Quality control methods in musculoskeletal tissue engineering: from imaging to biosensors

Abstract

Tissue engineering is rapidly progressing toward clinical application. In the musculoskeletal field, there has been an increasing necessity for bone and cartilage replacement. Despite the promising translational potential of tissue engineering approaches, careful attention should be given to the quality of developed constructs to increase the real applicability to patients. After a general introduction to musculoskeletal tissue engineering, this narrative review aims to offer an overview of methods, starting from classical techniques, such as gene expression analysis and histology, to less common methods, such as Raman spectroscopy, microcomputed tomography, and biosensors, that can be employed to assess the quality of constructs in terms of viability, morphology, or matrix deposition. A particular emphasis is given to standards and good practices (GXP), which can be applicable in different sectors. Moreover, a classification of the methods into destructive, noninvasive, or conservative based on the possible further development of a preimplant quality monitoring system is proposed. Biosensors in musculoskeletal tissue engineering have not yet been used but have been proposed as a novel technology that can be exploited with numerous advantages, including minimal invasiveness, making them suitable for the development of preimplant quality control systems.

Fig. 1

Analysis of cell viability in 2D and 3D cultures. a Comparison of viability and metabolic activity assessment via spectrophotometric readouts or (confocal) microscopy in 2D and 3D in vitro culture setups. b 2D cultures are shown as monolayers in well plates or tissue culture flasks where reagents have direct contact with the cells. c 3D samples are characterized by the presence of a biomaterial as a supportive structure (e.g., fabricated via 3D bioprinting) in which the cells are embedded. Reagents need to diffuse through the material to stain or be metabolized by the cells, and thus, a limitation in penetration depth may result in low or no positive staining toward the center of the 3D construct, as illustrated for live-dead staining. Furthermore, many biomaterials or compounds present in the materials can interfere and show an autofluorescent signal that, in the worst case, results in higher readout values or background staining than that derived from the cells only

Fig. 2

Quality monitoring via gene expression analysis. a Gene expression analysis starts with RNA isolation, which implies the destruction of the sample. Different techniques can then be employed to assess gene expression levels, including qPCR analysis, microarrays, and RNA sequencing. b Intracellular RNA can be imaged using fluorescent nucleic acid-based probes. The probe should be transfected into the cells; once inside, the probe anneals to the target sequence, leading to an increase in fluorescence, which can then be analyzed via confocal microscopy

Fig. 3

Summary of microscopy techniques for qualitative and quantitative assessment of cells, scaffolds and tissue engineered constructs. a With laser scanning confocal microscopy (LSCM), it is possible to analyze the expression of specific proteins and nucleic acids and to assess the biological responses and morphological organization of cells using fluorescent dyes. b With scanning electron microscopy (SEM), it is possible to gather high-resolution structural and analytical information about tissues and constructs. c Atomic force microscopy (AFM) allows us to analyze the surface and mechanical properties of constructs

Fig. 4

Simplified schematic of a Raman system. Reprinted by permission from Springer Nature Customer Service Center GmbH: Springer Nature. Springer eBook, Raman Micro-Spectroscopy as a Noninvasive Cell Viability Test, Verrier S. et al. © 2011

Fig. 5

Summary of Raman spectroscopy techniques: polarized Raman spectroscopy (PRS), suitable for highly oriented systems; Raman microscopy, able to couple chemical and morphological information; spatially offset Raman spectroscopy (SORS), used for collecting Raman signal from deep regions; coherent Raman spectroscopy (SRS & CARS), able to improve Raman signal intensity up to 10⁵ factor; and surface enhanced Raman spectroscopy, (SERS) able to enhance signal intensity up to 10¹⁰ factor, exploiting the plasmonic effect between the sample and metal particles. The processing of Raman data collected from biological samples is accomplished by spectral analysis and statistical treatments

Fig. 6

Schematic representation of the potential of microCT analysis for musculoskeletal tissue engineering. This nondestructive technique can be used for different applications for biomaterials per se or in combination with cells and tissue (both ex vivo and in vivo). The produced image dataset can be compared with that from different microscopy techniques, combined with biomechanical tests or used for 3D modeling

Fig. 7

Overview of the applicability of sensors to the field of tissue engineering. Critical parameters for construct quality control (e.g., glucose, lactate, and oxygen consumption) could be monitored in different types of in vitro culture systems (a) by using electrochemical (b) and optical (c) sensors/biosensors for real-time quality monitoring (d). These methods are conservative, allowing for longitudinal studies and further applicability of tissue engineered constructs

Fig. 8

The two-step reduction of oxygen allows the formation of hydrogen peroxide as a detectable intermediate

Fig. 9

The enzymatic oxidation of glucose produces hydrogen peroxide whose concentration is directly proportional to that of glucose