Bone healing outcome is highly dependent on the initial mechanical fracture environment [1]. In vivo, direct bone healing requires absolute stability and an interfragmentary strain (IFS) below 2% [2]. In the majority of cases, however, endochondral ossification is engaged where frequency and amplitude of IFS are key factors. Still, at the cellular level, the influence of those parameters remains unknown. Understanding the regulation of naïve hMSC differentiation is essential for developing effective bone healing strategies.

Human bone-marrow-derived MSC (KEK-ZH-NR: 2010–0444/0) were embedded in 8% gelatin methacryol. Samples (5mm Ø x 4mm) were subjected to 0, 10 and 30% compressive strain (5sec compression, 2hrs pause sequence for 14 days) using a multi-well uniaxial bioreactor (RISystem) and in presence of chondro-permissive medium (CP, DMEM HG, 1% NEAA, 10 µM ITS, 50 µg/mL ascorbic acid, and 100 mM Dex). Cell differentiation was assessed by qRT-PCR and histo-/immunohistology staining. Experiments were repeated 5 times with cells from 5 donors in duplicate. ANOVA with Tukey post-hoc correction or Kurskal-Wallis test with Dunn's correction was used.

Data showed a strong upregulation of hypertrophic related genes COMP, MMP13 and Type 10 collagen upon stimulation when compared to chondrogenic SOX9, ACAN, Type 2 collagen or to osteoblastic related genes Type 1 Collagen, Runx2. When compared to chondrogenic control medium, cells in CP with or without stimulation showed low proteoglycan synthesis as shown by Safranine-O-green staining. In addition, the cells were significantly larger in 10% and 30% strain compared to control medium with 0% strain. Type 1 and 10 collagens immunostaining showed stronger Coll 10 expression in the samples subjected to strain compared to control.

Uniaxial deformation seems to mainly promote hypertrophic-like chondrocyte differentiation of MSC. Osteogenic or potentially late hypertrophic related genes are also induced by strain.

Acknowledgments: Funded by the AO Foundation, StrainBot sponsored by RISystemAG & PERRENS 101 GmbH

Demineralized bone matrix (DBM) is a natural, collagen-based, well-established osteoinductive biomaterial. Nevertheless, there are conflicting reports on the efficacy of this product. The purpose of this study was to evaluate whether DBM collagen structure is affected by particle size and can influence DBM osteoinductivity.

Sheep cortical bone was ground and particles were divided in three fractions with different sizes, defined as large (L, 1–2 mm), medium (M, 0.5–1 mm), and small (S, < 0.5 mm). After demineralization, the three DBM samples were characterized by DTA analysis, XRD, ICP-OES, and FTIR. Data clearly showed a particle size-dependent alteration in collagen structure, with DBM-M being altered but not as much as DBM-S. The in vivo study showed that only DBM-M was able to induce new bone formation in a subcutaneous ectopic mouse model. When sheep MSC were seeded onto DBM particles before implantation, all DBM particles were able to induce new bone formation with the best incidence for DBM-M and DBM-S. Gene expression analysis performed on recovered implants supports the histological results and underlines the supportive role of MSC in DBM osteoinduction through the regulation of host cells. In conclusion, our results show a relation between DBM particle size, structural modification of the collagen and in vivo osteoinductivity. The medium particles represent a good compromise between no modification (largest particles) and excessive modification (smallest particles) of collagen structure, yielding highest osteoinduction. We believe that these results can guide researchers to use DBM particles of 0.5–1 mm size range in applications aimed at inducing new bone formation, obtaining results more comparable and reliable among different research groups. Furthermore, we suggest to carefully analyze the structure of the collagen when a collagen-based biomaterial is used alone or in association with cells to induce new bone formation.