Introduction and Background

• This study tackles the challenge of limb regeneration in adult vertebrates using adult Xenopus laevis frogs.
• Normally, after hindlimb amputation, adult frogs regenerate only a simple, underdeveloped cartilaginous spike.
• The goal is to improve this regenerative outcome by using a wearable bioreactor that delivers progesterone directly to the wound.
• Progesterone is a hormone known for its role in nerve repair and tissue remodeling; it can also influence the electrical state of cells (bioelectricity).

Device and Treatment Approach

• A wearable bioreactor is designed with a silk protein-based hydrogel loaded with progesterone.
• The device is applied locally to the amputated hindlimb for just 24 hours.
• This brief, targeted exposure increases progesterone levels only at the injury site, acting like a “kick-start” for the regeneration process.

Experimental Design and Methods

• Subjects: Adult Xenopus laevis frogs with hindlimb amputations.
• Groups:
  • Progesterone-device group (with drug),
  • Sham group (device only, no drug), and
  • Untreated control group.
• Assessments included molecular markers, X-ray imaging, immunofluorescence, histology, and behavioral assays.
• Multiple timepoints were examined from early stages (0.5 months) to late stages (up to 9.5 months) post-amputation.

Key Observations and Cellular Responses

• Progesterone receptors were confirmed in adult frog limb tissues, particularly in bone marrow cells.
• After 24 hours of device treatment, progesterone levels were significantly higher at the amputation site.
• Early cellular changes observed:
  • Reduced invasion of immune cells (leukocytes) at the wound edge, leading to scar-free healing.
  • Enhanced organization and increased numbers of regenerating nerves and blood vessels.

Anatomical and Morphological Outcomes

• In untreated frogs, regeneration resulted in a simple, hypomorphic cartilage spike.
• Frogs treated with the progesterone device developed complex, paddle-like structures with broader and more organized morphology.
• Key differences include:
  • Greater changes in tissue width and a larger unpigmented epithelial area,
  • Significant bone remodeling and reorganization that suggests the beginnings of joint-like structures.
• Morphometric analysis confirmed that treated regenerates had a distinct and improved shape compared to controls.

Functional Outcomes

• Behavioral tests showed that frogs with treated, paddle-like regenerates exhibited activity levels and swimming behaviors similar to uncut (normal) frogs.
• Specifically, treated frogs:
  • Were more active,
  • Displayed better coordinated movements, and
  • Utilized the regenerated limb effectively during swimming.

Molecular and Transcriptome Analysis

• RNA sequencing of the regeneration tissue (blastema) revealed:
  • Over 500 differentially expressed genes in the progesterone-device group compared to controls,
  • Upregulation of genes related to nuclear signaling, oxidative stress regulation, and ion channel modulation, and
  • Downregulation of genes involved in neurotransmission and cell ion flux, focusing the cellular response on regeneration.
• Pathway analysis indicated enrichment of regenerative processes including blood vessel formation, immune regulation, and nerve patterning.
• This suggests that the 24-hour progesterone treatment initiates a cascade of long-lasting transcriptional changes that support regeneration.

Discussion and Conclusions

• The brief, local application of progesterone via a wearable bioreactor dramatically improved the regenerative outcome in adult frogs.
• The treatment reactivates latent regenerative programs, leading to:
  • Complex, paddle-like anatomical structures instead of simple spikes, and
  • Enhanced functional recovery with improved movement and swimming.
• The molecular data support that a short exposure can trigger sustained, long-term regenerative responses.
• This approach offers promise for targeted regenerative therapies in non-regenerative animals and may inform future strategies in human regenerative medicine.
• Future work will focus on refining the device’s contact and understanding genetic factors that influence individual responses.

Step-by-Step Summary (Cooking Recipe Style)

• Step 1: Amputate the hindlimb of an adult frog and immediately attach the wearable bioreactor loaded with progesterone.
• Step 2: Leave the device in place for 24 hours to deliver a high concentration of progesterone directly to the injury.
• Step 3: Remove the device and observe early cellular responses, such as reduced immune cell infiltration and initiation of scar-free healing.
• Step 4: Over the following weeks to months, monitor the limb as it transforms from a simple spike to a complex, paddle-like structure.
• Step 5: Use imaging and molecular assays to measure changes in bone structure, nerve organization, and gene expression.
• Step 6: Evaluate functional recovery through behavioral tests (e.g., swimming activity) comparing treated frogs to untreated ones.
• Step 7: Analyze transcriptome data to identify key genes and pathways activated by the treatment.
• Step 8: Conclude that a brief, localized progesterone treatment successfully kick-starts a sustained regenerative process.

Key Terms Defined

• Progesterone: A hormone that promotes nerve repair and tissue remodeling, influencing cell behavior.
• Bioreactor: An engineered device that creates a controlled environment—in this case, for local drug delivery.
• Blastema: A cluster of cells at the wound site capable of growth and regeneration, acting like a repair kit.
• Hypomorphic spike: A rudimentary, underdeveloped structure that typically forms in untreated adult frog limb regeneration.
• Transcriptome: The complete set of RNA transcripts produced by the genome, used to study changes in gene expression.

