Customized MSCs to Enhance Healing of Bone Defects

创伤、感染、照射、磨粒病所致大段残存长骨缺损的修复 而其他情况仍然是一个悬而未决的临床挑战。这些骨缺损通常不会愈合，因为它们 大小，骨膜和骨内膜损伤，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黄金标准，然而这一来源可以是 在数量或质量上有限，并伴随着收获地点的发病。另一种方法是 将自体采集和浓缩的祖细胞与适当的支架结合起来，提供 促进骨骼愈合的元素。我们最近发现，脂多糖对骨髓间充质干细胞的预适应 和肿瘤坏死因子-α(肿瘤坏死因子-α)诱导NF-κB的急性瞬时激活，促进成骨，并 提高MSCs将巨噬细胞从促炎因子(肿瘤坏死因子-α)极化为更多 良好的抗炎促组织愈合(精氨酸酶1，CD206，IL1ra高)表型。我们还有 转基因间充质干细胞(以下简称GM-MSCs)过表达抗炎、促组织 修复分子白介素4(IL-4)，并进一步创建了一个首次检测NF-κB激活和 然后增加IL-4的产生(NF-κB感应和分泌IL-4的GM-MSCs)。这些GM MSC将是 对于也存在低度亚急性/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 在独特的可注射大孔中使用预条件MSCs和GM MSCs进行免疫调节 支架，直接移植到骨缺损处。 具体目标#1：确定移植预适应的MSCs、GM MSCs和 经新型可注射大孔水凝胶支架移植的GM MSCs优于自体骨移植 关于体内愈合：SA1a：一种急性临界大小的小鼠长骨缺损；SA1b：一种慢性临界大小 小鼠长骨缺损区的实验研究 具体目标2：论证上述促进急、慢性长寿愈合的原理 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：比较最有希望的骨髓间充质干细胞治疗与自体骨移植 挑战大型和老年动物的慢性临界尺寸缺陷：雄性和雌性老年新西兰大白兔。 我们预计，在新型支架中添加预适应或GM MSCs将导致类似的骨愈合 牺牲，与添加自体骨相比，但优于添加未改变的MSCs。通过以下方式治疗 预适应或转基因MSCs具有高度的创新性、机械性和直接转化为创伤性和 获得性急性和慢性长骨缺损，可能是一种有效的、侵入性较小的替代方法 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.