Oral Presentation 51st International Society for the Study of the Lumbar Spine Annual Meeting 2025

New minimally-invasive injectable biomaterials for the regeneration of the nucleus pulposus (115550)

Gabriela Graziani 1 , Marcia Muerner 2 , Chencheng Feng 2 , Restione Nicoletta 3 , Zonca Leonardo 3 , Sibylle Grad 2 , Silvia Fare' 1
  1. Department of Chemistry, Materials and Chemical Engineering "Giulio Natta", Polytechnic University of Milan, Milan, Italy
  2. Regenerative Orthopaedics, AO Research Institute, Davos, Switzerland
  3. Polytechnic University of Milan, Milan, Italy

INTRODUCTION

Intradiscal injections of hydrogels (with/without cells) have shown to be a promising approach for nucleus pulposus (NP) regeneration1. Several natural/synthetic polymeric biomaterials have been proposed1-6, but no fully satisfactory alternatives are currently clinically available.

Here, we propose new injectable hydrogels based on gelatine, GEL (for biomimicry and to provide motifs for cell adhesion) and silk fibroin, SF (to increase mechanical properties, oppose swelling and enhance long-term stability). A crosslinking route based on microbial transglutaminase (mTG), is selected to avoid toxicity and finely control crosslinking time.

Injectability, stability and degradation profile of the hydrogels are tested to select the optimal formulations. The regenerative potential of the hydrogels is evaluated in a bovine ex vivo NP defect model.

METHODS

Hydrogels are obtained from bovine GEL (type B, 10%) with/without the addition of SF (from Bombyx mori, 1 and 2% w/v). mTG is added at different concentrations (10-100 U/g). The optimal one is selected based on crosslinking time (target 5-15 minutes), measured by inversion test and rheology tests (time-sweep, 23 and 37°C). After selection, the stability of the hydrogels (PBS+0,02%NaN3, 30min, 1h, 2h, 3h, 4h, 6h, 24h, 48h, 72h, 96h, 1 week, 2 weeks) and absence of cytotoxicity are tested (L929 fibroblasts; 1,3,7 days; Alamar Blue).

After the selection of the optimal formulations (1 GEL and 1 GEL/SF), injectability through a 20G needle is assessed by a custom-made testing apparatus.

Bovine coccygeal discs are isolated and loaded in a bioreactor (0.02-0.2 MPa, 0.2 Hz, 2h/day). On day 2, defects are created using an endplate nucleotomy approach7. On D3, defects are filled with the hydrogels via the opposite endplate. Mechanical performance (stiffness, range of motion, hysteresis) is evaluated using recorded force and displacement; disc height changes are measured. Discs are harvested to assess disc cell viability (LDH/EtH) and hydrogel integration (SafO/Fast Green).

RESULTS

25 and 50 U/g mTG concentrations guarantee adequate crosslinking time (15 and 6 minutes, respectively, for GEL, 14/11 minutes for the GEL/SF), with negligible differences among different fibroin concentrations and temperatures.

GEL hydrogels show moderate swelling, limited to the first 24 hours, when a plateau is reached. Swelling is negligible for the blends. At 2 weeks, all formulations are still stable, although the GEL/SF at the lowest SF concentration starts degrading. No loss of integrity is observed for any formulation/timepoint.

G’ increases for increasing mTG concentration and, significantly, when SF is added, negligently of its concentrations. All samples are non-cytotoxic and injectable through 25 G needles.

Gelatine with 50U of mTG and GEL/SF 2% blend are selected for ex vivo tests. Results demonstrate that loading-induced height loss can be restored to a physiological range (~10%) after defect filling for all formulations.

DISCUSSION

The optimized hydrogels appear promising for NP regeneration showing: (i) suitable crosslinking time, (ii) adequate rheological behaviour, (iii) no excess swelling, (iv) high stability, (v) good injectability, (vi) no cytotoxicity and good height restoration capabilities ex vivo. Biomechanical and histological investigation will provide further insights. Future tests will entail cells and drugs loading.

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