Raman spectroscopic insight into osteoarthritic cartilage regeneration by mRNA therapeutics encoding cartilage-anabolic transcription factor Runx1

Abstract

While joint arthroplasty remains nowadays the most popular option available to repair chronically degenerated osteoarthritic joints, possibilities are recently emerging for regeneration of damaged cartilage rather than its replacement with artificial biomaterials. This latter strategy could allow avoiding the quite intrusive surgical procedures associated with total joint replacement. Building upon this notion, we first apply Raman spectroscopy to characterize diseased cartilage in a mice model of instability-induced knee osteoarthritis (OA) upon medial collateral ligament (MCL) and medial meniscus (MM) transections. Then, we examine the same OA model after cartilage regeneration by means of messenger RNA (mRNA) delivery of a cartilage-anabolic runt-related transcription factor 1 (RUNX1). Raman spectroscopy is shown to substantiate at the molecular scale the therapeutic effect of the Runx1 mRNA cartilage regeneration approach. This study demonstrates how the Raman spectroscopic method could support and accelerate the development of new therapies for cartilage diseases.

Keywords: Cartilage regeneration; Cartilage-anabolic transcription factor; Raman spectroscopy; mRNA therapeutics.

Graphical abstract

Fig. 1

Micro-CT and histological-section images of mice knee 4 weeks after MCL and MM transection. (a) Reconstituted images of sagittal and frontal planes from the raw micro-CT images. (b) Histological sections of the identical knee joint, stained with hematoxylin and eosin (left) or toluidine blue (right).

Fig. 2

Representative Raman spectra recorded ex vivo on (a) control healthy tibia cartilage, and those from knee joints at (b) 2 and (c) 4 weeks after MCL and MM transection. The main regions studied in this paper are emphasized with square insets (cf. labels).

Fig. 3

Raman analysis in the spectral zone at 1170–1500 ​cm⁻¹: (a) control healthy tibia cartilage, and those from knee joints at (b) 2 and (c) 4 weeks after MCL and MM transection. Deconvolution singled out 10 sub-bands whose frequencies and vibrational assignments are given in Table 1.

Fig. 4

Raman analysis in the spectral zone at 1000–1200 ​cm⁻¹: (a) control healthy tibia cartilage, and those from knee joints at (b) 2 and (c) 4 weeks after MCL and MM transection. Spectral frequencies are listed in Table 2 and assignments to specific molecules are given in inset.

Fig. 5

Raman analysis in the spectral zone at 1170–1500 ​cm⁻¹: (a) control healthy tibia cartilage, (b) diseased cartilage after 4 weeks since MCL and MM transection, and (c) regenerated cartilage after 4 weeks after MCL and MM transection and one successive week after Runx1 mRNA administration (deconvolution into 10 sub-bands with frequencies and vibrational assignments given in Table 1).

Fig. 6

Raman analysis in the spectral zone at 1000–1200 ​cm⁻¹: (a) control healthy tibia cartilage, (b) diseased cartilage after 4 weeks since MCL and MM transection, and (c) regenerated cartilage after 4 weeks after MCL and MM transection and one successive week after Runx1 mRNA administration. Spectral frequencies are given in Table 2 and assignments to specific molecules are given in inset.

Fig. 7

(a) Amide III ratio, I₁₂₄₀/I₁₂₇₀, and (b) hydration ratio, I_1312+1340/I₁₂₇₀, as functions of time after MCL and MM transection (standard deviations and details of statistical validations in inset); the green plots give the value of the two ratios one-week after Runx1 mRNA administration; in (c), schematic draft of the two-step proposed for disease development: Step 1 → gradual depletion of the internal α-helix hydrogen bonds with formation of external hydrogen bonds to water molecules and concurrent “loosening” toward a more open structure; and, Step 2 → α-helix structure irreversibly collapsing into a random coil configuration.

Fig. 8

(a) Glycosaminoglycan (GaGs) ratio, I₁₀₆₅/I₁₁₂₃, and (b) N-glycosylation ratio, I₁₁₆₅/I₈₅₀, as functions of time after MCL and MM transection (details of statistical validations in inset); the green shadowed areas give the value of the two ratios two-weeks after Runx1 mRNA administration; in (c), schematic draft of two successive steps in disease development from healthy cartilage: an initial step (biosynthetic phase) in which the chondrocytes yet attempt repairing the diseased extracellular matrix with promoting HA synthesis, and a successive step (degradative phase) in which chondrocytes start producing enzymes that inhibit matrix synthesis and undergo apoptosis. Note that the shown draft only represents what is, in the authors' opinion, the most plausible hypothesis, for explaining the obtained experimental data.

Fig. 9

(a) Putative therapeutic action of Runx1 mRNA in terms of chondrocyte activation; and, (b) the spectroscopic fingerprints of cartilage regeneration at the molecular scale.