Customized MSCs to Enhance Healing of Bone Defects

与创伤、感染、放射、磨粒病相关的大段残余长骨缺损的愈合 和其他病症仍然是未解决的临床挑战。这些骨缺损通常不会愈合，由于它们的 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金标准，然而，这种来源可以是 数量或质量有限，并伴有收获地点的发病率。另一种方法是 联合收割机将自体收获和浓缩的祖细胞与适当的支架结合， 骨愈合的元素。我们最近发现，用脂多糖（LPS）预处理MSCs， 和肿瘤坏死因子-α（TNF-α）诱导NF-κB的急性瞬时激活，增强骨生成， 提高了MSC对巨噬细胞的杀伤能力，从促炎性（TNF-α+，iNOS+）到更强的 有利的抗炎促组织愈合（精氨酸酶1+、CD 206+、IL 1 Ra高）表型。我们还 遗传修饰的MSC（下文称为GM MSC），以过表达抗炎的、促组织分化的MSC（下文称为GM MSC）。 愈合分子白细胞介素-4（IL-4），并进一步创建了一个构建体，以首先检测NF-κB激活， 然后增加IL-4的产生（NF-κB传感和IL-4分泌的GM MSC）。这些GM MSC将是 对于同时存在低度亚急性/慢性炎症的骨缺损非常有益， 临床上经常见到。该补助金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的免疫调节，在独特的可注射大孔 支架，直接移植到骨缺损部位。 具体目标#1：确定移植的预处理的MSC、GM MSC和预处理的MSC是否与移植的MSC、GM MSC和GM MSC的细胞相容。 通过新型可注射大孔水凝胶支架递送的GM MSC优于自体移植骨， 关于体内愈合：SA 1a：急性临界尺寸鼠长骨缺损; SA 1b：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创伤愈合的原则 骨缺损对以下小鼠有效：SA 2a：雄性和雌性小鼠; SA 2b：较年轻和较年长小鼠 具体目标#3：比较最有前途的MSC治疗与自体骨移植， 在较大和较老动物中挑战慢性临界尺寸缺陷：雄性和雌性新西兰白色老年兔。 我们希望在新的支架中加入预处理的或GM MSC将导致类似的骨愈合， 与添加自体移植骨相比，牺牲，但上级优于添加未改变的MSC。治疗 引发的预处理或GM MSC是高度创新的，机械的，并直接转化为创伤性的， 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.