Synergistic microenvironments for non-union bone defects

长骨骨折涉及周围组织和血管网络的损伤，因此，骨的自然愈合能力丧失，形成不愈合缺陷。在骨折中，这一临床问题的患病率为2.5%，但当有侧支软组织和血管损伤时，这一比例分别增加到15%和45%。目前的治疗方法包括自体骨移植、骨替代品和生长因子（GFs）的使用，特别是骨形态发生蛋白（BMP），但成功率有限，缺点明显。因此，对于骨不愈合骨缺损的骨再生和血管重建，开发新的治疗方法是一个尚未满足的临床需求。GFs在骨再生中的作用已得到广泛认可，但这些因子在维持其活性的同时增强组织愈合的递送尚未成功。可溶性给药或控制给药（包括水凝胶和支架）由于GFs从组织部位分解和清除而无法满足需要。然而，更重要的是，由于使用的高剂量，已经报告了灾难性的附带风险。这种不令人满意的生长因子临床转化需要强大、安全和有效的系统来控制输送。人骨形态发生蛋白-2 （BMP-2）是一种强大的生长因子，在组织形态发生中至关重要，并参与无数细胞过程，包括细胞募集、细胞分化和血管生成。重组BMP-2 （rhBMP-2）的使用已被推广到促进骨生长的广泛临床应用（脊柱，口腔颌面和创伤）。目前的临床给药是将蛋白以1.5 mg/cm3的浓度掺入胶原海绵载体中。然而，严重的临床并发症已被报道，甚至导致FDA发布了与rhBMP-2相关的危及生命的并发症（呼吸、神经、炎症）的公共卫生通知此后的报告包括观察到不受控制的骨形成和与高剂量使用相关的致癌风险。尽管在卫生机构、临床研究人员和工业界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增加。在这里，我们提出了一种简单而强大的基于材料的策略来诱导骨再生的不愈合缺陷。它基于调节GFs与其受体之间相互作用的工程结构，它允许使用非常小的rhBMP-2剂量（< 0.1 mg/cm3），同时保持细胞激活。这显然会使治疗更便宜、更安全、更有效。值得注意的是，我们的方法本身并没有调整GF的传递，而是调整了生长因子向细胞受体的有效呈现，从而防止了再生部位可溶性rhBMP-2传递相关的附带风险。最终的策略是植入一个无细胞的3D结构，该结构包含合成的可生物降解聚合物、细胞外基质蛋白和rhBMP-2。该系统的设计目的是在体内招募干细胞（不需要预先加载干细胞），促进骨不愈合缺陷的再生，以及帮助新组织的血管重建。

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.