[Show abstract][Hide abstract] ABSTRACT: Background: Sutureless microvascular anastomosis has great translational potential to simplify microvascular surgery, shorten operative times, and improve clinical outcomes. The authors developed a transient thermoreversible microvascular stent using a poloxamer to maintain vessel lumen patency before application of commercially available adhesives to seal the anastomosis instead of sutures. Despite technical success, human application necessitates bovine serum albumin removal from existing formulations; rapid poloxamer transition between states; and increased stiffness for reliable, reproducible, and precise microvascular approximation. Methods: Two commercially available poloxamers were used in this study (P407 and P188). After removing bovine serum albumin, each poloxamer was tested at varying concentrations either alone or in combination to determine the optimal preparation for sutureless microvascular anastomosis. Transition temperature and formulation stiffness were tested in vitro by rheometry, with the most promising combinations tested in an established in vivo model. Results: Increasing poloxamer concentration resulted in an increase in stiffness and decrease in transition temperature. Pure P188 without bovine serum albumin, dissolved in phosphate-buffered saline to a 45% concentration, demonstrated desirable rheologic behavior, with precise gel transition and increased gel stiffness compared with our previous formulation of 17% P407 (96 kPa versus 10 kPa). These characteristics were optimal for microsurgical intravascular use, offering surgical precision and control between liquid and solid states, depending on the surgically controlled local temperature. Conclusions: Use of 45% P188 without bovine serum albumin demonstrated optimal rheologic and translational properties as a microvascular stent for sutureless anastomosis. Rapid transition, increased stiffness, and safety profile demonstrate safe translational application for human clinical trials.
Full-text · Article · Feb 2016 · Plastic & Reconstructive Surgery
[Show abstract][Hide abstract] ABSTRACT: Adipose-derived mesenchymal stem cells (ASCs) are appealing for cell-based wound therapies because of their accessibility and ease of harvest, but their utility is limited by poor cell survival within the harsh wound microenvironment. In prior work, our laboratory has demonstrated that seeding ASCs within a soft pullulan-collagen hydrogel enhances ASC survival and improves wound healing. To more fully understand the mechanism of this therapy, we examined whether ASC-seeded hydrogels were able to modulate the recruitment and/or functionality of endogenous progenitor cells. Employing a parabiosis model and fluorescence-activated cell sorting (FACS) analysis, we demonstrate that application of ASC-seeded hydrogels to wounds, when compared to injected ASCs or a non-cell control, increased the recruitment of pro-vascular circulating bone marrow-derived mesenchymal progenitor cells (BM-MPCs). BM-MPCs comprised 23.0% of recruited circulating progenitor cells in wounds treated with ASC-seeded hydrogels, versus 8.4% and 2.1% in those treated with controls, p<0.05). Exploring the potential for functional modulation of BM-MPCs, we demonstrate a statistically significant increase in BM-MPC migration, proliferation, and tubulization when exposed to hydrogel-seeded ASC conditioned medium vs control ASC conditioned medium (73.8% vs. 51.4% scratch assay closure; 9.1% vs. 1.4% proliferation rate; 10.2 vs. 5.5 tubules/HPF; p<0.05 for all assays). BM-MPC expression of genes related to cell stemness and angiogenesis was also significantly increased following exposure to hydrogel-seeded ASC conditioned medium (p<0.05). These data suggest that ASC-seeded hydrogels improve both progenitor cell recruitment and functionality to effect greater neovascularization.
No preview · Article · Jan 2016 · Tissue Engineering Part A
[Show abstract][Hide abstract] ABSTRACT: Fibroblasts are the principle cell type responsible for secreting extracellular matrix and are a critical component of many organs and tissues. Fibroblast physiology and pathology underlie a spectrum of clinical entities, including fibroses in multiple organs, hypertrophic scarring following burns, loss of cardiac function following ischemia, and the formation of cancer stroma. However, fibroblasts remain a poorly characterized type of cell, largely due to their inherent heterogeneity. Existing methods for the isolation of fibroblasts require time in cell culture that profoundly influences cell phenotype and behavior. Consequently, many studies investigating fibroblast biology rely upon in vitro manipulation and do not accurately capture fibroblast behavior in vivo. To overcome this problem, we developed a FACS-based protocol for the isolation of fibroblasts from the dorsal skin of adult mice that does not require cell culture, thereby preserving the physiologic transcriptional and proteomic profile of each cell. Our strategy allows for exclusion of non-mesenchymal lineages via a lineage negative gate (Lin(-)) rather than a positive selection strategy to avoid pre-selection or enrichment of a subpopulation of fibroblasts expressing specific surface markers and be as inclusive as possible across this heterogeneous cell type.
