Sustained regulation of hypothalamus-pituitary-ovary hormones with tissue-engineered ovarian constructs as a treatment for osteoporosis in females

组织对下丘脑-垂体-卵巢激素的持续调节 基因工程卵巢构建治疗女性骨质疏松症 项目摘要/摘要 女性受骨质疏松症和骨质疏松性骨折的影响不成比例。在美国，大约 4000万妇女有或有患上骨质疏松症的风险，200万妇女中的大多数(~60%) 骨质疏松性骨折(直接费用&GT；每年200亿美元)发生在妇女身上。女性一生中只有1/2 患骨质疏松性骨折的几率是男性的4倍。一个关键的潜在因素 原因是由于更年期或其他导致卵巢激素丢失的情况而导致卵巢衰竭 伴随而来的是下丘脑-卵巢-垂体(HPO)轴的破坏。 直到2002年，传统的药物激素疗法(PHT)被广泛使用，因为它被认为有能力 减少骨质疏松的风险，更重要的是，降低骨质疏松性骨折的风险。妇女健康倡议(WHI)得到验证 这一由来已久的信念，表明PHT降低了骨质疏松性骨折的发生率。然而，WHI也 表明激素治疗的风险，如心血管疾病和某些类型的癌症， 超过了降低骨质疏松症水平的好处。后续研究表明，风险可能更高 在60岁以上和/或绝经后10年以上的女性中，表明激素 如果以更低、更安全的剂量并通过适当的给药平台进行治疗，治疗可能仍然有效。 我们提出了仿生的卵巢细胞结构作为一种组织工程方法来传递激素(细胞 激素疗法；CHT)治疗骨质疏松症。指导这项研究的假设是，红隧可以 获得比药物制剂更好的骨骼健康和安全结果，如PHT或 双磷酸盐，在卵巢衰竭中，因为它模仿了天然的卵巢结构和功能。 我们的CHT方法使用两种关键的卵巢细胞类型(颗粒细胞和卵泡膜细胞)的空间排列， 模拟天然卵泡结构，同时避免整个卵巢组织包裹的缺陷，如 随着时间的推移，卵母细胞(卵)排出和功能丧失。我们的系统已经实现了持续和生理上的- 相关水平的卵巢激素分泌。我们已经证明了被包裹的卵巢细胞 参与HPO轴，如卵泡刺激素(FSH)和黄体生成的调节 垂体前叶的激素水平。我们的CHT构造之间有根本的区别 和天然卵巢(例如，在我们的结构中缺乏卵母细胞)，但卵巢、脑垂体，可能还有下丘脑 荷尔蒙的调节方式类似于卵巢衰竭前的状态。这些基于细胞的构造也 释放其他由天然卵巢分泌但在PHT中不存在的激素。更重要的是，红隧 以一种比PHT更安全的方式获得有益的骨骼结果。 这项研究的长期目标是开发一种基于细胞的疗法，可以用于人类 预防与女性卵巢功能丧失相关的骨质疏松症。在我们的初步研究中，我们有 通过在大鼠模型中使用同基因细胞来评估CHT。然而，为了实现临床翻译，有必要 (1)了解CHT的剂量和植入时机如何影响安全性和骨骼健康；(2)确定合适的 人类、同种异体和/或异种细胞来源的CHT以及(3)了解CHT如何在 无论是细胞层面还是全身层面。我们还将确定CHT可以分泌的最长持续时间 卵巢激素和调节HPO轴的其他激素。 拟议的实验将主要在大鼠卵巢失败的手术模型中进行 骨质疏松症(卵巢切除；去卵巢)。然而，作为临床翻译的第一步，我们还建议使用 卵巢衰竭和骨质疏松症的较大兔模型(OVX加糖皮质激素注射)。红隧将直接 与PHT和阿伦磷酸钠相比，阿伦磷酸钠是一种被广泛开出的治疗骨质疏松症的双膦酸盐药物。我们会 也将CHT(和对照组)与未经治疗的OVX动物和未接受OVX治疗的大鼠(健康， 年龄匹配的大鼠)。我们将提出三个实验目标： 目的1.确定同种异体CHT剂量和植入时机对HPO激素水平、安全性和 去卵巢大鼠模型的骨健康状况。 目的2.评估两例卵巢衰竭患者的HPO激素水平、免疫反应、安全性和骨健康 同种、同种或人类CHT治疗的骨质疏松模型。 目的3.确定同基因、异基因和人类CHT的差异基因表达谱 在体外和大鼠OVX模型中，确定关键的功能通路和任何依赖时间的变化。 这些研究的完成将证明这些组织工程构建物作为一种 关于骨质疏松症的潜在治疗方法，并提供有关其作用机制的信息。

SUSTAINED REGULATION OF HYPOTHALAMUS-PITUITARY-OVARY HORMONES WITH TISSUE- ENGINEERED OVARIAN CONSTRUCTS AS A TREATMENT FOR OSTEOPOROSIS IN FEMALES PROJECT SUMMARY/ABSTRACT Females are disproportionately affected by osteoporosis and osteoporotic fracture. In the U.S., approximately 40 million women have or are at risk of developing osteoporosis and the majority (~ 60%) of the 2 million osteoporotic bone fractures (direct costs > $20 billion per year) occur in women. Women have a 1-in-2 lifetime chance of having an osteoporotic fracture and a 4-fold higher rate of osteoporosis than men. A key underlying cause is ovarian failure due to menopause or other conditions that lead to loss of ovarian hormones with concomitant disruption of the hypothalamus-ovary-pituitary (HPO) axis. Until 2002, traditional pharmacological hormone therapy (pHT) was widely used due to its perceived ability to reduce risk of osteoporosis and, importantly, osteoporotic fracture. The Women’s Health Initiative (WHI) verified this long-held belief, demonstrating that pHT reduced incidence of osteoporotic fracture. However, the WHI also indicated that the risks of hormone therapy, such as cardiovascular disease and certain types of cancer, outweighed the benefits of reduced levels of osteoporosis. Follow-on studies indicate that risks may be higher in women older than 60 years of age and/or more than 10 years post-menopause, suggesting that hormone therapy may still be effective if given at lower, safer doses and via suitable delivery platforms. We propose biomimetic, ovarian cell constructs as a tissue engineering approach to hormone delivery (cellular hormone