观察到的情况和研究背景 (中文简介)

• 本研究旨在解决成年脊椎动物肢体再生难题，采用成年非洲爪蟾 (Xenopus laevis) 作为实验对象。
• 通常情况下，成年蟾蜍在截肢后只能再生出简单的不完全软骨尖。
• 研究目标是利用可穿戴生物反应器局部输送孕酮，改善伤口的再生效果。
• 孕酮是一种促进神经修复和组织重塑的激素，同时还能影响细胞的生物电状态。

设备和治疗方法 (中文)

• 使用一种可穿戴生物反应器，该设备内含丝蛋白基水凝胶，并预加载孕酮。
• 在后肢截肢后立即将设备贴附于伤口部位，持续24小时。
• 设备将孕酮直接输送到伤口区域，不影响其他组织。
• 这种短暂而局部的治疗就像是给系统一个“启动信号”，激活长期的再生过程。

实验设计与方法 (中文)

• 实验对象：成年Xenopus laevis蟾蜍，其后肢被截肢。
• 分组：
  • 孕酮设备组（含药物）、
  • 假处理组（仅使用设备，无药物）以及
  • 未处理对照组。
• 评估方法包括分子标记、X光成像、免疫荧光、组织学及行为测试。
• 多个时间点（从截肢后0.5月至9.5月）被用来观察再生过程。

主要观察及细胞反应 (中文)

• 确认孕酮受体存在于成年蟾蜍肢体组织中，尤其集中在骨髓细胞中。
• 24小时治疗后，伤口处的孕酮水平显著升高。
• 早期细胞反应包括：
  • 伤口边缘免疫细胞（白细胞）浸润减少，促进无瘢痕愈合；
  • 再生神经及血管组织增多且排列更有序。

解剖和形态学结果 (中文)

• 对照组蟾蜍再生出的仅为简单的、发育不全的软骨尖。
• 孕酮设备处理组则再生出复杂的、类似桨状的结构，形态更宽且组织更整齐。
• 主要差异体现在：
  • 再生组织宽度变化更明显，无色素上皮比例更高；
  • 骨组织重塑与重新组织，暗示形成类似关节的结构。
• 形态测量分析证实处理组的再生形状显著优于对照组。

功能性结果 (中文)

• 行为测试显示，再生出桨状结构的处理组蟾蜍活动性和游泳表现接近未截肢的正常蟾蜍。
• 具体表现为：
  • 活动量增多，运动协调性更好，
  • 再生肢体被更有效地用于游泳等活动。

分子与转录组分析 (中文)

• RNA测序显示：
  • 与对照组相比，孕酮设备组的再生芽中有超过500个基因表达发生显著变化；
  • 与核信号传导、抗氧化应激及离子通道调控相关的基因上调；
  • 与神经传导和细胞离子流相关的基因下调，从而使细胞专注于再生。
• 通路分析发现多条再生相关通路被激活，包括血管生成、免疫调控及神经模式形成。
• 这些结果表明，短暂的孕酮治疗启动了一系列长期维持的转录变化，支持再生。

讨论与结论 (中文)

• 本研究证明，利用可穿戴生物反应器局部短暂输送孕酮，可显著改善成年蟾蜍肢体再生效果。
• 该治疗方法重新激活了潜在的再生程序，使得：
  • 再生出的结构由简单的软骨尖转变为复杂的桨状形态，
  • 功能上得到改善，运动和游泳能力提升。
• 分子数据显示短暂治疗可触发长期再生相关的转录变化，减少瘢痕形成。
• 这一方法为针对非再生动物的定向再生疗法提供了新思路，并对未来人类再生医学具有启示意义。
• 未来工作将进一步改进设备接触及探究影响个体响应的基因因素。

逐步总结 (烹饪食谱式) (中文)

• 步骤1: 对成年蟾蜍后肢进行截肢，并立即贴附预加载孕酮的可穿戴生物反应器。
• 步骤2: 保持设备贴附24小时，将高浓度孕酮直接输送到伤口。
• 步骤3: 移除设备，观察早期细胞变化，如白细胞减少及无瘢痕愈合迹象。
• 步骤4: 在接下来的几周到数月内，观察肢体从简单尖状结构向复杂桨状结构转变。
• 步骤5: 通过影像和分子检测，定量分析骨结构、神经组织及基因表达的变化。
• 步骤6: 进行行为测试，比较处理组与对照组在游泳和运动中的表现。
• 步骤7: 分析转录组数据，找出治疗后激活的关键基因和通路。
• 步骤8: 总结出短暂的局部孕酮治疗成功触发并维持了长期的再生过程。

关键术语定义 (中文)

• 孕酮: 促进神经修复和组织重塑的激素，可调控细胞行为。
• 生物反应器: 一种工程装置，用于提供受控环境，本研究中用于局部药物输送。
• 再生芽: 位于伤口处的一团具有再生能力的细胞，相当于体内的“修复工具包”。
• 发育不全的软骨尖: 成年蟾蜍截肢后通常再生出的简单、不完整的结构。
• 转录组: 基因组中所有RNA转录物的总和，用于研究基因表达的变化。