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

Abstract

Adipose tissue-derived microvascular fragments (ad-MVF) represent effective vascularisation units for the seeding of dermal substitutes. However, particularly in case of extensive skin defects, the required amounts of donor fat tissue for the harvesting of ad-MVF may not always be available. Therefore, we herein determined the lowest ad-MVF density needed to induce a sufficient vascularisation and incorporation of seeded implants. Collagen-glycosaminoglycan matrices (Integra ® ; diameter: 4 mm) were seeded with 15,000 (HD), 10,000 (MD) and 5,000 (LD) ad-MVF and implanted into full-thickness skin defects within mouse dorsal skinfold chambers, to analyse their in vivo vascularisation and incorporation. Intravital fluorescence microscopy showed a comparable vascularisation of HD and MD ad-MVF-seeded Integra ® , which was significantly higher when compared to LD ad-MVF-seeded Integra ® . As assessed by photoacoustic imaging, this was associated with an increased oxygenation of the implants. Additional histological and immunohistochemical analyses revealed an enhanced cellular infiltration, collagen content, microvessel density and epithelialisation of HD and MD ad-MVF-seeded Integra ® , indicating a better incorporation compared to LD ad-MVF-seeded implants. These findings demonstrate that 80,000 ad-MVF/cm² is the least required density to guarantee an effective vascularisation of the dermal substitute.

Enoxaparin does not affect network formation of adipose tissue–derived microvascular fragments

“…The Integra ® matrices were subsequently placed on a 500 µm cell strainer and 10 µL 0.9% NaCl containing approximately 10.000 ad‐MVF (Figure A) were transferred onto the matrices with a 20 µL pipette (Eppendorf, Wesseling‐Berzdorf, Germany). In addition, a 20 µL pipette was used to induce negative pressure from underneath the matrices to ensure a sufficient seeding depth of the ad‐MVF . Finally, the matrices were implanted into full‐thickness skin defects within dorsal skinfold chambers of recipient mice.…”

“…To identify epithelialized and nonepithelialized areas, the chamber tissue was visualized in epi‐illumination. Trans‐illumination was used to evaluate the extent of bleeding (given in % of implant surface) induced by the ad‐MVF‐seeded Integra ® matrices by means of a semiquantitative hemorrhagic score as follows—1: No bleeding, 2: 1–25%, 3: 26–50%, 4: 51–75%, 5: 76–100%, 6: Bleeding exceeding the implant surface . All microscopic images were recorded by a DVD system and analyzed with the computer‐assisted off‐line analysis system CapImage (Zeintl, Heidelberg, Germany).…”

“…Ad‐MVF represent fully functional vessel segments that are enzymatically isolated from fat samples . Hence, they rapidly reassemble into new microvascular networks within Integra ® and, thus, markedly improve the incorporation of the matrix at the defect site …”

The Crucial Role of Vascularization and Lymphangiogenesis in Skin Reconstruction

“…After transfer into a tissue defect, ad-MVF only have to develop interconnections with each other and the surrounding recipient microvessels to develop into blood-perfused microvascular networks, which avoids complex and timeconsuming in vitro or in situ incubation steps [53,55]. Accordingly, ad-MVF-seeded Integra exhibited markedly enhanced cellular infiltration, collagen content, microvessel density, and epithelialization compared to non-seeded controls [11,56]. Remarkably, prevascularization of Integra with ad-MVF allowed successful autologous skin grafting already on day 10 after implantation into murine skin defects, while coverage of non-seeded controls resulted in graft necrosis [57].…”

“…These shortcomings led to the development of tissue-engineered dermal substitutes [5]. The porous structure of these matrices provides mechanical support and promotes cellular infiltration and vascular ingrowth from the surrounding host tissue [6]. STSG coverage of the neodermis can be performed either in a one-stage procedure or 3 weeks after vascularization, depending on the substitute [7].…”