Hixon Katherine R, Katz Dakota B, McKenzie Jennifer A, Miller Anna N, Guilak Farshid, Silva Matthew J
Department of Orthopaedic Surgery, Musculoskeletal Research Center, Washington University, St. Louis, MO, United States.
Thayer School of Engineering, Dartmouth College, Hanover, NH, United States.
Front Bioeng Biotechnol. 2022 May 5;10:851904. doi: 10.3389/fbioe.2022.851904. eCollection 2022.
Non-union is defined as the permanent failure of a bone to heal and occurs clinically in 5% of fractures. Atrophic non-unions, characterized by absent/minimal callus formation, are poorly understood and difficult to treat. We recently demonstrated a novel murine model of atrophic non-union in the 3.6Col1A1-tk (Col1-tk) mouse, wherein dosing with the nucleoside analog ganciclovir (GCV) was used to deplete proliferating osteoprogenitor cells, leading to a radiographic and biomechanical non-union after the mid-shaft femur fracture. Using this Col1-tk atrophic non-union model, we hypothesized that the scaffold-mediated lentiviral delivery of doxycycline-inducible BMP-2 transgenes would induce osteogenesis at the fracture site. Cryogel scaffolds were used as a vehicle for GFP+ and BMP-2+ cell delivery to the site of non-union. Cryogel scaffolds were biofabricated through the cross-linking of a chitosan-gelatin polymer solution at subzero temperatures, which results in a macroporous, spongy structure that may be advantageous for a bone regeneration application. Murine adipose-derived stem cells were seeded onto the cryogel scaffolds, where they underwent lentiviral transduction. Following the establishment of atrophic non-unions in the femurs of Col1-tk mice (4 weeks post-fracture), transduced, seeded scaffolds were surgically placed around the site of non-union, and the animals were given doxycycline water to induce BMP-2 production. Controls included GFP+ cells on the cryogel scaffolds, acellular scaffolds, and sham (no scaffold). Weekly radiographs were taken, and endpoint analysis included micro-CT and histological staining. After 2 weeks of implantation, the BMP-2+ scaffolds were infiltrated with cartilage and woven bone at the non-union site, while GFP+ scaffolds had woven bone formation. Later, timepoints of 8 weeks had woven bone and vessel formation within the BMP-2+ and GFP + scaffolds with cortical bridging of the original fracture site in both groups. Overall, the cell-seeded cryogels promoted osseous healing. However, while the addition of BMP-2 promoted the endochondral ossification, it may provide a slower route to healing. This proof-of-concept study demonstrates the potential for cellularized cryogel scaffolds to enhance the healing of non-unions.
骨不连被定义为骨折后骨的永久性愈合失败，临床上发生率为5%。萎缩性骨不连的特征是骨痂形成缺失或极少，目前对其了解甚少且治疗困难。我们最近在3.6Col1A1 - tk（Col1 - tk）小鼠中建立了一种新型的萎缩性骨不连小鼠模型，其中通过给予核苷类似物更昔洛韦（GCV）来消耗增殖的骨祖细胞，导致股骨中段骨折后出现影像学和生物力学上的骨不连。利用这个Col1 - tk萎缩性骨不连模型，我们推测通过支架介导的强力霉素诱导型BMP - 2转基因慢病毒递送将在骨折部位诱导成骨。冷冻凝胶支架被用作将GFP +和BMP - 2 +细胞递送至骨不连部位的载体。冷冻凝胶支架是通过壳聚糖 - 明胶聚合物溶液在零下温度下交联生物制造而成，形成一种大孔海绵状结构，这可能有利于骨再生应用。将小鼠脂肪来源的干细胞接种到冷冻凝胶支架上，在那里它们接受慢病毒转导。在Col1 - tk小鼠股骨中建立萎缩性骨不连（骨折后4周）后，将转导并接种了细胞的支架手术放置在骨不连部位周围，并给动物饮用强力霉素水以诱导BMP - 2产生。对照组包括冷冻凝胶支架上的GFP +细胞、无细胞支架和假手术组（无支架）。每周进行X线摄影，终点分析包括微型计算机断层扫描（micro - CT）和组织学染色。植入2周后，BMP - 2 +支架在骨不连部位有软骨和编织骨浸润，而GFP +支架有编织骨形成。后来，在8周的时间点，BMP - 2 +和GFP +支架内均有编织骨和血管形成，两组原骨折部位均有皮质桥接。总体而言，接种了细胞的冷冻凝胶促进了骨愈合。然而，虽然添加BMP - 2促进了软骨内成骨，但它可能提供了一条较慢的愈合途径。这项概念验证研究证明了细胞化冷冻凝胶支架增强骨不连愈合的潜力。
