain. We will promote the use of this model over animal testing through dissemination at national and International conferences and workshops (e.g. Orthopaedic Research Society Preclinical Models Section, British Orthopaedic Research Society, Osteoarthritis Research Society International), and by validation with drugs from our current collaborators in the pharmaceutical industry.