In Vivo Assessment of Bone Regeneration in Alginate/Bone ECM Hydrogels with Incorporated Skeletal Stem Cells and Single Growth Factors

Abstract

The current study has investigated the use of decellularised, demineralised bone extracellular matrix (ECM) hydrogel constructs for in vivo tissue mineralisation and bone formation. Stro-1-enriched human bone marrow stromal cells were incorporated together with select growth factors including VEGF, TGF-β3, BMP-2, PTHrP and VitD3, to augment bone formation, and mixed with alginate for structural support. Growth factors were delivered through fast (non-osteogenic factors) and slow (osteogenic factors) release PLGA microparticles. Constructs of 5 mm length were implanted in vivo for 28 days within mice. Dense tissue assessed by micro-CT correlated with histologically assessed mineralised bone formation in all constructs. Exogenous growth factor addition did not enhance bone formation further compared to alginate/bone ECM (ALG/ECM) hydrogels alone. UV irradiation reduced bone formation through degradation of intrinsic growth factors within the bone ECM component and possibly also ECM cross-linking. BMP-2 and VitD3 rescued osteogenic induction. ALG/ECM hydrogels appeared highly osteoinductive and delivery of angiogenic or chondrogenic growth factors led to altered bone formation. All constructs demonstrated extensive host tissue invasion and vascularisation aiding integration and implant longevity. The proposed hydrogel system functioned without the need for growth factor incorporation or an exogenous inducible cell source. Optimal growth factor concentrations and spatiotemporal release profiles require further assessment, as the bone ECM component may suffer batch variability between donor materials. In summary, ALG/ECM hydrogels provide a versatile biomaterial scaffold for utilisation within regenerative medicine which may be tailored, ultimately, to form the tissue of choice through incorporation of select growth factors.

Fig 1. Micro CT analysis of hydrogels following 28 days in vivo implantation.

Tissue volume (A), percentage bone volume (B), bone surface to volume ratio (C), trabecular number (D), trabecular thickness (E) and trabecular separation (F) were all assessed between growth factor groups. Emboldened columns depict statistically significant intragroup differences between those with and without Stro-1+ cell incorporation. Asterisks depict statistical difference between the group above which the asterisk is positioned and all the other groups; if positioned centrally above both groups with and without Stro-1+ cell incorporation, statistical difference was observed for both compared across all groups. Error bars are S.D. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001.

Fig 2. Hydrogel mineralisation between growth factor groups following 28 days in vivo implantation.

Groups included irradiated ALG/ECM (A), ALG/Col (B), ALG/ECM (C), ALG/ECM HSA (D), ALG/ECM HSA/VEGF (E), ALG/ECM HSA/TGF-β3 (F), ALG/ECM HSA/BMP-2 (G), ALG/ECM HSA/PTHrP (H) and ALG/ECM HSA/VitD3 (I). Images were taken at high (scale bar is 500 μm) and low magnification (box inserts–scale bar is 50 μm).

Fig 3. Histological analysis of hydrogels stained with Alcian blue/Sirius red.

Hydrogels were subcutaneously implanted within immunodeficient for 28 days. Colour quantification was through the use of an optimised Image J macro (S2 Fig). Blue indicated proteoglycan deposition and residual hydrogel (A), purple indicated collagen deposition within the hydrogel (B) and red indicated tissue invasion (C). Emboldened columns depict statistically significant intragroup differences between those with and without Stro-1+ cell incorporation. Asterisks depict statistical difference between the group above which the asterisk is positioned and all the other groups; if positioned centrally above both groups with and without Stro-1+ cell incorporation, statistical difference was observed for both compared across all groups. Error bars are S.D. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001.

Fig 4. Histological analysis of hydrogels stained with Von Kossa.

Hydrogels were subcutaneously implanted within immunodeficient mice for 28 days. Colour quantification was through the use of an optimised Image J macro (S2 Fig). Black indicates mineralised tissue (A) and pink indicates cell invasion (B). Emboldened columns depict statistically significant intragroup differences between those with and without Stro-1+ cell incorporation. Asterisks depict statistical difference between the group above which the asterisk is positioned and all the other groups; if positioned centrally above both groups with and without Stro-1+ cell incorporation, statistical difference was observed for both compared across all groups. Error bars are S.D. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001.

Fig 5. Histological analysis of hydrogels stained with Goldner’s Trichrome.

Hydrogels were subcutaneously implanted within immunodeficient mice for 28 days. A square grid (200 μm) overlay was used to quantify erythrocytes (A) and assess vascularisation (B). Error bars are S.D. * P ≤ 0.05.