No preview · Article · Jan 2016 · Journal of Visualized Experiments
[Show abstract][Hide abstract] ABSTRACT: Significance:
Adipose-derived mesenchymal stem cells (ASCs) are an appealing source of therapeutic progenitor cells because of their multipotency, diverse cytokine profile, and ease of harvest via liposuction. Alternative approaches to classical suction-assisted liposuction are gaining popularity; however, little evidence exists regarding the impact of different liposuction methods on the regenerative functionality of ASCs. Human ASC characteristics and regenerative capacity were assessed when harvested via ultrasound-assisted (UAL) versus standard suction-assisted liposuction. ASCs obtained via UAL were of equal quality when directly compared with the current gold standard harvest method. UAL is an adjunctive source of fully functional mesenchymal stem cells for applications in basic research and clinical therapy.
No preview · Article · Dec 2015 · STEM CELLS TRANSLATIONAL MEDICINE
[Show abstract][Hide abstract] ABSTRACT: Advanced age is characterized by impairments in wound healing and evidence is accumulating that this may be due in part to a concomitant increase in oxidative stress. Extended exposure to reactive oxygen species (ROS) is thought to lead to cellular dysfunction and organismal death via the destructive oxidation of intracellular proteins, lipids, and nucleic acids. Extracellular superoxide dismutase (ecSOD/ SOD3) is a prime antioxidant enzyme in the extracellular space that eliminates ROS. Here, we demonstrate that reduced SOD3 levels contribute to healing impairments in aged mice. These impairments include delayed wound closure, reduced neovascularization, impaired fibroblast proliferation, and increased neutrophil recruitment. We further establish that SOD3 KO and aged fibroblasts both display reduced production of TGFβ1, leading to decreased differentiation of fibroblasts into myofibroblasts. Taken together these results suggest that wound healing impairments in aging are associated with increased levels of ROS, decreased SOD3 expression and impaired extracellular oxidative stress regulation. Our results identify SOD3 as a possible target to correct age-related cellular dysfunction in wound healing. This article is protected by copyright. All rights reserved.
No preview · Article · Dec 2015 · Experimental Dermatology
[Show abstract][Hide abstract] ABSTRACT: The superior aesthetic outcomes of nipple-sparing mastectomies (NSM) explain their increased use and rising popularity. Fortunately, cancer recurrences involving the nipple-areolar complex (NAC) have been reassuringly low in the range of 1%. Technical considerations and challenges of this procedure are centered on nipple ischemia and necrosis. Patient selection, reconstructive strategies and incision placement have lowered ischemic complications. In this context, rates of full NAC necrosis are 3% or less. The emergence of noninvasive tissue angiography provides surgeons with a practical tool to assess real-time breast skin and NAC perfusion. Herein, we review our classification system of NAC perfusion patterns defined as V1 (from subjacent breast), V2 (surrounding skin), and V3 (combination of V1 + V2). Additionally, we describe the benefits of a first stage operation to devascularize the NAC as a means of improving blood flow to the NAC in preparation for NSM, helping extend the use of NSM to more women. Intraoperative evaluation of skin perfusion allows surgeons to detect ischemia and modify the operative approach to optimize outcomes.
[Show abstract][Hide abstract] ABSTRACT: Radiation therapy is a mainstay in the treatment of many malignancies, but also results in collateral obliteration of microvasculature and dermal/subcutaneous fibrosis. Soft tissue reconstruction of hypovascular, irradiated recipient sites through fat grafting remains challenging, however a coincident improvement in surrounding skin quality has been noted. Cell-assisted lipotransfer (CAL), the enrichment of fat with additional adipose-derived stem cells (ASCs) from the stromal vascular fraction, has been shown to improve fat volume retention, and enhanced outcomes may also be achieved with CAL at irradiated sites. Supplementing fat grafts with additional ASCs may also augment the regenerative effect on radiation-damaged skin. In this study, we demonstrate the ability for CAL to enhance fat graft volume retention when placed beneath the irradiated scalps of immunocompromised mice. Histologic metrics of fat graft survival were also appreciated, with improved structural qualities and vascularity. Finally, rehabilitation of radiation-induced soft tissue changes were also noted, as enhanced amelioration of dermal thickness, collagen content, skin vascularity, and biomechanical measures were all observed with CAL compared to unsupplemented fat grafts. Supplementation of fat grafts with ASCs therefore shows promise for reconstruction of complex soft tissue defects following adjuvant radiotherapy. This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: 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 (BMPR)-IA(+) subpopulation of ASCs to enhance de novo adipogenesis.
Human lipoaspirate was enzymatically digested to isolate SVF and magnetic-activated cell separation was utilized to obtain BMPRIA(+) and BMPRIA(-) 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 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 formation of mature adipocytes by transplanted cells.
