Cell-Assisted Lipotransfer Improves Volume Retention in Irradiated Recipient Sites and Rescues Radiation-Induced Skin Changes.

Radiation therapy is not only 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.

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.

Autologous Fat Grafting: The Science Behind the Surgery.

An invaluable part of the plastic surgeon's technical arsenal for soft tissue contouring, fat grafting continues to be plagued by unpredictable outcomes, resulting in either reoperation and/or patient dissatisfaction. Thus, extensive research has been conducted into the effects of adipose tissue procurement, processing, and placement on fat graft quality at both the cellular level and in terms of overall volume retention. Herein, we present an overview of the vast body of literature in these areas, with additional discussion of cell-assisted lipotransfer as a therapy to improve volume retention, and on the controversial use of autologous fat in the setting of prior irradiation.

Emerging drugs for the treatment of wound healing.

INTRODUCTION: Wound healing can be characterized as underhealing, as in the setting of chronic wounds, or overhealing, occurring with hypertrophic scar formation after burn injury. Topical therapies targeting specific biochemical and molecular pathways represent a promising avenue for improving and, in some cases normalizing, the healing process. AREAS COVERED: A brief overview of both normal and pathological wound healing has been provided, along with a review of the current clinical guidelines and treatment modalities for chronic wounds, burn wounds and scar formation. Next, the major avenues for wound healing drugs, along with drugs currently in development, are discussed. Finally, potential challenges to further drug development, and future research directions are discussed. EXPERT OPINION: The large body of research concerning wound healing pathophysiology has provided multiple targets for topical therapies. Growth factor therapies with the ability to be targeted for localized release in the wound microenvironment are most promising, particularly when they modulate processes in the proliferative phase of wound healing.

Gene expression in fetal murine keratinocytes and fibroblasts.

BACKGROUND: Early fetuses heal wounds without the formation of a scar. Many studies have attempted to explain this remarkable phenomenon. However, the exact mechanism remains unknown. Herein, we examine the predominant cell types of the epidermis and dermis--the keratinocyte and fibroblast--during different stages of fetal development to better understand the changes that lead to scarring wound repair versus regeneration. MATERIALS AND METHODS: Keratinocytes and fibroblasts were harvested and cultured from the dorsal skin of time-dated BALB/c fetuses. Total RNA was isolated and microarray analysis was performed using chips with 42,000 genes. Significance analysis of microarrays was used to select genes with >2-fold expression differences with a false discovery rate<2. Enrichment analysis was performed on significant genes to identify differentially expressed pathways. RESULTS: By comparing the gene expression profile of keratinocytes from E16 versus E18 fetuses, we identified 24 genes that were downregulated at E16. Analysis of E16 and E18 fibroblasts revealed 522 differentially expressed genes. Enrichment analysis showed the top 20 signaling pathways that were downregulated in E16 keratinocytes and upregulated or downregulated in E16 fibroblasts. CONCLUSIONS: Our data reveal 546 differentially expressed genes in keratinocytes and fibroblasts between the scarless and scarring transition. In addition, a total of 60 signaling pathways have been identified to be either upregulated or downregulated in these cell types. The genes and pathways recognized by our study may prove to be essential targets that may discriminate between fetal wound regeneration and adult wound repair.

Critical-size defect ossification: effect of leporid age in a cranioplasty model.

The ability of the human cranium to ossify full-thickness defects depends on the size of the area and the age of the patient. An adult leporid cranioplasty model is commonly used to study inlay cranioplasty materials; the influence of age on ossification is unknown in this model. The purpose of this study was to determine the effect of age on healing of a rabbit critical-size defect. Nineteen rabbits were divided into 4 groups: group 1 (n = 5) aged 4 months, group 2 (n = 4) aged 8 months, group 3 (n = 5) aged 12 months, and group 4 (n = 5) aged 16 months. A 17 × 17-mm defect was created in the parietal bones with preservation of the underlying dura. Animals underwent micro-computed tomography 4 months postoperatively to determine ossification of the defect. Group 1 defects healed by 28.5% (SD, 12.5%), group 2 defects ossified by 37.2% (SD, 5.7%), group 3 defects closed by 28.2% (SD, 11.9%), and group 4 defects healed by 39.4% (SD, 11.0%). No difference in ossification was found between groups (P = 0.31).Leporids as young as 4 months do not close a 17 × 17-mm defect; ossification is similar to animals as old as 16 months. Rabbits 4 months or older are suitable for a calvarial critical-size defect model.

Kinetics of bacterial fluorescence staining with 3,3'-diethylthiacyanine.

