Customized MSCs to Enhance Healing of Bone Defects

与创伤、感染、辐射、磨损颗粒病相关的大残留长骨缺损的愈合 和其他条件仍然是一个未解决的临床挑战。这些骨缺损通常不会愈合，因为 大小、骨膜和内膜损伤以及骨祖细胞和血管数量不足 祖细胞。自体骨移植是治疗的金标准，但是这种来源可以 数量或质量有限，并伴有收获现场的发病率。另一种方法是 将自体收获和浓缩的祖细胞与适当的支架结合起来，以提供 骨骼愈合的元素。我们最近表明，用脂多糖（LPS）预处理 MSC 和肿瘤坏死因子-α (TNF-α) 诱导 NF-κB 的急性瞬时激活，增强成骨作用，并且 提高间充质干细胞将巨噬细胞从促炎细胞（TNF-α、iNOS）极化为更强大的细胞的能力 有利的抗炎促组织愈合（精氨酸酶 1、CD206、IL1Ra 高）表型。我们还有 转基因 MSC（以下称为 GM MSC）过度表达抗炎、促组织细胞 修复分子白细胞介素 4 (IL-4)，此外还创建了一个构建体来首先感知 NF-κB 激活并 然后增加 IL-4 的产生（NF-κB 感应和分泌 IL-4 的 GM MSC）。这些 GM MSC 将是 对于还存在低度亚急性/慢性炎症的骨缺损非常有益，这是一种情况 临床上常见。这笔赠款的目的是通过以下方式加速长骨缺损的愈合 使用预处理的 MSC 和 GM MSC 在独特的可注射大孔中进行免疫调节 支架，直接移植到骨缺损部位。 具体目标#1：确定移植的预处理 MSC、GM MSC 和预处理是否 通过新型可注射大孔水凝胶支架输送的 GM MSC 比自体移植骨更好 关于以下体内愈合： SA1a：急性临界大小的小鼠长骨缺损； SA1b：慢性临界尺寸 小鼠长骨缺损 具体目标#2：证明上述促进急性和慢性长期疾病愈合的原则 骨缺陷适用于： SA2a：雄性和雌性小鼠； SA2b：更年轻和更年长的小鼠 具体目标#3：以更广泛的方式比较最有前途的 MSC 治疗与自体移植骨 大型和老年动物中具有挑战性的慢性临界尺寸缺陷：雄性和雌性老年新西兰白兔。 我们预计，在新型支架中添加预处理或 GM MSC 将导致类似的骨愈合 与添加自体移植骨相比，牺牲的效果更好，但优于添加未改变的 MSC。治疗用 经引发的预处理或 GM MSC 具有高度创新性、机械性并可直接转化为创伤性和 人类获得性急性和慢性长骨缺陷，可能是一种有效的、侵入性较小的替代方案 传统的骨移植技术。

Healing of large residual long bone defects associated with trauma, infection, irradiation, wear particle disease and other conditions is still an unsolved clinical challenge. These bone defects often will not heal due to their size, injury to the periosteum and endosteum, and insufficient numbers of osteoprogenitor and vascular progenitor cells. Autologous bone grafting is the gold standard for treatment, however this source can be limited in quantity or quality, and accompanied by morbidity at the harvest site. Another approach is to combine autologous harvested and concentrated progenitor cells with an appropriate scaffold to provide the elements for bone healing. We recently showed that preconditioning of MSCs with lipopolysaccharide (LPS) and tumor necrosis factor-α (TNF-α) to induce acute transient activation of NF-κB enhances osteogenesis, and improves MSCs' ability to polarize macrophages from a pro-inflammatory (TNF-α+, iNOS+) to a more favorable anti-inflammatory pro-tissue healing (Arginase 1+, CD206+, IL1Ra high) phenotype. We have also genetically modified MSCs (hereafter termed GM MSCs) to over express the anti-inflammatory, pro-tissue healing molecule Interleukin-4 (IL-4), and furthermore created a construct to first sense NF-κB activation and then increase production of IL-4 (NF-κB sensing and IL-4 secreting GM MSCs). These GM MSC would be very beneficial for bone defects in which low-grade subacute/chronic inflammation is also present, a scenario frequently seen clinically. The purpose of this grant is to accelerate the healing of long bone defects via immune modulation using preconditioned MSCs and GM MSCs, in a unique injectable macroporous scaffold, transplanted directly to the bone defect site. Specific Aim #1: To determine whether transplanted preconditioned MSCs, GM MSCs, and preconditioned GM MSCs delivered via a novel injectable macroporous hydrogel scaffold are better than autograft bone with respect to in vivo healing of: SA1a: an acute critical size murine long bone defect; SA1b: a chronic critical size murine long bone defect Specific Aim #2: To demonstrate that the above principles of enhancing the healing of acute and chronic long bone defects are valid for: SA2a: male and female mice; SA2b: younger and more elderly mice Specific Aim #3: To compare the most promising of the MSC treatments with autograft bone in a more challenging chronic critical size defect in larger and older animals: male and female elderly NZ white rabbits. We expect that addition of preconditioned or GM MSCs in a novel scaffold will lead to similar bone healing at sacrifice, compared to addition of autograft bone, but superior to addition of unaltered MSCs. Treatment with primed preconditioned or GM MSCs is highly innovative, mechanistic and directly translational to traumatic and acquired acute and chronic long bone defects in humans, and may be an effective, less invasive alternative to conventional bone graft techniques.