In comparison to BMPR1A(-) and unenriched ASCs, BMPRIA(+) cells demonstrated significantly enhanced adipogenesis when cultured in adipogenic differentiation medium, as evidenced by increased staining with Oil Red O and increased expression of PPAR-γ and FABP4. BMPRIA(+) cells also formed significantly more adipocytes in vivo, as demonstrated by quantification of GFP+ adipocytes and Image J analysis. Minimal formation of mature adipocytes was appreciated by BMPRIA(-) cells.
BMPRIA(+) ASCs show an enhanced ability for adipogenesis in vitro, as shown by gene expression and histological staining. Furthermore, within an adipogenic niche, BMPRIA(+) cells possessed an increased capacity to generate de novo fat compared to BMPRIA(-) and unenriched cells. This suggests utility for the BMPRIA(+) subpopulation in cell-based strategies for soft tissue reconstruction.
No preview · Article · Nov 2015 · Tissue Engineering Part A
[Show abstract][Hide abstract] ABSTRACT: Fibroblasts have a central role in wound healing via matrix production, remodeling, and contraction. Their role as mechanoresponsive cells during tissue repair is evident, but the molecular mechanisms of this process remain uncertain. Filamin A, an intracellular protein that stabilizes the actin cytoskeleton regulates fibroblast-matrix interactions. Fibroblast defects in cytoskeletal dynamics may underlie key aspects of chronic wound pathophysiology.
No preview · Article · Nov 2015 · Journal of Investigative Dermatology
[Show abstract][Hide abstract] ABSTRACT: Background:
Diabetes and aging are known risk factors for impaired neovascularization in response to ischemic insult, resulting in chronic wounds, and poor outcomes following myocardial infarction and cerebrovascular injury. Hypoxia-inducible factor (HIF)-1α, has been identified as a critical regulator of the response to ischemic injury and is dysfunctional in diabetic and elderly patients. To better understand the role of this master hypoxia regulator within cutaneous tissue, the authors generated and evaluated a fibroblast-specific HIF-1α knockout mouse model.
The authors generated floxed HIF-1 mice (HIF-1) by introducing loxP sites around exon 1 of the HIF-1 allele in C57BL/6J mice. Fibroblast-restricted HIF-1α knockout (FbKO) mice were generated by breeding our HIF-1 with tamoxifen-inducible Col1a2-Cre mice (Col1a2-CreER). HIF-1α knockout was evaluated on a DNA, RNA, and protein level. Knockout and wild-type mice were subjected to ischemic flap and wound healing models, and CD31 immunohistochemistry was performed to assess vascularity of healed wounds.
Quantitative real-time polymerase chain reaction of FbKO skin demonstrated significantly reduced Hif1 and Vegfa expression compared with wild-type. This finding was confirmed at the protein level (p < 0.05). HIF-1α knockout mice showed significantly impaired revascularization of ischemic tissue and wound closure and vascularity (p < 0.05).
Loss of HIF-1α from fibroblasts results in delayed wound healing, reduced wound vascularity, and significant impairment in the ischemic neovascular response. These findings provide new insight into the importance of cell-specific responses to hypoxia during cutaneous neovascularization.
No preview · Article · Oct 2015 · Plastic and Reconstructive Surgery
[Show abstract][Hide abstract] ABSTRACT: Cell based therapy is an emerging paradigm in skeletal regenerative medicine. However, the primary means by which transplanted cells contribute to bone repair and regeneration remain controversial. To gain insight into the mechanisms of how both transplanted and endogenous cells mediate skeletal healing, we utilized a transgenic mouse strain expressing both the topaz variant of green fluorescent protein under the control of the collagen type I alpha 1 promoter/enhancer sequence (Col1a1GFP) and membrane-bound tomato red constitutively in all cells types (R26mTmG). A comparison of healing in parietal versus frontal calvarial defects in these mice revealed that frontal osteoblasts express Col1a1 to a greater degree than parietal osteoblasts. Furthermore, the scaffold-based application of adipose-derived stromal cells (ASCs), bone marrow-derived mesenchymal stem cells (BM-MSCs), and osteoblasts derived from these mice to critical sized calvarial defects allowed for investigation of cell survival and function following transplantation. We found that ASCs led to significantly faster rates of bone healing in comparison to BM-MSCs and osteoblasts. ASCs displayed both increased survival and increased Col1a1 expression compared to BM-MSCs and osteoblasts following calvarial defect transplantation, which may explain their superior regenerative capacity in the context of bone healing. By utilizing this novel reporter system we were able to elucidate how cell-based therapies impact bone healing and identify ASCs as an attractive candidate for cell-based skeletal regenerative therapy. These insights potentially influence stem cell selection in translational clinical trials evaluating cell-based therapeutics for osseous repair and regeneration.
Full-text · Article · Oct 2015 · Tissue Engineering Part A