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

下丘脑-垂体-卵巢激素与组织- 卵巢分泌物治疗女性骨质疏松症 项目概要/摘要 女性患骨质疏松症和骨质疏松性骨折的比例过高。在美国，约 4000万女性患有骨质疏松症或有患骨质疏松症的风险，200万女性中的大多数（约60%） 骨质疏松性骨折（每年直接成本> 200亿美元）发生在妇女身上。女性的一生中 患骨质疏松性骨折的几率比男性高4倍。一把钥匙 原因是卵巢衰竭，由于更年期或其他条件，导致卵巢激素的损失， 伴随下丘脑-卵巢-垂体（HPO）轴的破坏。 直到2002年，传统的药理学激素治疗（pHT）被广泛使用，因为它被认为具有 降低骨质疏松症的风险，更重要的是，降低骨质疏松性骨折的风险。妇女健康倡议（WHI） 这一长期持有的信念，证明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， 在60岁以上和/或绝经后10年以上的妇女中，表明激素 如果以较低、较安全的剂量并通过合适的递送平台给予，治疗仍然有效。 我们提出仿生卵巢细胞构建作为激素递送的组织工程方法（细胞 激素疗法（cHT）治疗骨质疏松症。指导这项研究的假设是，cHT可以 实现比药物制剂（如pHT或 双膦酸盐，在卵巢衰竭，因为它模仿天然卵巢的结构和功能。 我们的cHT方法使用两种关键卵巢细胞类型（颗粒细胞和卵泡膜细胞）的空间排列， 模仿天然卵泡结构，同时避免整个卵巢组织包裹的缺陷， 卵母细胞（卵子）排出和功能随时间丧失。我们的系统已经达到了持续的生理- 卵巢激素分泌的相关水平。我们已经证明了被包裹的卵巢细胞 参与HPO轴，如通过调节促卵泡激素（FSH）和促黄体生成 激素（LH）水平从垂体前叶。我们的cHT结构之间存在根本差异 而天然卵巢（例如，我们的构建体中缺乏卵母细胞），但卵巢、垂体和可能的下丘脑 激素以类似于卵巢衰竭前状态的方式调节。这些基于细胞的构建体还 释放由天然卵巢自然分泌但不存在于pHT中的其他激素。更重要的是， 以比pHT更安全的方式实现了有益的骨结局。 这项研究的长期目标是开发一种基于细胞的疗法，可用于人类， 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， 通过在大鼠模型中使用同基因细胞评估cHT。然而，为了实现临床翻译， (1)了解cHT剂量和植入时间如何影响安全性和骨骼健康，（2）确定合适的 cHT的人、同种异体和/或异种细胞来源，以及（3）了解cHT如何在 细胞和系统层面都是如此我们还将确定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卵巢衰竭的大鼠手术模型中进行，以诱导 骨质疏松症（卵巢切除术; OVX）。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更大兔子模型（OVX联合糖皮质激素递送）。cHT将直接 与pHT和阿仑膦酸钠（一种广泛用于治疗骨质疏松症的双膦酸盐药物）相比。我们将 还将cHT（和对照）与未处理的OVX动物和未接受OVX的大鼠（健康， 年龄匹配的大鼠）。我们将提出三个实验目标： 目标1。确定等基因cHT剂量和植入时间对HPO激素水平、安全性和 大鼠OVX模型的骨健康。 目标2.评估两例卵巢功能衰竭患者的HPO激素水平、免疫反应、安全性和骨健康 用同基因、同种异体或人cHT治疗的骨质疏松症模型。 目标3。确定同基因、同种异体和人cHT的差异基因表达（DGE）谱 在体外和大鼠OVX模型中鉴定关键功能途径和任何时间依赖性变化。 这些研究的完成将证明这些组织工程构建物作为一种 潜在的治疗骨质疏松症，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.