Isolation of CD248-expressing stromal vascular fraction for targeted improvement of wound healing.

Wound healing remains a global issue of disability, cost, and health. Addition of cells from the stromal vascular fraction (SVF) of adipose tissue has been shown to increase the rate of full thickness wound closure. This study aimed to investigate the angiogenic mechanisms of CD248+ SVF cells in the context of full thickness excisional wounds. Single cell transcriptional analysis was used to identify and cluster angiogenic gene-expressing cells, which was then correlated with surface marker expression. SVF cells isolated from human lipoaspirate were FACS sorted based on the presence of CD248. Cells were analyzed for angiogenic gene expression and ability to promote microvascular tubule formation in vitro. Following this, 6mm full thickness dermal wounds were created on the dorsa of immunocompromised mice and then treated with CD248+, CD248-, or unsorted SVF cells delivered in a pullalan-collagen hydrogel or the hydrogel alone. Wounds were measured every other day photometrically until closure. Wounds were also evaluated histologically at 7 and 14 days post-wounding and when fully healed to assess for reepithelialization and development of neovasculature. Wounds treated with CD248+ cells healed significantly faster than other treatment groups, and at 7 days, had quantitatively more reepithelialization. Concurrently, immunohistochemistry of CD31 revealed a much higher presence of vascularity in the CD248+ SVF cells treated group at the time of healing and at 14 days post-op, consistent with a pro-angiogenic effect of CD248+ cells in vivo. Therefore, using CD248+ pro-angiogenic cells obtained from SVF presents a viable strategy in wound healing by promoting increased vessel growth in the wound.

Cell-Based Soft Tissue Reconstruction in a Hydrogel Scaffold.

BACKGROUND: Renevia is a hyaluronin-gelatin crosslinked matrix scaffold that has been studied as an alternative to adipose transfer in soft tissue reconstruction. It is designed to emulate the native extracellular matrix environment by supporting stromal vascular fraction (SVF) cell attachment, survival, and proliferation, thus promoting cell-based volume restoration. However, the concentration of incorporated cells for a clinically relevant result has yet to be determined. METHODS: Five experimental groups of seven CD-1 nude immunodeficient mice were given 250 µL grafts of the following composition: 1 million human SVF cells per mL of Renevia scaffold, 6 million human SVF cells per mL scaffold, 12 million human SVF cells per mL scaffold, Renevia scaffold-alone or human adipose tissue-alone. Volumetric analysis was conducted at discrete time points over 16 weeks using 3-dimensional ultrasound, after which time the grafts were explanted for histologic analysis. RESULTS: At the conclusion of the study at week 16, the Renevia scaffold group incorporating the highest concentration of human SVF cells (12 million cells per mL scaffold) had significantly greater volume retention compared with the 2 lower concentrations, scaffold-alone and fat-alone groups. Histology of the 12 million scaffold group revealed abundant adipocyte formation within the scaffold, exceeding that observed in the 6 million, 1 million, and scaffold-alone groups. The 12 million group also demonstrated significantly increased vascularity per CD31 staining. CONCLUSIONS: Stromal vascular fraction cells coupled with Renevia hydrogel scaffold can enhance soft tissue volume reconstruction. In this study, we observed the greatest effect with 12 million cells per mL. From the perspective of volume retention, incorporation of higher concentrations of SVF cells with Renevia may be an alternative to conventional adipose tissue grafting.

Elucidating the fundamental fibrotic processes driving abdominal adhesion formation.

Adhesions are fibrotic scars that form between abdominal organs following surgery or infection, and may cause bowel obstruction, chronic pain, or infertility. Our understanding of adhesion biology is limited, which explains the paucity of anti-adhesion treatments. Here we present a systematic analysis of mouse and human adhesion tissues. First, we show that adhesions derive primarily from the visceral peritoneum, consistent with our clinical experience that adhesions form primarily following laparotomy rather than laparoscopy. Second, adhesions are formed by poly-clonal proliferating tissue-resident fibroblasts. Third, using single cell RNA-sequencing, we identify heterogeneity among adhesion fibroblasts, which is more pronounced at early timepoints. Fourth, JUN promotes adhesion formation and results in upregulation of PDGFRA expression. With JUN suppression, adhesion formation is diminished. Our findings support JUN as a therapeutic target to prevent adhesions. An anti-JUN therapy that could be applied intra-operatively to prevent adhesion formation could dramatically improve the lives of surgical patients.

