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Conditioning the microenvironment for soft tissue regeneration in a cell free scaffold

The use of cell-free scaffolds for the regeneration of clinically relevant volumes of soft tissue has been challenged, particularly in the case of synthetic biomaterials, by the difficulty of reconciling the manufacturing and biological performance requirements. Here, they investigated in vivo the importance of biomechanical and biochemical cues for conditioning the 3D regenerative microenvironment towards soft tissue formation. In particular, they evaluated the adipogenesis changes related to 3D mechanical properties by creating a gradient of 3D microenvironments with different stiffnesses using 3D Poly(Urethane-Ester-ether) PUEt scaffolds. Their results showed a significant increase in adipose tissue proportions while decreasing the stiffness of the 3D mechanical microenvironment. This mechanical conditioning effect was also compared with biochemical manipulation by loading extracellular matrices (ECMs) with a PPAR-γ activating molecule. Notably, results showed mechanical and biochemical conditioning equivalency in promoting adipose tissue formation in the conditions tested, suggesting that adequate mechanical signaling could be sufficient to boost adipogenesis by influencing tissue remodeling. Overall, this work could open a new avenue in the design of synthetic 3D scaffolds for microenvironment conditioning towards the regeneration of large volumes of soft and adipose tissue, with practical and direct implications in reconstructive and cosmetic surgery.


(A) Schematic representation of the synthetic route of 3 PUEt scaffolds of different stiffnesses, starting from 3 polyester triols: P(CL-co-GL) of different CL:GL ratios and accordingly of different crystallinities. Physicochemical properties of the 3 PUEt scaffolds formulations and the related precursors: (B) 1HNMR spectra of the 3 polyesters P(CL-co-GL) 4:1, P(CL-co-GL) 10:1, P(CL-co-GL) 20:1 showing correspondence between the ratio between CL and GL in the final products compared to the reactants; (C) DSC traces overlay of the 3 polyesters showing different thermal properties as a functions of the ratio CL: GL; (D) µCT scan micrograph (up), graphical rendering of the local thickness (middle) and of the pore size (bottom) of the 3 scaffolds formulations PUEt 4:1, PUEt 10:1 and PUEt 20:1; (E) graphical representation of the compression elastic moduli (Ec) of the 3 scaffolds formulations, showing statistically significant difference between: PUEt 10:1 and PUEt 20:1 (*p < 0.05), PUEt 4:1 and PUEt 10:1 (** = p < 0.025) and PUEt 4:1 and PUEt 20:1 (**p < 0.025).


Current soft tissue reconstruction approaches depend on inert fillers or autologous grafts to replace the lost volume. Despite the clinical success of these techniques, patients suffer from multiple drawbacks such as donor-site morbidity and volume loss over time. Therefore, an unmet clinical need still exists for efficacious solutions for soft tissue restoration after trauma or surgical removal of lesioned tissues. In particular, breast cancer reconstructive treatments, which have one of medicine's highest reoperation rates, would greatly benefit from a successful regeneration of relevant soft tissue volumes. In this context, cell-free scaffold-based approaches are emerging as a promising solution due to their biocompatibility, properties modulation/adaptability to that of the target tissue, cost-effectiveness, and compliance with international manufacturing standardS. Moreover, synthetic scaffolds also represent a more scalable solution in clinics because they can avoid regulatory and manufacturing hurdles typical of the cell-based therapies.


(A) representative H&E stained histological images of PUEt 4:1, PUEt 10:1, and PUEt 20:1. In the middle: low-magnitude images showing the entire explanted scaffolds after 3 months of subcutaneous implantation. Subregions were selected to show peripheral inflammation and adipose tissue (dashed blue and dotted gray, respectively) (scale bar, 2.5 mm). Left and right columns display higher magnification images representing the peripheral inflammation and adipose tissue in each group, respectively. Vascular tissue is indicated by black arrowheads, scale bar = 250 µm. (B) graphical representation of peripheral inflammation of the 3 groups: PUEt 4:1, PUEt 10:1, and PUEt 20:1 . No significant difference between the three groups was found, in terms of peripheral inflammation. (C) Graphical representation of the adipose tissue % for the 3 groups: PUEt 4:1, PUEt 10:1, and PUEt 20:1. Statistically relevant differences in terms of % of adipose tissue was found between PUEt 4:1 vs PUEt 10:1 (**p < 0.025) and PUEt 4:1 vs PUEt 20:1 (***p < 0.01).

