Two-step Gelatin Embedding Preserves Fibrocartilage Morphology, Enthesis, and Cartilage Stain of Bone Joints

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Abstract
Maintaining the native morphology and characteristic staining patterns of dissected tissues is critical for histological analysis. Reliable preservation enables accurate assessment of structural integrity and cellular components, which is fundamental to identifying viable avenues for tissue regeneration and improving clinical outcomes. Acknowledging the constraints of conventional paraffin embedding, we propose an alternative method employing gelatin as an embedding medium. Gelatin is a natural, water-soluble protein derived from collagen, known for its biocompatibility. As a hydrogel, it provides a supportive matrix that closely mimics the natural extracellular matrix of soft tissues facilitating excellent preservation of structural and molecular features during the histological process. Here we demonstrate that this strategy offers superior support for fragile tissues, including fibrocartilage and hyaline cartilage across various anatomical sites, such as the temporomandibular joint, the knee joint, the long bone growth plate, and the intervertebral disc. To assess the maintenance of tissue morphology and matrix composition, histological sections were subjected to various staining techniques, including hematoxylin and eosin, Masson’s trichome, safranin O, immunofluorescence, and von Kossa staining. Gelatin embedding resulted in superior maintenance of fibrocartilage architecture, as evidenced by histological evaluation. In addition, quantification of safranin O staining showed significantly greater glycosaminoglycan content in gelatin-embedded samples.
Funding Information
  • DOD | Department of Defense Education Activity (W81XWH2010572)
  • HHS | NIH | National Institute of Dental and Craniofacial Research (R01DE027662)
  • HHS | NIH | National Institute of Dental and Craniofacial Research (R01DE020843)
  • National Heart, Lung, and Blood Institute (R01HL162939)
  • Fulbright Brazil (88881.625411/2021-01)

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