Fat Grafting into Younger Recipients Improves Volume Retention in an Animal Model.

BACKGROUND: Soft-tissue deficits associated with various craniofacial anomalies can be addressed by fat grafting, although outcomes remain unpredictable. Furthermore, consensus does not exist for timing of these procedures. Whereas some advocate approaching soft-tissue reconstruction after the underlying skeletal foundation has been corrected, other studies have suggested that earlier grafting may exploit a younger recipient niche that is more conducive to fat graft survival. As there is a dearth of research investigating effects of recipient age on fat graft volume retention, this study compared the effectiveness of fat grafting in younger versus older animals through a longitudinal, in vivo analysis. METHODS: Human lipoaspirate from three healthy female donors was grafted subcutaneously over the calvaria of immunocompromised mice. Volume retention over 8 weeks was evaluated using micro-computed tomography at three experimental ages: 3 weeks, 6 months, and 1 year. Histologic examination was performed on explanted grafts to evaluate graft health and vascularity. Recipient-site vascularity was also evaluated by confocal microscopy. RESULTS: The greatest retention of fat graft volume was noted in the youngest group compared with both older groups (p < 0.05) at 6 and 8 weeks after grafting. Histologic and immunohistochemical analyses revealed that improved retention in younger groups was associated with greater fat graft integrity and more robust vascularization. CONCLUSION: The authors' study provides evidence that grafting fat into a younger recipient site correlates with improved volume retention over time, suggesting that beginning soft-tissue reconstruction with fat grafting in patients at an earlier age may be preferable to late correction.

Deferoxamine Preconditioning of Irradiated Tissue Improves Perfusion and Fat Graft Retention.

BACKGROUND: Radiation therapy is a mainstay in the treatment of many malignancies, but collateral damage to surrounding tissue, with resultant hypovascularity, fibrosis, and atrophy, can be difficult to reconstruct. Fat grafting has been shown to improve the quality of irradiated skin, but volume retention of the graft is significantly decreased. Deferoxamine is a U.S. Food and Drug Administration-approved iron-chelating medication for acute iron intoxication and chronic iron overload that has also been shown to increase angiogenesis. The present study evaluates the effects of deferoxamine treatment on irradiated skin and subsequent fat graft volume retention. METHODS: Mice underwent irradiation to the scalp followed by treatment with deferoxamine or saline and perfusion and were analyzed using laser Doppler analysis. Human fat grafts were then placed beneath the scalp and retention was also followed up to 8 weeks radiographically. Finally, histologic evaluation of overlying skin was performed to evaluate the effects of deferoxamine preconditioning. RESULTS: Treatment with deferoxamine resulted in significantly increased perfusion, as demonstrated by laser Doppler analysis and CD31 immunofluorescent staining (p < 0.05). Increased dermal thickness and collagen content secondary to irradiation, however, were not affected by deferoxamine (p > 0.05). Importantly, fat graft volume retention was significantly increased when the irradiated recipient site was preconditioned with deferoxamine (p < 0.05). CONCLUSIONS: The authors' results demonstrated increased perfusion with deferoxamine treatment, which was also associated with improved fat graft volume retention. Preconditioning with deferoxamine may thus enhance fat graft outcomes for soft-tissue reconstruction following radiation therapy.

Utilizing Confocal Microscopy to Characterize Human and Mouse Adipose Tissue.