therapy; cHT) in the treatment of osteoporosis. The hypothesis guiding this research is that cHT can achieve better bone health and safety outcomes than pharmacological agents, such as pHT or the bisphosphonates, in ovarian failure because it mimics native ovarian structure and function. Our cHT approach uses a spatial arrangement of two key ovarian cell types (granulosa and theca cells) that mimics native ovarian follicle structure while avoiding pitfalls of whole ovarian tissue encapsulation such as oocyte (egg) expulsion and loss of function with time. Our system has achieved sustained and physiologically- relevant levels of ovarian hormone secretion. We have demonstrated that the encapsulated ovarian cells participate in the HPO axis, as evidenced by regulation of follicle stimulating hormone (FSH) and luteinizing hormone (LH) levels from the anterior pituitary. There are fundamental differences between our cHT constructs and the native ovary (e.g., lack of oocytes in our constructs) but ovarian, pituitary, and possibly hypothalamic hormones are regulated in a manner similar to a pre-ovarian failure state. These cell-based constructs also release other hormones naturally secreted by native ovaries but not present in pHT. More importantly, cHT achieved beneficial bone outcomes in a manner that was safer than pHT. The long-term goal of this research is to develop a cell-based therapy that can be used in humans for the prevention of osteoporosis associated with loss of ovarian function in women. In our preliminary studies, we have assessed cHT by using isogeneic cells in a rat model. However, to achieve clinical translation it is necessary to (1) understand how the cHT dose and timing of implantation affects safety and bone health, (2) identify suitable human, allogeneic, and/or xenogeneic cell sources for cHT and (3) understand how cHT achieves its effects at both a cellular and systemic level. We will also determine the maximum duration of time that cHT can secrete ovarian hormones and regulate other hormones of the HPO axis. The proposed experiments will be conducted primarily in a rat surgical model of ovarian failure to induce osteoporosis (ovariectomy; ovx). However, as a first step towards clinical translation we also propose the use of a larger, rabbit model of ovarian failure and osteoporosis (ovx with glucocorticoid delivery). cHT will be directly compared to pHT and to alendronate, a bisphosphonate drug widely prescribed to treat osteoporosis. We will also compare cHT (and controls) to untreated ovx animals and to rats that have not received an ovx (healthy, age-matched rats). We will propose three experimental aims: Aim 1. Determine effect of isogeneic cHT dose and implant timing on HPO hormone levels, safety and bone health in rat ovx model. Aim 2. Assess HPO hormone levels, immune response, safety and bone health in two ovarian failure models of osteoporosis with isogeneic, allogeneic or human cHT treatment. Aim 3. Determine differential gene expression (DGE) profiles for isogeneic, allogeneic, and human cHT to identify key functional pathways and any time-dependent changes in vitro and in a rat ovx model. Completion of these studies will demonstrate the safety and efficacy of these tissue-engineered constructs as a potential treatment for osteoporosis and provide information on their mechanisms of action.