Charles P. Blackshear

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‐β‐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

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.

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.

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.

Biomimetics of Bone Implants: The Regenerative Road

Abstract The current strategies for healing bone defects are numerous and varied. At the core of each bone healing therapy is a biomimetic mechanism, which works to enhance bone growth. These range from porous scaffolds, bone mineral usage, collagen, and glycosaminoglycan substitutes to transplanted cell populations. Bone defects face a range of difficulty in their healing, given the composite of dense outer compact bone and blood-rich inner trabecular bone. As such, the tissue possesses a number of inherent characteristics, which may be clinically harnessed as promoters of bone healing. These include mechanical characteristics, mineral composition, native collagen content, and cellular fraction of bone. This review charts multiple biomimetic strategies to help heal bony defects in large and small osseous injury sites, with a special focus on cell transplantation.

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.

Abstract 99: Isolation of CD248 Expressing Adipose Derived Stromal Cells for Targeted Improvement of Wound Healing

PURPOSE: Large surface area wounds, resulting from burns, trauma, or iatrogenic injury, often result in significant scarring and wound contracture. Despite treatment with any of the currently available gold-standard therapies, contracture can still develop as a sequelae of the process of normal wound healing, specifically, myofibroblast activity. Photochemical Tissue Passivation (PTP) is a process that induces collagen cross-linking after a tissue is painted with photosensitizing dye and then exposed to visible light. PTP has also been shown to limit myofibroblast activity in healing surgical wounds. PTP is easy to perform and can be complete in minutes, and has been established as a safe treatment modality in animal and human studies. We hypothesize that PTP treatment to wounds can significantly decrease the morbidities associated with wound contracture by reinforcing the wound bed with collagen cross-linking and limiting the fibrotic response during wound healing.

Abstract 68: Deferoxamine Preconditioning of Irradiated Tissue Increases Fat Graft Volume Retention

PURPOSE: Inflammatory breast cancer (IBC) is an aggressive disease characterized by the formation of tumor emboli, rapid local invasion, and lymphatic dissemination. Furthermore, IBC rapidly develops therapeutic resistance and evades immune surveillance and attack. For these reasons, the treatment of inflammatory breast cancer is extremely challenging and new therapeutic approaches are needed. Numerous studies have shown that adipose derived stem cells (ASCs), which are abundant in breast tissue, are recruited to the tumor microenvironment where they influence tumor progression. We have previously demonstrated the feasibility of using nanoparticles in conjunction with ASCs in treatmentresistant breast cancer. In this study, we show that ASCs localize to IBC tumor emboli and can be used as a targeted delivery vehicle for cancer nanotherapeutics.

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 patients own body. The ability to grow bone de novo using a patients 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 patients 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.

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 &mgr;L 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.