Raman Spectroscopy-Based 3D Analysis of Odontogenic Differentiation of Human Dental Pulp Stem Cell Spheroids

Abstract

Numerous efforts have been made to establish three-dimensional (3D) cell cultures that mimic the structure, cell composition, and functions of actual tissues and organs in vitro; however, owing to its intrinsic complexity, precise characterization of 3D differentiation remains challenging and often results in high variations in the resulting differentiated spheroids. This study reports a 3D Raman mapping-based analytical method that helps us to identify the crucial factors responsible for inducing variability in differentiated stem cell spheroids. Human dental pulp stem cell spheroids were generated at various cell densities using the hanging drop (HD) and molded parafilm-based (MP) methods and were then further subjected to odontogenic differentiation. Thereafter, the 3D cellular differentiation in these spheroids was analyzed based on three different Raman peaks, namely, 960, 1156/1528, and 2935 cm −1 , which correspond to hydroxyapatite (HA, odontogenic differentiation marker), β-carotene (precursor of HA), and proteins/cellular components (cell reference). By correlating such cell differentiation-related peaks and water/medium peaks (3400 cm −1 ), we discovered that the diffusion of the medium containing various nutrients and differentiation factors was crucial in determining the variations in 3D differentiation of stem cell spheroids. Odontogenic differentiation was majorly induced at the outer shell of HD spheroids (up to ∼20 μm), whereas odontogenic differentiation was markedly induced in MP spheroids (up to 40−50 μm). Considering the challenges associated with high variations in spheroid and organoid differentiation, we conclude that the proposed Raman-based 3D analysis plays a pivotal role in stem cell-based regenerative therapy and drug screening.

Tunable Nanomaterials of Intracellular Crystallization for In Situ Biolabeling and Biomedical Imaging

“…Bacteria play an important role in the process of biocalcification, biosilication and biomineralization. In organisms, bacteria-mediated calcification can be found in crustaceans, marine organisms, plants and human organs. − Calcification in tumor tissue is supposed to be related to bacteria. , The appearance of dental plaque is the cause of bacteria, and the appearance of magnetosomes in bacteria is also the cause of biosynthesis. , A large number of bacteria can produce acidic polysaccharide matrix on the surface of bacteria, provide nucleation sites for mineral deposition, and contribute to biomineralization. − This provides a synthetic basis for the intrabacterial biosynthesis of nanoparticles with physicochemical properties.…”

Hypes and Hopes of Stem Cell Therapies in Dentistry: a Review

“…In 2021, RAMAN spectroscopy was used to find the factors responsible for stem cell differentiation in spheroids formed by aggregation of human DPSCs. This research showed that the differentiation and acquisition of the 3D structure is mainly related to the diffusion of nutrients, morphogens, and growth factors within the culture medium [ 119 ].…”

Therapeutic potential of dental pulp stem cells and their derivatives: Insights from basic research toward clinical applications

“…Some of these methods are still in the research stage and are not ready to be adopted for large-scale manufacturing. The manufacturing methods of DPSC spheroids have also emerged in an endless series, including but not limited to serum-free medium culture[ 144 , 145 ], culturing on Matrigel[ 146 ], special 3D Petri dish culture[ 135 , 147 ], low or ultralow attachment culture plates[ 62 , 148 ], culturing on gelatin methacrylamine/poly(ethylene glycol) diacrylate (GelMA/PEGDA) composite hydrogels[ 149 ], coculturing with microparticles with a leaf-stacked structure based on polycaprolactone[ 150 ], HD or molded parafilm-based methods[ 151 ]. Most of these methods belong to the classification of the aforementioned technologies.…”