Three-Dimensional Culture of Cartilage Tissue on Nanogel-Cross-Linked Porous Freeze-Dried Gel Scaffold for Regenerative Cartilage Therapy: A Vibrational Spectroscopy Evaluation

Abstract

This study presents a set of vibrational characterizations on a nanogel-cross-linked porous freeze-dried gel (NanoCliP-FD gel) scaffold for tissue engineering and regenerative therapy. This scaffold is designed for the in vitro culture of high-quality cartilage tissue to be then transplanted in vivo to enable recovery from congenital malformations in the maxillofacial area or crippling jaw disease. The three-dimensional scaffold for in-plate culture is designed with interface chemistry capable of stimulating cartilage formation and maintaining its structure through counteracting the dedifferentiation of mesenchymal stem cells (MSCs) during the formation of cartilage tissue. The developed interface chemistry enabled high efficiency in both growth rate and tissue quality, thus satisfying the requirements of large volumes, high matrix quality, and superior mechanical properties needed in cartilage transplants. We characterized the cartilage tissue in vitro grown on a NanoCliP-FD gel scaffold by human periodontal ligament-derived stem cells (a type of MSC) with cartilage grown by the same cells and under the same conditions on a conventional (porous) atelocollagen scaffold. The cartilage tissues produced by the MSCs on different scaffolds were comparatively evaluated by immunohistochemical and spectroscopic analyses. Cartilage differentiation occurred at a higher rate when MSCs were cultured on the NanoCliP-FD gel scaffold compared to the atelocollagen scaffold, and produced a tissue richer in cartilage matrix. In situ spectroscopic analyses revealed the cell/scaffold interactive mechanisms by which the NanoCliP-FD gel scaffold stimulated such increased efficiency in cartilage matrix formation. In addition to demonstrating the high potential of human periodontal ligament-derived stem cell cultures on NanoCliP-FD gel scaffolds in regenerative cartilage therapy, the present study also highlights the novelty of Raman spectroscopy as a non-destructive method for the concurrent evaluation of matrix quality and cell metabolic response. In situ Raman analyses on living cells unveiled for the first time the underlying physiological mechanisms behind such improved chondrocyte performance.

Keywords: NanoCliP-FD gel scaffold; human periodontal ligament-derived stem cell; in situ Raman spectroscopy; regenerative therapy; tissue engineering.

Figure 1

Laser microscope images of cartilage tissue formed after culturing for 14 days onto (a) NanoCliP-FD and (b) atelocollagen scaffolds; (c,d) represent their respective 3D views.

Figure 2

Light microscopy images of Safranin O staining in chondrogenic medium ((a,b) for NanoCliP-FD gel and atelocollagen, respectively) and of Picrosirius red staining ((c,d) for NanoCliP-FD gel and atelocollagen, respectively). In (e), the results of MIA concentrations in culture supernatant at 14 days culture are compared between NanoCliP-FD gel and atelocollagen scaffolds. The (-) symbol represents the negative control (no scaffold).

Figure 3

Light microscopy images of biotinylated hyaluronan-binding protein staining of cartilage tissue grown in CM onto (a) NanoCliP-FD gel and (b) atelocollagen scaffolds; (c) ELISA measurements to determine the soluble hyaluronan supernatant concentration at 14 days of culture (statistical results of Student’s t-test showed significance between data collected on different scaffolds; the abbreviation n.s. means non-significant); (d,e) show light microscopy images are shown of stained type II collagen in the cartilage tissue grown in CM onto NanoCliP-FD gel and atelocollagen, respectively.

Figure 4

Average Raman spectra collected on (a) NanoCliP-FD gel and (b) atelocollagen scaffolds before exposure to PDLSCs; the labels of the main spectroscopic bands correspond to those listed in Table 1 and Table 2, respectively. The red arrow in (b) represents a characteristic signal from α-helical polypeptide poly (γ-benzyl glutamate). In (c,d), Raman spectra are shown of cartilage tissue grown by PDLSCs on NanoCliP-FD gel and atelocollagen scaffolds, respectively. Both spectra are normalized to the amide I band intensity (labels are explained in the text).

Figure 5

The Raman spectral zone between 1000 and 1200 cm⁻¹ (characteristic of GAGs) is deconvoluted into sub-bands related to CS, HA, and HS (cf. labels): cartilage grown onto (a) NanoCliP-FD gel and (b) atelocollagen scaffolds. Structure and fingerprint bands for HA (bending vibrations of C-OH groups) and CS (O-S-O₃^– symmetric stretching) are given in (c,d), respectively.

Figure 6

Deconvoluted spectra in the amide III wavenumber region are shown for cartilage tissue grown onto (a) NanoCliP-FD gel and (b) atelocollagen scaffolds. In (c), schematic drafts are given for α-helix, its reversible hydrated configuration, and its irreversibly disordered random coil structures (from top to bottom).

Figure 7

Raman maps collected on cartilage tissue grown onto NanoCliP-FD gel (a,c) and atelocollagen (d–f) scaffolds: (a,d) are optical micrographs, (b,e) represent the spatial distribution of the ratio I₁₁₂₃/I₁₀₆₃ (measuring the fractional ratio of HA to CS), (c,f) show the spatial distribution of the ratio I_1312+1340/I₁₂₆₃ (measuring the degree of α-helix hydration).

Figure 8

FTIR spectra collected in the spectral region 1400 to 1800 cm⁻¹ for cartilage tissue grown on (a) NanoCliP-FD gel and (b) atelocollagen scaffolds. In (c,d), the enlarged spectral zone 1500–1600 cm⁻¹ of the amide II vibrations is shown as collected on cartilage samples grown onto NanoCliP-FD gel and atelocollagen scaffolds, respectively.

Figure 9

The spectrum in (a) shows the average SR-FTIR spectrum obtained on the cross-section of the cartilage sample grown onto the NanoCliP-FD gel scaffold given in the bright-field optical micrograph (b). In (c–g), related SR-FTIR images are given for proteoglycan, collagen, GAGs, amide I, and lipids, respectively (cf. mapping wavenumbers in inset to each figure).

Figure 10

Schematic of metabolic effects of the pullulan molecules of the NanoCliP-FD gel scaffold on chondrocytes.