For more than a century, colorimetric and fluorescence staining have been the foundation of a broad range of key bioanalytical techniques. The dynamics of such staining processes, however, still remains largely unexplored. We investigated the kinetics of fluorescence staining of two gram-negative and two gram-positive species with 3,3'-diethylthiacyanine (THIA) iodide. An increase in the THIA fluorescence quantum yield, induced by the bacterial dye uptake, was the principal reason for the observed emission enhancement. The fluorescence quantum yield of THIA depended on the media viscosity and not on the media polarity, which suggested that the microenvironment of the dye molecules taken up by the cells was restrictive. The kinetics of fluorescence staining did not manifest a statistically significant dependence neither on the dye concentration, nor on the cell count. In the presence of surfactant additives, however, the fluorescence-enhancement kinetic patterns manifested species specificity with statistically significant discernibility.

Prrx1 Fibroblasts Represent a Pro-fibrotic Lineage in the Mouse Ventral Dermis.

Fibroblast heterogeneity has been shown within the unwounded mouse dorsal dermis, with fibroblast subpopulations being identified according to anatomical location and embryonic lineage. Using lineage tracing, we demonstrate that paired related homeobox 1 (Prrx1)-expressing fibroblasts are responsible for acute and chronic fibroses in the ventral dermis. Single-cell transcriptomics further corroborated the inherent fibrotic characteristics of Prrx1 fibroblasts during wound repair. In summary, we identify and characterize a fibroblast subpopulation in the mouse ventral dermis with intrinsic scar-forming potential.

An Improved Humanized Mouse Model for Excisional Wound Healing Using Double Transgenic Mice.

Objective: Splinting full-thickness cutaneous wounds in mice has allowed for a humanized model of wound healing. Delineating the epithelial edge and assessing time to closure of these healing wounds via macroscopic visualization have remained a challenge. Approach: Double transgenic mice were created by crossbreeding K14-Cre and ROSAmT/mG reporter mice. Full-thickness excisional wounds were created in K14-Cre/ROSAmT/mG mice (n = 5) and imaged using both normal and fluorescent light on the day of surgery, and every other postoperative day (POD) until wound healing was complete. Ten blinded observers analyzed a series of images from a single representative healing wound, taken using normal or fluorescent light, to decide the POD when healing was complete. K14-Cre/ROSAmT/mG mice (n = 4) were subsequently sacrificed at the four potential days of rated wound closure to accurately determine the histological point of wound closure using microscopic fluorescence imaging. Results: Average time to wound closure was rated significantly longer in the wound series images taken using normal light, compared with fluorescent light (mean POD 13.6 vs. 11.6, *p = 0.008). Fluorescence imaging of histological samples indicated that reepithelialization was complete at 12 days postwounding. Innovation: We describe a novel technique, using double transgenic mice K14-Cre/ROSAmT/mG and fluorescence imaging, to more accurately determine the healing time of wounds in mice upon macroscopic evaluation. Conclusion: The accuracy by which wound healing can be macroscopically determined in vivo in mouse models of wound healing is significantly enhanced using K14-Cre/ROSAmT/mG double transgenic mice and fluorescence imaging.

Excess Dermal Tissue Remodeling In Vivo: Does It Settle?

BACKGROUND: Surgical manipulation of skin may result in undesired puckering of excess tissue, which is generally assumed to settle over time. In this article, the authors address the novel question of how this excess tissue remodels. METHODS: Purse-string sutures (6-0 nylon) were placed at the midline dorsum of 22 wild-type BALB/c mice in a circular pattern marked with tattoo ink. Sutures were cinched and tied under tension in the treatment group, creating an excess tissue deformity, whereas control group sutures were tied without tension. After 2 or 4 weeks, sutures were removed. The area of tattooed skin was measured up to 56 days after suture removal. Histologic analysis was performed on samples harvested 14 days after suture removal. RESULTS: The majority of excess tissue deformities flattened within 2 days after suture removal. However, the sutured skin in the treatment group decreased in area by an average of 18 percent from baseline (n = 9), compared to a 1 percent increase in the control group (n = 10) at 14 days after suture removal (p < 0.05). This was similarly observed at 28 days (treatment, -11.7 percent; control, 4.5 percent; n = 5; p = 0.0243). Despite flattening, deformation with purse-string suture correlated with increased collagen content of skin, in addition to increased numbers of myofibroblasts. Change in area did not correlate with duration of suture placement. CONCLUSIONS: Excess dermal tissue deformities demonstrate the ability to remodel with gross flattening of the skin, increased collagen deposition, and incomplete reexpansion to baseline area. Further studies will reveal whether our findings in this mouse model translate to humans.

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.

Magnetic Nanoparticle-Based Upregulation of B-Cell Lymphoma 2 Enhances Bone Regeneration.

