A Nondestructive Raman Spectral Method for Temporal Tracking of Articular Cartilage Maturation

Abstract

Because articular cartilage (AC) lacks inherent repair capacity, research has focused on translating tissue-engineered cartilage to the clinic. Toward this, rapid and nondestructive methods would be useful for determining in-process and release characteristics during the manufacture of tissue-engineered products. The current work aims to introduce a Raman-based methodology for nondestructive qualitative and quantitative characterization of tissue development using AC. First, Raman shifts associated with critical biochemical components of AC, with particular emphasis on DNA, glycosaminoglycans (long chains of sugar molecules and a key component of cartilage), total collagen, as well as pyridinoline (a marker of collagen crosslinking and maturation), were collated. Next, verification of the molecular spectroscopic biomarkers was conducted by temporally tracking tissue maturation/development of nascent and mature AC, establishing a temporal reference dataset. Finally, validation was performed by correlating the spectroscopic biomarkers with traditional photometric biochemical assays and mass spectrometry. The results presented here include a Maturity Index for quantification of tissue development/maturation. Strong correlations were found between nondestructive spectroscopic-based measurements and destructive (photometric and mass spectrometric) measurements with high linearity for both nondestructive Raman (R² > 0.96) and destructive biochemical (R² > 0.97) assays, respectively. Uses of the proposed rapid and nondestructive method include in-line quality assessment (in which the sample is not removed from the process stream) to monitor the manufacturing of tissue-engineered medical products. This study shows that Raman spectroscopy has the capacity of being a powerful tool for nondestructive quality control and assurance in traditional biomanufacturing workflows, and the approach taken here may also be utilized as a template and research tool for studies on the development of other native and engineered tissues.

Impact Statement

This work introduces a rapid and nondestructive Raman spectroscopy method for biochemical analysis of native cartilage, validated against gold-standard destructive assays. This approach enabled the development of a novel cartilage-specific Maturity Index, which integrates collagen, glycosaminoglycan, and crosslink content into a single nondestructive metric of tissue development. By eliminating destructive sampling and by yielding data rapidly, Raman spectroscopy allows continuous monitoring and quality assurance/quality control of tissue-engineered medical products, conserving resources and enabling fast and reliable release assays. This methodology establishes a scalable, multiplexable platform with strong translational potential for tissue engineering and regenerative medicine.