In the last decade, 3D scaffolds for the regeneration of clinically relevant soft tissue volumes have seen considerable progress, especially at the preclinical level, as exemplified by manufacturing of poly(D,L)-lactide and medical grade polycaprolactone scaffolds by fused deposition modelling. However, their use is challenged by the high local stiffness of the polymer filaments which fails to match that of the target tissue despite reductions in the filament thickness to a few hundred microns. This is not surprising since the adipose tissue regeneration process is one of the most challenging among connective tissues due to its structural and mechanical complexity and sensitivity to signals, such as the ones from hormone and nervous systems. Therefore, developing scaffolds for adipose tissue regeneration required control over biomechanical cues to achieve appropriate cell/biomaterial interactions, which is fundamental to drive the differentiation of mesenchymal stem cells towards adipogenesis.


(A) representative H&E stained histological images of PUEt + PLL (control) and PUEt + PLL + RG. In the middle: low-magnitude images showing the entire explanted scaffolds after 3 months of subcutaneous implantation. Rectangular regions were selected to show peripheral inflammation and adipose tissue (dashed blue and dotted gray, respectively), scale bar = 2.5 mm. Left and right columns are high-magnitude selected images representing the peripheral inflammation and adipose tissue in each group, respectively. Vascular tissue is indicated by black arrowheads (scale bar, 250 µm). (B) graphical representation of peripheral inflammation of PUEt + PLL (control) and PUEt + PLL + RG. No significant difference between the 2 groups was found, in terms of peripheral inflammation. (C) Graphical representation of the adipose tissue %. Statistically relevant differences in terms of % of adipose tissue was found between PUEt + PLL + RG and PUEt + PLL (control) (***p < 0.01). (D) Graphical representation of the in vitro cumulative release of RG from PUEt + PLL + RG, under physiological conditions. The release profile is characterized by a 9.35% initial bust effect and 5.50% daily release. The in vitro release proceeded for 16 days until complete release of RG from the scaffold.

The effect of mechanical cues on human adipocyte function was previously demonstrated in vitro by Pellegrinelli et. al.. In Young et. al., adult adipose-derived stem cells cultured on soft hydrogels that mimicked the stiffness of adipose tissue (2 kPa) showed significant upregulation of adipogenic markers in vitro. However, in a more physiologically relevant 3D in vivo scenario, the evaluation of the impact of mechanical cues on adipogenesis is more complex, mainly due to the interdependence of cell viability and differentiation with the foreign body response. In their previous studies, they addressed key factors impacting the biological performance of polyurethane-based crosslinked porous biomaterials as scaffolds for soft tissue regeneration, focusing the attention on the role of polymer chemistry and the microarchitecture.These findings enabled their group to develop 3D scaffolds with physicochemical and morphological properties guiding cell infiltration to the scaffold core and rapid recruitment of vascular tissue. In the present work, by modifying the composition of polyester triol segments co-polymerized in the polyurethane network, they synthetized a gradient of Poly(Urethane-Ester-ether) PUEt porous scaffolds sharing similar physicochemical and morphological properties but displaying different substrate stiffnesses. This experimental configuration allowed us to investigate the effects of mechanical cues on adipogenesis. They also compared the effects of mechanical and biochemical conditioning on the regenerative microenvironment using a PUEt scaffold loaded with a peroxisome proliferator–activated receptor γ (PPAR- γ) agonist (Rosiglitazone, RG) for the induction of adipocyte differentiation. The effects of PPAR- γ-loaded scaffolds were compared with unloaded control. The findings from this study are discussed in view of the emerging trends for developing implantable devices for adipose tissue regeneration, with particular emphasis on breast reconstruction surgeries1.


1. Gerges, I., Tamplenizza, M., Martello, F. et al. Conditioning the microenvironment for soft tissue regeneration in a cell free scaffold. Sci Rep11, 13310 (2021). https://doi.org/10.1038/s41598-021-92732-9