Pre-cultivation of adipose tissue-derived microvascular fragments in porous scaffolds does not improve their in vivo vascularisation potential

Abstract

Adipose tissue-derived microvascular fragments represent promising vascularisation units for implanted tissue constructs. However, their reassembly into functional microvascular networks takes several days, during which the cells inside the implants are exposed to hypoxia. In the present study, we analysed whether this critical phase may be overcome by pre-cultivation of fragment-seeded scaffolds prior to their implantation. Green fluorescent protein (GFP)-positive microvascular fragments were isolated from epididymal fat pads of male C57BL/6-TgN (ACTB-EGFP) 1Osb/J mice. Nano-size hydroxyapatite particles/poly (ester-urethane) scaffolds were seeded with these fragments and cultivated for 28 days. Subsequently, these scaffolds or control scaffolds, which were freshly seeded with GFP-positive microvascular fragments, were implanted into the dorsal skinfold chamber of C57BL/6 wild-type mice to study their vascularisation and incorporation by means of intravital fluorescence microscopy, histology and immunohistochemistry over 2 weeks. Pre-cultivation of microvascular fragments resulted in the loss of their native vessel morphology. Accordingly, pre-cultivated scaffolds contained a network of individual CD31/GFP-positive endothelial cells with filigrane cell protuberances. After implantation into the dorsal skinfold chamber, these scaffolds exhibited an impaired vascularisation, as indicated by a significantly reduced functional microvessel density and lower fraction of GFPpositive microvessels in their centre when compared to freshly seeded control implants. This was associated with a deteriorated incorporation into the surrounding host tissue. These findings indicate that freshly isolated, noncultivated microvascular fragments should be preferred as vascularisation units. This would also facilitate their use in clinical practice during intra-operative one-step procedures.

Microvascular Fragment Transplantation Improves Rat Dorsal Skin Flap Survival

“…12 In fact, a recent publication demonstrated the superiority of utilizing freshly isolated MVFs compared with those cultured in vitro. 11 In agreement with our hypothesis, we show that MVFs did improve flap survival 14 days after surgery; however, the mechanisms whereby this improvement occurred remain to be determined.…”

“…MVFs consist of adipose tissue–derived arterioles, venules, and capillaries, 7 which improve tissue perfusion in a variety of tissue injury models. 8,9,11,12,28 An additional factor that provides significant motivation for understanding the potential of MVFs for flap survival is that they can be delivered without prior culturing: that is, fragments can be harvested, implanted on the day of surgery, and inosculated with the host within days. 12 In fact, a recent publication demonstrated the superiority of utilizing freshly isolated MVFs compared with those cultured in vitro.…”

“…MVFs have been used as a means to (1) improve tissue perfusion after myocardial infarction, 8 (2) improve vascularity and tissue perfusion for the treatment of large muscle defects, 9 (3) aid in islet implant survival, 10 and (4) provide an implant that retains its native structure when implanted into a dorsal skinfold chamber in mice. 11 The findings that they inosculate within a day when implanted subcutaneously in collagen 12 and that they are capable of extensive remodeling in vivo over time 8,12 make them particularly suited for improving flap survival. We hypothesized that application of MVFs into the flap would serve as a means for rapid improvements in tissue perfusion and thereby improve flap survival.…”

Seeding density is a crucial determinant for the in vivo vascularisation capacity of adipose tissue-derived microvascular fragments

“…The observation window of the chamber was positioned under a Zeiss Axiotech fluorescent microscope (Zeiss, Oberkochen, Germany) and the microscopic images were recorded by a charge-coupled device video camera (FK6990; Pieper, Schwerte, Germany) and a DVD system for off-line analyses (Laschke et al, 2015).…”

“…Of interest, ad-MVF can be isolated in large amounts from fat tissue within a short time and represent fully functional vessel segments (Frueh et al, 2017b). Hence, they bear the major advantage that their reassembly into new microvascular networks induces a rapid implant vascularisation and blood perfusion (Laschke et al, 2012;Laschke and Menger, 2015). For this purpose, angiogenic sprouts, growing out of the vessel www.ecmjournal.org T Später et al ad-MVF density affects implant vascularisation segments, have to develop interconnections (Nunes et al, 2010).…”