Adipose tissue and the vascularization of biomaterials: Stem cells, microvascular fragments and nanofat-a review.

Tissue defects in the human body after trauma and injury require precise reconstruction to regain function. Hence, there is a great demand for clinically translatable approaches with materials that are both biocompatible and biodegradable. They should also be able to adequately integrate within the tissue through sufficient vascularization. Adipose tissue is abundant and easily accessible. It is a valuable tissue source in regenerative medicine and tissue engineering, especially with regard to its angiogenic potential. Derivatives of adipose tissue, such as microfat, nanofat, microvascular fragments, stromal vascular fraction and stem cells, are commonly used in research, but also clinically to enhance the vascularization of implants and grafts at defect sites. In plastic surgery, adipose tissue is harvested via liposuction and can be manipulated in three ways (macro-, micro- and nanofat) in the operating room, depending on its ultimate use. Whereas macro- and microfat are used as a filling material for soft tissue injuries, nanofat is an injectable viscous extract that primarily induces tissue remodeling because it is rich in growth factors and stem cells. In contrast to microfat that adds volume to a defect site, nanofat has the potential to be easily combined with scaffold materials due to its liquid and homogenous consistency and is particularly attractive for blood vessel formation. The same is true for microvascular fragments that are easily isolated from adipose tissue through collagenase digestion. In preclinical animal models, it has been convincingly shown that these vascular fragments inosculate with host vessels and subsequently accelerate scaffold perfusion and host tissue integration. Adipose tissue is also an ideal source of stem cells. It yields larger quantities of cells than any other source and is easier to access for both the patient and doctor compared with other sources such as bone marrow. They are often used for tissue regeneration in combination with biomaterials. Adipose-derived stem cells can be applied unmodified or as single cell suspensions. However, certain pretreatments, such as cultivation under hypoxic conditions or three-dimensional spheroids production, may provide substantial benefit with regard to subsequent vascularization in vivo due to induced growth factor production. In this narrative review, derivatives of adipose tissue and the vascularization of biomaterials are addressed in a comprehensive approach, including several sizes of derivatives, such as whole fat flaps for soft tissue engineering, nanofat or stem cells, their secretome and exosomes. Taken together, it can be concluded that adipose tissue and its fractions down to the molecular level promote, enhance and support vascularization of biomaterials. Therefore, there is a high potential of the individual fat component to be used in regenerative medicine.

The Crucial Role of Vascularization and Lymphangiogenesis in Skin Reconstruction.

BACKGROUND: The treatment of extensive skin defects and bradytrophic wounds remains a challenge in clinical practice. Despite emerging tissue engineering approaches, skin grafts and dermal substitutes are still the routine procedure for the majority of skin defects. Here, we review the role of vascularization and lymphangiogenesis for skin grafting and dermal substitutes from the clinician's perspective. SUMMARY: Graft revascularization is a dynamic combination of inosculation, angiogenesis, and vasculogenesis. The majority of a graft's microvasculature regresses and is replaced by ingrowing microvessels from the wound bed, finally resulting in a chimeric microvascular network. After inosculation within 48-72 h, the graft is re-oxygenated. In contrast to skin grafts, the vascularization of dermal substitutes is slow and dependent on the ingrowth of vessel-forming angiogenic cells. Preclinical angiogenic strategies with adipose tissue-derived isolates are appealing for the treatment of difficult wounds and may markedly accelerate skin reconstruction in the future. However, their translation from bench to bedside is still restricted by major regulatory restrictions. Finally, the lymphatic system contributes to edema reduction and the removal of local wound debris. Therapeutic lymphangiogenesis is an emerging field of research in skin reconstruction. Key Messages: For the successful engraftment of skin grafts and dermal substitutes, the rapid formation of a microvascular network is of pivotal importance. Hence, to understand the biological processes behind revascularization of skin substitutes and to implement this knowledge into clinical practice is a prerequisite when treating skin defects. Furthermore, a functional lymphatic drainage crucially contributes to the engraftment of skin substitutes.

Nanofat Grafting for Scar Treatment and Skin Quality Improvement.

