Synergistic microenvironments for non-union bone defects

长骨骨折涉及对周围组织和血管网络的损伤，因此，自然骨愈合能力丧失，形成骨不连缺陷。这种临床问题的患病率在骨折中为2.5%，但当有侧支软组织和血管损伤时，这一比率分别高达15%和45%。目前的治疗方法包括自体骨移植、骨替代品和使用生长因子（GF），特别是骨形态发生蛋白（BMP），成功有限，缺点明显。因此，开发新的治疗方法用于骨再生和血管化的临床需求尚未得到满足骨不连骨缺损。GF在骨再生中的作用被广泛认可，但递送这些因子以增强组织愈合，同时保持其活性，尚未成功。可溶性施用或受控递送（包括水凝胶和支架）由于GF从组织部位的分解和清除而未能满足需要。然而，更重要的是，由于使用了高剂量，已经报告了灾难性的附带风险。生长因子的这种不令人满意的临床转化需要稳健、安全和有效的系统来控制递送。人骨形态发生蛋白-2（BMP-2）是一种强大的生长因子，在组织形态发生中是必不可少的，并参与无数的细胞过程，包括细胞募集、细胞分化和血管生成。重组BMP-2（rhBMP-2）的使用已被广泛用于促进广泛临床应用（脊柱、口腔颌面和创伤）中的骨生长。目前的临床递送涉及以1.5mg/cm 3的浓度将蛋白质掺入胶原海绵载体中。然而，严重的临床并发症已经报告，甚至导致FDA发布了与rhBMP-2相关的危及生命的并发症（呼吸系统、神经系统、炎症）的公共卫生通知。7此后的报告包括与使用高剂量相关的不受控制的骨形成和致癌风险的观察结果。尽管卫生机构、临床研究人员和行业之间存在附带风险和争议，但rhBMP-2在临床实践中的使用仍在继续增加。8在此，我们提出了一种简单而稳健的基于材料的策略，用于诱导骨不连缺损的骨再生。它是基于工程结构，调整GF和它们的受体之间的相互作用，它允许使用非常小的rhBMP-2剂量（< 0.1 mg/cm 3），同时保持细胞活化。这显然将使治疗更便宜，更安全，更有效。重要的是要注意，我们的方法本身并不调节GF递送，而是调节生长因子对细胞受体的有效呈递，从而防止与在再生部位递送可溶性rhBMP-2相关的附带风险。最终的策略包括植入无细胞的3D构建体，该构建体包含合成的可生物降解的聚合物、细胞外基质蛋白和rhBMP-2。该系统旨在体内招募干细胞（无需用干细胞预加载构建体），并促进骨不连缺损的骨再生，以及帮助新组织的血运重建。

Long bone fractures involve damage to the surrounding tissues and vascular networks and, as a result, the natural bone healing capacity is lost and non-union defects are formed. The prevalence of this clinical problem is 2.5% for bone fractures but this rate increases up to 15% and 45% when there is collateral soft tissue and vascular injuries respectively. Current treatments comprise bone autografts, bone substitutes and the use of growth factors (GFs), specially bone morphogenetic proteins (BMP) with limited success and significant drawbacks.Thus, there is an unmet clinical need to develop new therapeutic approaches for bone regeneration and vascularisation in non-union bone defects. The role of GFs in bone regeneration is broadly recognised but the delivery of these factors to enhance tissue healing, while maintaining their activity, has not been successful. Soluble administration or controlled delivery (including hydrogels and scaffolds) have failed to meet the need due to the breakdown and clearance of GFs from tissue sites. More importantly, however, catastrophic collateral risks have been reported due to the high dose used. This unsatisfactory clinical translation of growth factors demands robust, safe and effective systems that control delivery. Human bone morphogenetic protein-2 (BMP-2) is a powerful growth factor that is essential in tissue morphogenesis and is involved in a myriad of cellular processes, including cell recruitment, cell differentiation, and angiogenesis. The use of recombinant BMP-2 (rhBMP-2) has been generalised to promote bone growth in a broad range of clinical applications (spine, oral-maxillofacial and trauma). Current clinical delivery involves the incorporation of the protein in a collagen sponge carrier at a concentration of 1.5 mg/cm3. However, serious clinical complications have been reported that even led the FDA to issue a Public Health Notification of life threatening complications associated with rhBMP-2 (respiratory, neurological, inflammatory).7 Reports since then include observations of uncontrolled bone formation and carcinogenic risks associated to the high doses used. Notwithstanding collateral risks and controversy between health agencies, clinical researchers and industry, the use of rhBMP-2 continues to increase in clinical practice.8Here we present a simple and robust materials-based strategy to induce bone regeneration in non-union defects. It is based on engineered constructs that tune the interaction between GFs and their receptors and it allows very small rhBMP-2 doses to be used (< 0.1 mg/cm3) while maintaining cell activation. This will clearly make treatments cheaper, safer and more effective. It is important to note that our approach does not tune GF delivery per se, but rather it tunes the effective presentation of growth factors to cell receptors, and hence prevents collateral risks associated with the delivery of soluble rhBMP-2 at the regenerating site. The final strategy involves the implant of a cell-free 3D construct that incorporates synthetic-biodegradable polymers, extracellular matrix proteins and rhBMP-2. The system is designed to recruit stem cells in vivo (no pre-loading the construct with stem cells needed) and to promote bone regeneration in non-union defects, as well as aiding in revascularisation of the new tissue.