Injectable prevascularized mature adipose tissues (iPAT) to achieve long-term survival in soft tissues regeneration

Abstract

Soft tissue regeneration remains a challenge in reconstructive surgery. Current autologous fat implantations lead to high fat absorption ratios, while artificial implants can be associated with lymphoma occurrence. To overcome these limitations, our aim was to reproduce adipose tissue vasculature structure before implantation. Here, we developed injectable prevascularized adipose tissues (iPAT), using physiological collagen microfibers (CMF) mixed with human mature adipocytes, adipose-derived stem cells (ADSC) and human umbilical vein endothelial cells (HUVEC). Following murine subcutaneous implantation, higher cell survival (84±6% viability) and volume maintenance were shown after 3 months for the iPAT (up to twice heavier than the non-prevascularized balls). This higher survival can be explained by the greater amount of blood vessels (up to 1.6 folds increase), with balanced host anastomosis (51±1% of human/mouse lumens), also involving infiltration by the lymphatic and neural vasculature networks. These iPAT tissues allowed non-invasive soft tissue reconstruction for long-term outcomes, and the ability to cryopreserve them with maintained viability and functionality also enables a later reinjection usually required before reaching the final patient desired graft volume.

Bioprinted Vascularized Mature Adipose Tissue with Collagen Microfibers for Soft Tissue Regeneration

“…The lumen structure of these blood capillaries was then confirmed using the 3D surface reconstruction of the bioprinted vascularized mature adipose tissue ( Figure 6(a) ) which allowed to navigate inside the vasculature lumen, with an example of a branching section ( Figure 6(b) ) and a longitudinal section where aligned nuclei can be observed ( Figure 6(c) ). Immunohistochemistry on manually seeded samples [ 39 ] (Supplementary Figure 2 ) also confirmed the lumen structures of different sizes in sectioned tissues, being observed in the vicinity (Supplementary Figure 2 a) and surrounding (Supplementary Figure 2 b) the mature adipocytes, structures which are expected to be the same in the bioprinted samples.…”

“…Our CMF-based bioprinting method, dispensing tissues on the surface of a culture dish, was therefore thought to be suitable for the production of an in vitro vascularized adipose tissue regeneration. First, similar line patterns, containing the cells required for the angiogenesis induction (ADSC and HUVEC) [ 39 ], were printed on a surface, incorporating a larger line in between containing the mature adipocytes in a fully cocultured scaffold (Figures 1(a) and 2 ). The tissue structure was maintained in the culture, even up to 7 days, but showed a settlement of the thickness, appearing like a 2D structure.…”

“…The final advantage is that it should be possible to get a full noninvasive process for the patient from the liposuction which involves a small incision up to the reconstructed graft implantation. An interesting last point could be the possibility to cryopreserve the bioprinted tissues for additional subsequent implantations in case of long-term adjustments of the final graft volume following its resorption [ 39 ].…”

“…To construct the fat tissues, the CMF were first weighted and washed in DMEM without FBS before being centrifuged for 1 minute at 10,000 rpm to adjust the final concentration depending on the volume added to resuspend it (MiniSpin, Thermo Fisher Scientific, Waltham, MA, USA). When needed, the ADSC and the HUVEC were added after trypsin detachment (always used at passages 1-6) and centrifuged for 1 minute at 3500 rpm (MiniSpin, Thermo Fisher Scientific, Waltham, MA, USA) to get a final cell concentration of 2.5 × 10 6 ADSC/mL and 1.25 × 10 6 HUVEC/mL, as already reported in our previous study [ 39 ]. The pellet containing CMF, ADSC, and HUVEC was then mixed with the fibrinogen solution (to get a final concentration at 6 mg/mL, the stock solution was prepared in DMEM without FBS 1% antibiotics, to avoid the early gelation of the fibrinogen solution due to the FBS calcium ion content [ 44 ], then filtrated using a 0.2 μ m filter).…”

“…Moreover, the CMF also provides a scaffold to induce the formation of the vascular network by endothelial cells [ 37 ]. Altogether, this method enables the fabrication of fully vascularized reconstructed adipose tissue showing host anastomosis following implantation for a higher graft survival rate [ 39 ].…”