BACKGROUND: Fat grafting has been gaining attention in tissue augmentation over the past decade, not only for lipofilling, but also for its observed regenerative properties and overall skin texture improvement. OBJECTIVES: The aim of this study was to analyze the effect of nanofat grafting on scars, wrinkles, and skin discolorations in our clinic. METHODS: Nanofat was prepared by a standard emulsification and filtration protocol. The resulting liquid was injected intradermally or directly into the scar tissue. Skin quality was evaluated based on a scoring system, and patient satisfaction was documented. Three physicians compared and analyzed standardized pre- and posttreatment photographs in respect to general improvement of skin aesthetics. RESULTS: Fifty-two patients were treated with nanofat from November 2013 to April 2016. The mean (± standard deviation) posttreatment follow up was 155 ± 49 days and average volume of harvested fat amounted to 165 cc. The primary harvesting areas were the abdomen and flanks, and the injected volume of nanofat ranged from 1 to 25 mL (mean, 4.6 mL). A total of 40 scars (76% of all patient defects) were effectively treated as well as 6 patients with wrinkles, and 6 patients with discoloration. Posttreatment clinical evaluations showed a marked improvement of scar quality and a high patient satisfaction. The results in our clinic showed that nanofat grafting softened the scars, made discolorations less pronounced, and wrinkles appeared less prominent. CONCLUSIONS: Nanofat grafting has been shown to have beneficial effects in the treatment of scars, wrinkles, and skin discolorations.

Lipidomic analysis of microfat and nanofat reveal different lipid mediator compositions.

BACKGROUND: Microfat and nanofat are two commonly used techniques in various surgical procedures from skin rejuvenation to scar correction that are known to contribute to tissue regeneration. While microfat mainly contains adipocytes and is well suited for tissue augmentation, nanofat is rich in lipids, adipose derived stem cells, microvascular fragments, and growth factors, making it attractive for esthetic use. We have previously demonstrated that the mechanical processing of microfat into nanofat significantly changes its proteomic profile. Considering that mechanical fractionation leads to adipocyte disruption and lipid release, we aimed to analyze the lipidomic profile for its regenerative properties. METHODS: Microfat and nanofat samples were isolated from fourteen healthy patients. Lipidomic profiling was performed by liquid chromatography tandem mass spectrometry. Resulting data was compared against the Human Metabolome and LIPID MAPS® Structure Database. Metaboanalyst was used to analyze metabolic pathways and lipids of interest. RESULTS: From 2,388 mass-to-charge ratio features, metabolic pathway enrichment analysis between microfat and nanofat samples revealed 109 pathways that were significantly enriched. While microfat samples revealed higher intensity levels of sphingosines, different eicosanoids and fat-soluble vitamins, increased levels of coumaric acids and prostacyclin were found in nanofat. CONCLUSIONS: This is the first study that has analyzed the lipidomic profile of micro- and nanofat, providing evidence that mechanical emulsification of microfat into nanofat leads to changes in their lipid profile. From 109 biological pathways, anti-inflammatory, anti-fibrotic and anti-melanogenic lipid mediators were particularly enriched in nanofat samples when compared to microfat. Although further studies are necessary to have a deeper understanding on the composition of these specific lipid mediators in nanofat samples, we propose that they might contribute to its regenerative effects on tissue.

Protein Profiling of Mechanically Processed Lipoaspirates: Discovering Wound Healing and Antifibrotic Biomarkers in Nanofat.

BACKGROUND: Nanofat is an injectable oily emulsion, rich in adipose-derived stem cells and growth factors. It is prepared from lipoaspirates through mechanical emulsification and filtration. Despite being successfully used in several procedures in regenerative medicine such as scar attenuation, skin rejuvenation, and treatment of chronic wounds, little is known about exactly how nanofat induces regeneration in treated skin at the molecular level. METHODS: Microfat and nanofat samples were isolated from 18 healthy patients. Proteomic profiling was performed through untargeted mass spectrometry proteomics and multiplex antibody arrays. Pathway enrichment analysis of differentially expressed proteins between microfat and nanofat was performed using Gene Ontology, Reactome, and Kyoto Encyclopaedia of Genes and Genomes as reference databases. RESULTS: Untargeted proteomics showed that up-regulated genes in nanofat are involved in innate immunity responses, coagulation, and wound healing, whereas down-regulated genes were linked to cellular migration and extracellular matrix production. Secretome array screening of microfat and nanofat samples showed no significantly different expression, which strongly suggests that the mechanical emulsification step does not affect the concentration of tissue regeneration biomarkers. The identified proteins are involved in wound healing, cellular migration, extracellular matrix remodeling, angiogenesis, stress response, and immune response. CONCLUSIONS: Mechanical processing of lipoaspirates into nanofat significantly influences the proteome profile by enhancing inflammation, antimicrobial, and wound healing pathways. Nanofat is extremely rich in tissue repair and tissue remodeling factors. This study shows that the effects of microfat and nanofat treatment are based on up-regulated inflammation, antimicrobial, and wound healing pathways. Mechanical emulsification does not alter the concentration of tissue regeneration biomarkers. CLINICAL RELEVANCE STATEMENT: In addition to adipose-derived stems cells, nanofat contains distinct tissue repair and remodelling factors, which explains its beneficial effects on tissue regeneration.

