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Purpose <p>To develop and validate a cartilage organoid (CO)-laden digital light processing (DLP) bioprinting strategy for auricular reconstruction and to compare its performance with conventional chondrocyte-laden prints.</p> Materials and methods <p>Rat bone-marrow stromal cells (BMSCs) were aggregated into spheroid and chondrogenically induced to form CO. Organoid construction and characterization included EdU proliferation assay, CD73/CD90 immunofluorescence (IF), and real-time quantitative polymerase chain reaction (qPCR) of chondrogenic genes. <i>O</i>-nitrobenzyl functionalized gelatin (GelNB)/ methacrylated hyaluronic acid (HAMA) bioinks were screened by gross morphology, tensile/ compressive mechanics, enzymatic degradability (0.1% collagenase II), and swelling, identifying 10% (w/v) GelNB + 1% (w/v) HAMA as the working bioink. Full-scale ears were DLP-printed from acellular, chondrocyte-laden, or CO-laden inks. In vitro, whole-mount Live/Dead imaging was performed at 7/14/21 days and qPCR quantified COL2A1, aggrecan (ACAN), COL10A1. In vivo, constructs were implanted subcutaneously in nude mice and analyzed at 4/8 weeks by Hematoxylin–eosin (H&amp;E), Safranin O/Fast Green, ACAN and COL II IHC staining, and compressive Young’s modulus measurement on implants.</p> Results <p>The 10% GelNB + 1% HAMA ink balanced print fidelity, mechanical robustness, and controlled degradability. Loading CO did not impair printability. During 21-day culture, viability remained high; at day 21, CO-laden ears showed higher COL2A1/ACAN and lower COL10A1 than chondrocyte-laden controls. After implantation, CO-laden explants exhibited more abundant lacuna-like cartilage morphology, stronger ACAN and COL II staining, increased EVG-positive elastic fiber-associated matrix, and a higher compressive Young’s modulus at 8 weeks.</p> Conclusions <p>CO-laden DLP bioprinting enhances chondrogenesis and mitigates hypertrophy versus chondrocyte-laden printing, offering an exploratory high-fidelity strategy for auricular cartilage-like tissue engineering rather than definitive mature elastic cartilage regeneration.</p>

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DLP bioprinting of cartilage organoid-laden bioinks yields high-fidelity auricular constructs with enhanced chondrogenesis

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年7月3日

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  • Yuxuan Chen,
  • Yifan Zhang,
  • Pengxiang Luo,
  • Shanshan Su,
  • Wenbin Jiang,
  • Chao Luo,
  • Xia Cai,
  • Jiaming Sun,
  • Zhenxing Wang

摘要

Purpose

To develop and validate a cartilage organoid (CO)-laden digital light processing (DLP) bioprinting strategy for auricular reconstruction and to compare its performance with conventional chondrocyte-laden prints.

Materials and methods

Rat bone-marrow stromal cells (BMSCs) were aggregated into spheroid and chondrogenically induced to form CO. Organoid construction and characterization included EdU proliferation assay, CD73/CD90 immunofluorescence (IF), and real-time quantitative polymerase chain reaction (qPCR) of chondrogenic genes. O-nitrobenzyl functionalized gelatin (GelNB)/ methacrylated hyaluronic acid (HAMA) bioinks were screened by gross morphology, tensile/ compressive mechanics, enzymatic degradability (0.1% collagenase II), and swelling, identifying 10% (w/v) GelNB + 1% (w/v) HAMA as the working bioink. Full-scale ears were DLP-printed from acellular, chondrocyte-laden, or CO-laden inks. In vitro, whole-mount Live/Dead imaging was performed at 7/14/21 days and qPCR quantified COL2A1, aggrecan (ACAN), COL10A1. In vivo, constructs were implanted subcutaneously in nude mice and analyzed at 4/8 weeks by Hematoxylin–eosin (H&E), Safranin O/Fast Green, ACAN and COL II IHC staining, and compressive Young’s modulus measurement on implants.

Results

The 10% GelNB + 1% HAMA ink balanced print fidelity, mechanical robustness, and controlled degradability. Loading CO did not impair printability. During 21-day culture, viability remained high; at day 21, CO-laden ears showed higher COL2A1/ACAN and lower COL10A1 than chondrocyte-laden controls. After implantation, CO-laden explants exhibited more abundant lacuna-like cartilage morphology, stronger ACAN and COL II staining, increased EVG-positive elastic fiber-associated matrix, and a higher compressive Young’s modulus at 8 weeks.

Conclusions

CO-laden DLP bioprinting enhances chondrogenesis and mitigates hypertrophy versus chondrocyte-laden printing, offering an exploratory high-fidelity strategy for auricular cartilage-like tissue engineering rather than definitive mature elastic cartilage regeneration.