Significant advances in our understanding of human obesity, endocrinology, and metabolism have been made possible by murine comparative models, in which anatomically analogous fat depots are utilized; however, current research has questioned how truly analogous these depots are. In this study, we assess the validity of the analogy from the perspective of cellular architecture. Whole tissue mounting, confocal microscopy, and image reconstruction software were used to characterize the three-dimensional structure of the inguinal fat pad in mice, gluteofemoral fat in humans, and subcutaneous adipose tissue of the human abdominal wall. Abdominal and gluteofemoral adipose tissue specimens from 12 human patients and bilateral inguinal fat pads from 12 mice were stained for adipocytes, blood vessels, and a putative marker for adipose-derived multipotent progenitor cells, cluster of differentiation 34 (CD34). Samples were whole-mounted and imaged with laser scanning confocal microscopy. Expectedly, human adipocytes were larger and demonstrated greater size heterogeneity. Mouse fat displayed significantly higher vascular density compared with human fat when normalized to adipocyte count. There was no significant difference in the concentration of CD34-positive (CD34+) stromal cells from either species. However, the mean distance between CD34+ stromal cells and blood vessels was significantly greater in human fat. Finally, mouse inguinal fat contained larger numbers of brown adipocytes than did human gluteofemoral or human abdominal fat. Overall, the basic architecture of human adipose tissue differs significantly from that of mice. Insofar as human gluteofemoral fat differs from human abdominal adipose tissue, it was closer to mouse inguinal fat, being its comparative developmental analog. These differences likely confer variance in functional properties between the two sources and thus must be considered when designing murine models of human disease.

Genetic dissection of clonal lineage relationships with hydroxytamoxifen liposomes.

Targeted genetic dissection of tissues to identify precise cell populations has vast biological and therapeutic applications. Here we develop an approach, through the packaging and delivery of 4-hydroxytamoxifen liposomes (LiTMX), that enables localized induction of CreERT2 recombinase in mice. Our method permits precise, in vivo, tissue-specific clonal analysis with both spatial and temporal control. This technology is effective using mice with both specific and ubiquitous Cre drivers in a variety of tissue types, under conditions of homeostasis and post-injury repair, and is highly efficient for lineage tracing and genetic analysis. This methodology is directly and immediately applicable to the developmental biology, stem cell biology and regenerative medicine, and cancer biology fields.

Author Correction: Genetic dissection of clonal lineage relationships with hydroxytamoxifen liposomes.

In the original version of this Article, the authors inadvertently omitted Elizabeth A. Brett, who contributed to the generation of the histology figures, from the author list.This has now been corrected in both the PDF and HTML versions of the Article.

Purified Adipose-Derived Stromal Cells Provide Superior Fat Graft Retention Compared with Unenriched Stromal Vascular Fraction.

Cell-assisted lipotransfer has shown much promise as a technique to improve fat graft retention in both mouse and human studies. However, the literature varies as to whether fresh stromal vascular fraction or culture-expanded adipose-derived stromal cells are used to augment volume retention. The authors' study sought to determine whether there was a significant advantage to using adipose-derived stromal cells over unpurified stromal vascular fraction cells in a mouse model of cell-assisted lipotransfer.

Rapid Isolation of BMPR-IB+ Adipose-Derived Stromal Cells for Use in a Calvarial Defect Healing Model.

Invasive cancers, major injuries, and infection can cause bone defects that are too large to be reconstructed with preexisting bone from the patient's own body. The ability to grow bone de novo using a patient's own cells would allow bony defects to be filled with adequate tissue without the morbidity of harvesting native bone. There is interest in the use of adipose-derived stromal cells (ASCs) as a source for tissue engineering because these are obtained from an abundant source: the patient's own adipose tissue. However, ASCs are a heterogeneous population and some subpopulations may be more effective in this application than others. Isolation of the most osteogenic population of ASCs could improve the efficiency and effectiveness of a bone engineering process. In this protocol, ASCs are obtained from subcutaneous fat tissue from a human donor. The subpopulation of ASCs expressing the marker BMPR-IB is isolated using FACS. These cells are then applied to an in vivo calvarial defect healing assay and are found to have improved osteogenic regenerative potential compared with unsorted cells.

