[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: Scar formation, a physiologic process in adult wound healing, can have devastating effects for patients; a multitude of pathologic outcomes, affecting all organ systems, stems from an amplification of this process. In contrast to adult wound repair, the early gestation fetal skin wound heals without scar formation, a phenomenon that appears to be intrinsic to fetal skin. Interestingly, there is strong evidence to suggest healing of oral mucosal wounds parallels that of fetal skin wound repair, and that the relative scarless repair of oral mucosa is also derived primarily from the intrinsic differences in oral mucosal tissue, rather than from the intraoral environment. An intensive research effort has focused on unraveling the mechanisms that underlie scarless fetal wound healing in an attempt to improve the quality of healing in both children and adults. Unique properties of fetal skin and oral mucosal cells, extracellular matrix, cytokine profile, and gene expression contribute to this scarless repair. Despite the great increase in knowledge gained over the past decades, the precise mechanisms regulating scarless wound healing remain unknown. Herein, we describe the current models and proposed mechanisms underlying scarless wound healing in an effort to better understand this privileged phenotype.
[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: Issues surrounding wound healing have garnered deep scientific interest as well as booming financial markets invested in novel wound therapies. Much progress has been made in the field, but it is unsurprising to find that recent successes reveal new challenges to be addressed. With regard to wound healing, large tissue deficits, recalcitrant wounds, and pathological scar formation remain but a few of our most pressing challenges. Stem cell-based therapies have been heralded as a promising means by which to surpass current limitations in wound management. The wide differentiation potential of stem cells allows for the possibility of restoring lost or damaged tissue, while their ability to immunomodulate the wound bed from afar suggests that their clinical applications need not be restricted to direct tissue formation. The clinical utility of stem cells has been demonstrated across dozens of clinical trials in chronic wound therapy, but there is hope that other aspects of wound care will inherit similar benefit. Scientific inquiry into stem cell-based wound therapy abounds in research labs around the world. While their clinical applications remain in their infancy, the heavy investment in their potential makes it a worthwhile subject to review for plastic surgeons, in terms of both their current and future applications.
[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: 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
[Show abstract][Hide abstract] ABSTRACT: Bone formation and skeletal repair are dynamic processes involving a fine-tuned balance between osteoblast proliferation and differentiation orchestrated by multiple signaling pathways. Canonical Wnt (cWnt) signaling is known to playing a key role in these processes. In the current study, using a transgenic mouse model with targeted disruption of axin2, a negative regulator of cWnt signaling, we investigated the impact of enhanced activation of cWnt signaling on the osteogenic capacity and skeletal repair. Specifically, we looked at two calvarial bones of different embryonic tissue origin: the neural crest-derived frontal bone and the mesoderm-derived parietal bone, and we investigated the proliferation and apoptotic activity of frontal and parietal bones and derived osteoblasts. We found dramatic differences in cell proliferation and apoptotic activity between Axin2-/- and wild type calvarial bones, with Axin2-/- showing increased proliferative activity and reduced levels of apoptosis. Furthermore, we compared osteoblast differentiation and bone regeneration in Axin2-/- and wild type neural crest-derived frontal and mesoderm-derived parietal bones, respectively. Our results demonstrate a significant increase either in osteoblast differentiation or bone regeneration in Axin2-/- mice as compared to wild type, with Axin2-/- parietal bone and derived osteoblasts displaying a "neural crest-derived frontal bone-like" profile, which is typically characterized by higher osteogenic capacity and skeletal repair than parietal bone. Taken together, our results strongly suggest that enhanced activation of cWnt signaling increases the skeletal potential of a calvarial bone of mesoderm origin, such as the parietial bone to a degree similar to that of a neural crest origin bone, like the frontal bone. Thus, providing further evidence for the central role played by the cWnt signaling in osteogenesis and skeletal-bone regeneration.
[Show abstract][Hide abstract] ABSTRACT: The postnatal skeleton undergoes growth, remodeling, and repair. We hypothesized that skeletal progenitor cells active during these disparate phases are genetically and phenotypically distinct. We identified a highly potent regenerative cell type that we term the fracture-induced bone, cartilage, stromal progenitor (f-BCSP) in the fracture callus of adult mice. The f-BCSP possesses significantly enhanced skeletogenic potential compared with BCSPs harvested from uninjured bone. It also recapitulates many gene expression patterns involved in perinatal skeletogenesis. Our results indicate that the skeletal progenitor population is functionally stratified, containing distinct subsets responsible for growth, regeneration, and repair. Furthermore, our findings suggest that injury-induced changes to the skeletal stem and progenitor microenvironments could activate these cells and enhance their regenerative potential.
No preview · Article · Aug 2015 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: Background:
Plastic surgery is among the most competitive specialties in medicine, but little is known about the attributes of programs that are most attractive to successful applicants. This study aimed to understand and provide insights regarding program characteristics that are most influential to students when ranking plastic surgery programs.
An anonymous online survey was conducted with newly matched plastic surgery residents for the integrated and combined Match in 2012 and 2013. Subjects were queried regarding their demographics, qualifications, application experiences, and motivations for residency program selection.
A total of 92 of 245 matched plastic surgery residents (38 percent) responded to the survey. The perception of resident happiness was the most positive factor influencing program ranking, followed by high operative volume, faculty mentorship, and strong research infrastructure. Perception of a program as "malignant" was the most negative attribute. Applicants with Step 1 scores greater than 245 received significantly more interviews (p =0.001) and considered resident benefits less important (p < 0.05), but geographic location more important (p =0.005). Applicants who published more than two articles also received more interviews (p =0.001) and ranked a strong research infrastructure and program reputation as significantly more important (p < 0.05). Forty-two percent of applicants completed an away rotation at the program with which they matched, and these applicants were more likely to match at their number one ranked program (p = 0.001).
Plastic surgery applicants have differing preferences regarding the ideal training program, but some attributes resonate. These trends can guide programs for improvement in attracting the best applicants.
No preview · Article · Jun 2015 · Plastic and Reconstructive Surgery