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

Abstract

Dermal substitutes are frequently used for the initial coverage of extensive skin defects. The seeding of these implants with adipose tissue-derived microvascular fragments (ad-MVF) has recently been shown to accelerate their vascularization and incorporation. In the present study we analyzed whether these processes are affected by a thromboprophylactic therapy with the low molecular weight heparin (LMWH) enoxaparin (enox). Green fluorescent protein (GFP) ad-MVF were isolated from enox- (8 mg/kg s.c.) and vehicle-treated (0.9% NaCl s.c.) (C57BL/6-Tg(CAG-EGFP)1Osb/J mice and seeded onto Integra matrices. Subsequently, these were implanted into full-thickness skin defects within dorsal skinfold chambers of enox- and vehicle-treated C57BL/6 wild-type mice. Repetitive stereomicroscopy and intravital fluorescence microscopy over 2 weeks as well as histological and immunohistochemical analyses on day 14 revealed that enox does not inhibit the reassembly of ad-MVF into new microvascular networks. In addition, treatment with the anticoagulative compound did not promote implant-induced hemorrhage formation. Accordingly, Integra matrices in enox- and vehicle-treated animals exhibited a comparable final microvessel density, fraction of GFP blood vessels originating from seeded ad-MVF, collagen fiber content, and epithelialization. These novel findings demonstrate that the seeding of dermal substitutes with ad-MVF may be applied also during thromboprophylactic therapy without affecting implant vascularization and bleeding risk.

Prevascularization of collagen-glycosaminoglycan scaffolds: stromal vascular fraction versus adipose tissue-derived microvascular fragments

“…As previously reported [ 26 , 27 ], the vascularization of ad-MVF-seeded implants induces hemorrhagic bleeding, which is most pronounced between day 6 and 10 of microvascular network formation. These findings were confirmed in the present study (Fig.…”

“…To measure both epithelialization and implant-induced bleeding of the implanted scaffolds by planimetry, the anesthetized animals were fixed on a Plexiglas® stage and the dorsal skinfold chambers were positioned under a stereomicroscope (Leica M651, Wetzlar, Germany) on day 0 (day of implantation), 3, 6, 10 and 14. Epithelialized and non-epithelialized implant areas (given in %) were visualized by epi-illumination and epithelialization was calculated by the equation: (total implant area – non-epithelialized implant area) / (total implant area) × 100 [ 27 ]. In addition, trans-illumination was used to determine the extent of implant-induced bleeding (given in % of implant surface) by means of a semi-quantitative hemorrhagic score as follows: 1: no bleeding, 2: 1–25%, 3: 26–50%, 4: 51–75%, 5: 76–100%, 6: bleeding exceeding implant surface.…”

“…The observation window of the dorsal skinfold chamber was then 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 the analysis by means of the computer-assisted off-line analysis system CapImage. As previously described [ 27 ], the vascularization of the implanted scaffolds was assessed in 4 ROIs in their center and in 4 ROIs in their border zones (Fig. 1i ).…”

Macrophages promote network formation and maturation of transplanted adipose tissue–derived microvascular fragments

“…Trans-illumination was further used to evaluate the extent of bleeding (given in % of implant surface) induced by the MVF-seeded 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% of the implant surface with bleeding, and 6: bleeding exceeding the implant surface. 22 All microscopic images were recorded by a DVD system and analyzed with the computer-assisted off-line analysis system CapImage (Version 8.5, Zeintl, Heidelberg, Germany). The epithelialized area (given in %) was calculated by the equation as follows: (total implant area − non-epithelialized implant area)/(total implant area) × 100.…”

Altered Response to Total Body Irradiation of C57BL/6-Tg (CAG-EGFP) Mice

“…To verify if there are any altered response of GFP transgenic mice to TBI, a mouse model for RAR (Yonezawa Effect) was adopted and applied to this work using C57BL/6N-GFP mice, the first engineered GFP transgenic mice in the world with applications in a wide range of research fields including radiation biology. 25 , 51 - 63 RAR is a phenomenon that pre-exposure to a low priming dose reduces the biological effects of a subsequent higher challenge dose. Since the discovery of RAR, 64 it has been demonstrated in a variety of biosystems from simple prokaryotes to higher eukaryotes including mammalian animals with varied endpoints including cell death and bone marrow death.…”