Forskolin-enhanced microfracture to regenerate hyaline cartilage in chondral defect

毛喉素增强微骨折再生软骨缺损中的透明软骨

• 批准号: 10727123
• 负责人: Hang Lin
• 金额: $ 38.48万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10727123
• 关键词: Actins; Address; Adenylate Cyclase; Animal Model; Biochemical; Blood; Bone Marrow; Cartilage; Cartilage injury; Cells; Chondrocytes; Chondrogenesis; Clinic; Clinical; Clinical Research; Collagen Type I; Collagen Type II; Collagen Type X; Comparative Study; Defect; Deposition; Diameter; Disease; Dose; Encapsulated; Enhancement Technology; Exposure to; Fibrocartilages; Fibrosis; Forskolin; Fracture; Generations; Genetic Transcription; Glycolates; Glycosaminoglycans; Histologic; Human; Hyaline Cartilage; Hypertrophy; Implantable Infusion Pumps; In Situ; In Vitro; Infection; Injections; Injury; Intra-Articular Injections; Joints; Knee; Knee joint; Magnetic Resonance Imaging; Mechanics; Methods; Modeling; Modification; Natural regeneration; Nature; Operative Surgical Procedures; Outcome; Pharmaceutical Preparations; Phenotype; Polymers; Procedures; Property; Proteins; Publications; Publishing; Rattus; Reporting; Side; Site; Smooth Muscle; Testing; Time; Tissues; Treatment Efficacy; Visit; angiogenesis; articular cartilage; bone; controlled release; cost; efficacy testing; healing; inhibitor; joint stiffness; mechanical properties; minimally invasive; next generation; osteochondral tissue; particle; preclinical study; premature; prevent; protein activation; repaired; screening; severe injury; stem cells; subchondral bone; tissue regeneration; wound

Abstract When critically injured, articular cartilage has an extremely limited capacity for self-healing. Currently, there are several surgical procedures available to treat knee cartilage defects. Given its minimally invasive nature, low cost, and beneficial short-term outcomes, microfracture (MFx), a procedure creating small fractures in the bone, is often considered as the first‐line procedure. However, neotissue in the defect site post-MFx is ultimately remodeled into fibrocartilage, which possesses different biochemical and mechanical properties compared with normal hyaline cartilage, predisposing the repair site to degeneration. Consequently, MFx repairs often fail within several years. Although some MFx procedural modifications have been proposed, evidence regarding their efficacy in humans is lacking. Recently, we found Yes-associated protein (YAP) is highly expressed in the regenerated tissue in the rat knee joint after osteochondral injury, but not in the middle zone of undamaged native cartilage. We then conducted an in vitro screening and found that forskolin, a biosafe herbal compound that stimulates adenylate cyclase activity and indirectly inhibits YAP activation, was able to significantly suppress the transcription and generation of collagen type I (COL1) and alpha-smooth muscle actin (α-SMA) (fibrocartilage markers) , collagen type X (COL10, chondrocyte hypertrophy marker), without compromising the generation of collagen type II (COL2) and glycosaminoglycans (major cartilage components). Therefore, herein we hypothesize that post-MFx forskolin introduction will significantly promote hyaline cartilage formation, thus enhancing the long-term chondral repair outcome after MFx. In this study, we will test the efficacy of two different methods to administer forskolin: repeated injection of forskolin solution (Aim 1), and one-time injection of forskolin preloaded in microparticles for long-term and controlled release (Aim 2). Specifically, in Aim 1, after creating MFx in the knee joint of rats, different doses of forskolin will be injected into the knee joint at weeks 0, 2, and 4 (a total of 3 injections). Eight weeks after surgery, the quality of newly formed tissues will be characterized. In Aim 2, we will generate poly(lactic-co-glycolic acid) (PLGA) microparticles for long-term and controlled release of forskolin, which avoids complications due to repeated injections, such as infection and unnecessary systemic exposure to high dose of drugs. Lastly, several potential MFx-enhancing methods had been previously reported in different publications, but a comparative study to side-by-side assess their efficacy has not been reported. As the first step to address the issue, we will also compare the outcome of the best forskolin treatment conditions, defined from studies in Aims 1&2, to that of a previously published MFx-enhancing technology, specifically the angiogenesis-targeting strategy. By repurposing the biosafe forskolin, the next generation of MFx developed here would provide a minimally invasive and cost-affordable procedure to achieve robust and long-term chondral defect repair, with the neocartilage possessing native tissue cellular phenotype, biochemical composition, and mechanical properties.

摘要严重损伤时，关节软骨的自我修复能力极其有限。目前， 有几种外科手术可用于治疗膝关节软骨缺陷。鉴于其微创的性质， 微骨折(MFX)是一种成本低、短期效果好的微骨折治疗方法。 骨移植，通常被认为是第一线手术。然而，MFX术后缺损处的肿瘤组织最终是 改建成纤维软骨，其具有不同的生化和力学性能 正常的透明软骨，易使修复部位变性。因此，MFX修复通常在 好几年了。尽管已经提出了一些最惠国待遇的程序修改，但关于其 在人类身上缺乏疗效。最近，我们发现YAP(Yes-Associated Protein，YAP)在血管内皮细胞中高表达。 大鼠膝关节骨软骨损伤后再生组织，但不在未损伤的中段 天然软骨。然后我们进行了体外筛选，发现Forsklin，一种生物安全草本化合物 刺激腺苷环化酶活性并间接抑制YAP激活，能够显著抑制 I型胶原(COL1)和α-平滑肌肌动蛋白(α-SMA)(纤维软骨)的转录和生成 标志物)、X型胶原(COL10，软骨细胞肥大标志物)，而不影响 II型胶原(COL2)和糖胺多糖(软骨的主要成分)。因此，在此，我们 假设MFX术后Forsklin的引入将显著促进透明软骨的形成，因此 提高MFX术后软骨修复的远期疗效。在这项研究中，我们将测试两种不同的 福斯科林给药方法：多次注射福斯科林溶液(目标1)，一次性注射福斯科林 预装在微粒中，用于长期和受控释放(目标2)。具体地说，在目标1中，在创建MFX之后 在大鼠膝关节内，于第0、2、4周(共4周)向膝关节内注射不同剂量的Forsklin 3次注射)。手术8周后，新形成的组织的质量将得到鉴定。在目标2中，我们 将产生聚(乳酸-乙醇酸)(PLGA)微粒，用于Forsklin的长期和受控释放， 避免因反复注射而引起的并发症，如感染和不必要的全身暴露 大剂量的毒品。最后，几种潜在的增强MFX的方法以前曾在不同的 但一项并列评估其疗效的比较研究尚未见报道。作为第一个 为解决这一问题，我们还将比较福斯科林最佳治疗条件的结果，确定 从AIMS 1和AIMS 2中的研究，到之前发布的MFX增强技术的研究，特别是 血管生成靶向策略。通过改变生物安全Forsklin的用途，开发出了下一代MFX 这将提供一个最小的创伤和成本负担得起的程序，以实现强大和长期的软骨 缺陷修复，新生组织具有天然的组织细胞表型、生化成分和 机械性能。