Dynamic Rheology for the Prediction of Surgical Outcomes in Autologous Fat Grafting.

BACKGROUND: Because of the abundance and biocompatibility of fat, lipotransfer has become an attractive method for treating soft-tissue deficits. However, it is limited by unpredictable graft survival and retention. Currently, little is known about the viscoelastic properties of fat after various injection methods. Here, the authors assess the effects of cannula diameter, length, and shape on the viscoelastic properties, structure, and retention of fat. METHODS: Human lipoaspirate was harvested using suction-assisted liposuction and prepared for grafting. A syringe pump was used to inject fat at a controlled flow rate through cannulas of varying gauges, lengths, and shapes. Processed samples were tested in triplicate on an oscillatory rheometer to measure their viscoelastic properties. Fat grafts from each group were placed into the scalps of immunocompromised mice. After 8 weeks, graft retention was measured using micro-computed tomography and grafts were explanted for histologic analysis. RESULTS: Lipoaspirate injected through narrower, longer, and bent cannulas exhibited more shear thinning with diminished quality. The storage modulus (G') of fat processed with 18-gauge cannulas was significantly lower than when processed with 14-gauge or larger cannulas, which also corresponded with inferior in vivo histologic structure. Similarly, the longer cannula group had a significantly lower storage modulus than the shorter cannula, and was associated with decreased graft retention. CONCLUSIONS: Discrete modifications in the methods used for fat placement can have a significant impact on immediate graft integrity, and ultimately on graft survival and quality. Respecting these biomechanical influences during the placement phase of lipotransfer may allow surgeons to optimize outcomes. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, V.

Enrichment of Adipose-Derived Stromal Cells for BMPR1A Facilitates Enhanced Adipogenesis.

BACKGROUND: Reconstruction of soft tissue defects has traditionally relied on the use of grafts and flaps, which may be associated with variable resorption and/or significant donor site morbidity. Cell-based strategies employing adipose-derived stromal cells (ASCs), found within the stromal vascular fraction (SVF) of adipose tissue, may offer an alternative strategy for soft tissue reconstruction. In this study, we investigated the potential of a bone morphogenetic protein receptor type 1A (BMPR1A)(+) subpopulation of ASCs to enhance de novo adipogenesis. METHODS: Human lipoaspirate was enzymatically digested to isolate SVF and magnetic-activated cell separation was utilized to obtain BMPR1A(+) and BMPR1A(-) cells. These cells, along with unenriched cells, were expanded in culture and evaluated for adipogenic gene expression and in vitro adipocyte formation. Cells from each group were also labeled with a green fluorescent protein (GFP) lentivirus and transplanted into the inguinal fat pads, an adipogenic niche, of immunocompromised mice to determine their potential for de novo adipogenesis. Confocal microscopy along with staining of lipid droplets and vasculature was performed to evaluate the formation of mature adipocytes by transplanted cells. RESULTS: In comparison to BMPR1A(-) and unenriched ASCs, BMPR1A(+) cells demonstrated significantly enhanced adipogenesis when cultured in an adipogenic differentiation medium, as evidenced by increased staining with Oil Red O and increased expression of peroxisome proliferator-activating receptor gamma (PPAR-γ) and fatty acid-binding protein 4 (FABP4). BMPR1A(+) cells also formed significantly more adipocytes in vivo, as demonstrated by quantification of GFP+ adipocytes. Minimal formation of mature adipocytes was appreciated by BMPR1A(-) cells. CONCLUSIONS: BMPR1A(+) ASCs show an enhanced ability for adipogenesis in vitro, as shown by gene expression and histological staining. Furthermore, within an adipogenic niche, BMPR1A(+) cells possessed an increased capacity to generate de novo fat compared to BMPR1A(-) and unenriched cells. This suggests utility for the BMPR1A(+) subpopulation in cell-based strategies for soft tissue reconstruction.