Lipoconstruct surface topography grating size influences vascularization onset in the dorsal skinfold chamber model.

After skin tissue injury or pathological removal, vascularization timing is paramount in graft survival. As full thickness skin grafts often fail to become perfused over larger surfaces, split-thickness grafts are preferred and can be used together with biomaterials, which themselves are non-angiogenic. One way of promoting vascular ingrowth is to "pre-vascularize" an engineered substitute by introducing endothelial cells (ECs). Since it has been previously demonstrated that surface structured biomaterials have an effect on wound healing, skin regeneration, and fibrosis reduction, we proposed that a microvascular-rich lipoconstruct with anisotropic topographical cues could be a clinically translatable vascularization approach. Murine lipofragments were formed with three polydimethylsiloxane molds (flat, 5 µm, and 50 µm parallel gratings) and implanted into the dorsal skinfold chamber of male C57BL/6 mice. Vascular ingrowth was observed through intravital microscopy over 21 days and further assessed by histology and protein identification. Our investigation revealed that topographical feature size influenced the commencement of neovascular ingrowth, with 5 µm gratings exhibiting early construct perfusion at 3 days post-operation, and 50 µm being delayed until day 5. We therefore postulate that surface structured lipoconstructs may serve as an easily obtained and produced construct suitable for providing soft tissue and ECs to tissue defects. STATEMENT OF SIGNIFICANCE: Skin graft failures due to inadequate or uneven perfusion frequently occur and can be even more complicated in deep, difficult to heal wounds, or bone coverage. In complex injuries, biomaterials are often used to cover bone structures with a standard split thickness graft; however, perfusion can take up to 3 weeks. Thus, any means to promote faster and uniform vascularization could significantly reduce healing time, as well as lower patient down-time. As pre-vascularized constructs have reported success in research, we created a cost-efficient, translatable method with no additional laboratory time as adipose tissue can be harvested and used immediately. We further used surface topography as an aspect to modulate construct perfusion, which has been reported for the first time here.

High heparin content surface-modified polyurethane discs promote rapid and stable angiogenesis in full thickness skin defects through VEGF immobilization.

Three-dimensional scaffolds have the capacity to serve as an architectural framework to guide and promote tissue regeneration. Parameters such as the type of material, growth factors, and pore dimensions are therefore critical in the scaffold's success. In this study, heparin has been covalently bound to the surface of macroporous polyurethane (PU) discs via two different loading methods to determine if the amount of heparin content had an influence on the therapeutic affinity loading and release of (VEGF165 ) in full thickness skin defects. PU discs (5.4 mm diameter, 300 µm thickness, and interconnected pore size of 150 µm) were produced with either a low (2.5 mg/g) or high (6.6 mg/g) heparin content (LC and HC respectively), and were implanted into the modified dorsal skin chamber (MDSC) of C57BL/6 J mice with and without VEGF. Both low- and high-content discs with immobilized VEGF165 (LCV and HCV, respectively) presented accelerated neovascularization and tissue repair in comparison to heparin discs alone. However, the highest angiogenetic peak was on day 7 with subsequent stabilization for HCV, whereas other groups displayed a delayed peak on day 14. We therefore attribute the superior performance of HCV due to its ability to hold more VEGF165, based on its increased heparin surface coverage, as also demonstrated in VEGF elution dynamics. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2543-2550, 2017.