Clinical translation of cell-based strategies for tissue regeneration remains challenging because survival of implanted cells within hostile, hypoxic wound environments is uncertain. Overexpression of B-cell lymphoma 2 (Bcl-2) has been shown to inhibit apoptosis in implanted cells. The present study describes an "off the shelf" prefabricated scaffold integrated with magnetic nanoparticles (MNPs) used to upregulate Bcl-2 expression in implanted adipose-derived stromal cells for bone regeneration. Iron oxide cores were sequentially coated with branched polyethyleneimine, minicircle plasmid encoding green fluorescent protein and Bcl-2, and poly-ß-amino ester. Through in vitro assays, increased osteogenic potential and biological resilience were demonstrated in the magnetofected group over control and nucleofected groups. Similarly, our in vivo calvarial defect study showed that magnetofection had an efficiency rate of 30%, which in turn resulted in significantly more healing compared with control group and nucleofected group. Our novel, prefabricated MNP-integrated scaffold allows for in situ postimplant temporospatial control of cell transfection to augment bone regeneration. Stem Cells Translational Medicine 2017;6:151-160.

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.

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.

The role of stem cells in limb regeneration.

Limb regeneration is a complex yet fascinating process observed to some extent in many animal species, though seen in its entirety in urodele amphibians. Accomplished by formation of a morphologically uniform intermediate, the blastema, scientists have long attempted to define the cellular constituents that enable regrowth of a functional appendage. Today, we know that the blastema consists of a variety of multipotent progenitor cells originating from a variety of tissues, and which contribute to limb tissue regeneration in a lineage-restricted manner. By continuing to dissect the role of stem cells in limb regeneration, we can hope to one day modulate the human response to limb amputation and facilitate regrowth of a working replacement.

Stem Cells in Bone Regeneration.

Bone has the capacity to regenerate and repair itself. However, this capacity may be impaired or lost depending on the size of the defect or the presence of certain disease states. In this review, we discuss the key principles underlying bone healing, efforts to characterize bone stem and progenitor cell populations, and the current status of translational and clinical studies in cell-based bone tissue engineering. Though barriers to clinical implementation still exist, the application of stem and progenitor cell populations to bone engineering strategies has the potential to profoundly impact regenerative medicine.

Scaffold-mediated BMP-2 minicircle DNA delivery accelerated bone repair in a mouse critical-size calvarial defect model.

Scaffold-mediated gene delivery holds great promise for tissue regeneration. However, previous attempts to induce bone regeneration using scaffold-mediated non-viral gene delivery rarely resulted in satisfactory healing. We report a novel platform with sustained release of minicircle DNA (MC) from PLGA scaffolds to accelerate bone repair. MC was encapsulated inside PLGA scaffolds using supercritical CO2 , which showed prolonged release of MC. Skull-derived osteoblasts transfected with BMP-2 MC in vitro result in higher osteocalcin gene expression and mineralized bone formation. When implanted in a critical-size mouse calvarial defect, scaffolds containing luciferase MC lead to robust in situ protein production up to at least 60 days. Scaffold-mediated BMP-2 MC delivery leads to substantially accelerated bone repair as early as two weeks, which continues to progress over 12 weeks. This platform represents an efficient, long-term nonviral gene delivery system, and may be applicable for enhancing repair of a broad range of tissues types. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2099-2107, 2016.

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.

Surveillance of Stem Cell Fate and Function: A System for Assessing Cell Survival and Collagen Expression In Situ.

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 an insight into the mechanisms of how both transplanted and endogenous cells mediate skeletal healing, we used 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 (Col1a1(GFP)) and membrane-bound tomato red fluorescent protein constitutively in all cell types (R26(mTmG)). 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. Using 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.

Short Hairpin RNA Silencing of PHD-2 Improves Neovascularization and Functional Outcomes in Diabetic Wounds and Ischemic Limbs.

The transcription factor hypoxia-inducible factor 1-alpha (HIF-1α) is responsible for the downstream expression of over 60 genes that regulate cell survival and metabolism in hypoxic conditions as well as those that enhance angiogenesis to alleviate hypoxia. However, under normoxic conditions, HIF-1α is hydroxylated by prolyl hydroxylase 2, and subsequently degraded, with a biological half-life of less than five minutes. Here we investigated the therapeutic potential of inhibiting HIF-1α degradation through short hairpin RNA silencing of PHD-2 in the setting of diabetic wounds and limb ischemia. Treatment of diabetic mouse fibroblasts with shPHD-2 in vitro resulted in decreased levels of PHD-2 transcript demonstrated by qRT-PCR, higher levels of HIF-1α as measured by western blot, and higher expression of the downstream angiogenic genes SDF-1 and VEGFα, as measured by qRT-PCR. In vivo, shPHD-2 accelerated healing of full thickness excisional wounds in diabetic mice compared to shScr control, (14.33 ± 0.45 days vs. 19 ± 0.33 days) and was associated with an increased vascular density. Delivery of shPHD-2 also resulted in improved perfusion of ischemic hind limbs compared to shScr, prevention of distal digit tip necrosis, and increased survival of muscle tissue. Knockdown of PHD-2 through shRNA treatment has the potential to stimulate angiogenesis through overexpression of HIF-1α and upregulation of pro-angiogenic genes downstream of HIF-1α, and may represent a viable, non-viral approach to gene therapy for ischemia related applications.