Evolving Role of Exosomes in Plastic and Reconstructive Surgery and Dermatology.

Exosomes, or extracellular vesicles, represent the latest cell-free addition to the regenerative medicine toolkit. In vitro preclinical studies have demonstrated the safety and efficacy of exosomes, which vary based on source and biomanufacturing, for a myriad of potential therapeutic applications relevant to skin and soft tissue reconstruction. Primary search was performed in September 2021 on the MEDLINE database via PubMed and Ovid, with focus on articles about therapeutic application of exosomes or extracellular vesicles. In total, 130 articles met criteria for applicability, including early-stage clinical trials, preclinical research studies with in vivo application, and articles applicable to plastic and reconstructive surgery and dermatology. Most studies used animal models of human disease processes, using either animal donor cells to isolate exosomes, or human donor cells in animal models. Exosome technology has catapulted as an acellular therapeutic vehicle with off-the-shelf accessibility. These features eliminate prior threshold for broad adoption of regenerative cell-based therapies into surgical and medical practice. To date, there are no exosome products approved by the US Food and Drug Administration. This review highlights exosomes as the new frontier in regenerative medicine and outlines its preclinical therapeutic applications for cutaneous repair and restoration.

Nebulized platelet-derived extracellular vesicles attenuate chronic cigarette smoke-induced murine emphysema.

Chronic obstructive pulmonary disease (COPD) is a prevalent lung disease usually resulting from cigarette smoking (CS). Cigarette smoking induces oxidative stress, which causes inflammation and alveolar epithelial cell apoptosis and represents a compelling therapeutic target for COPD. Purified human platelet-derived exosome product (PEP) is endowed with antioxidant enzymes and immunomodulatory molecules that mediate tissue repair. In this study, a murine model of CS-induced emphysema was used to determine whether nebulized PEP can influence the development of CS-induced emphysema through the mitigation of oxidative stress and inflammation in the lung. Nebulization of PEP effectively delivered the PEP vesicles into the alveolar region, with evidence of their uptake by type I and type II alveolar epithelial cells and macrophages. Lung function testing and morphometric assessment showed a significant attenuation of CS-induced emphysema in mice treated with nebulized PEP thrice weekly for 4 weeks. Whole lung immuno-oncology RNA sequencing analysis revealed that PEP suppressed several CS-induced cell injuries and inflammatory pathways. Validation of inflammatory cytokines and apoptotic protein expression on the lung tissue revealed that mice treated with PEP had significantly lower levels of S100A8/A9 expressing macrophages, higher levels of CD4+/FOXP3+ Treg cells, and reduced NF-κB activation, inflammatory cytokine production, and apoptotic proteins expression. Further validation using in vitro cell culture showed that pretreatment of alveolar epithelial cells with PEP significantly attenuated CS extract-induced apoptotic cell death. These data show that nebulization of exosomes like PEP can effectively deliver exosome cargo into the lung, mitigate CS-induced emphysema in mice, and suppress oxidative lung injury, inflammation, and apoptotic alveolar epithelial cell death.

The lysine methyltransferases SET and MYND domain containing 2 (Smyd2) and Enhancer of Zeste 2 (Ezh2) co-regulate osteoblast proliferation and mineralization.

Bone formation is controlled by histone modifying enzymes that regulate post-translational modifications on nucleosomal histone proteins and control accessibility of transcription factors to gene promoters required for osteogenesis. Enhancer of Zeste homolog 2 (EZH2/Ezh2), a histone H3 lysine 27 (H3K27) methyl transferase, is a suppressor of osteoblast differentiation. Ezh2 is regulated by SET and MYND domain-containing protein 2 (SMYD2/Smyd2), a lysine methyltransferase that modifies both histone and non-histone proteins. Here, we examined whether Smyd2 modulates Ezh2 suppression of osteoblast differentiation. Musculoskeletal RNA-seq data show that SMYD2/Smyd2 is the most highly expressed SMYD/Smyd member in human bone tissues and mouse osteoblasts. Smyd2 loss of function analysis in mouse MC3T3 osteoblasts using siRNA depletion enhances proliferation and calcium deposition. Loss of Smyd2 protein does not affect alkaline phosphatase activity nor does it result in a unified expression response for standard osteoblast-related mRNA markers (e.g., Bglap, Ibsp, Spp1, Sp7), indicating that Smyd2 does not directly control osteoblast differentiation. Smyd2 protein depletion enhances levels of the osteo-suppressive Ezh2 protein and H3K27 trimethylation (H3K27me3), as expected from increased cell proliferation, while elevating the osteo-inductive Runx2 protein. Combined siRNA depletion of both Smyd2 and Ezh2 protein is more effective in promoting calcium deposition when compared to loss of either protein. Collectively, our results indicate that Smyd2 inhibits proliferation and indirectly the subsequent mineral deposition by osteoblasts. Mechanistically, Smyd2 represents a functional epigenetic regulator that operates in parallel to the suppressive effects of Ezh2 and H3K27 trimethylation on osteoblast differentiation.

Exosome biopotentiated hydrogel restores damaged skeletal muscle in a porcine model of stress urinary incontinence.

Urinary incontinence afflicts up to 40% of adult women in the United States. Stress urinary incontinence (SUI) accounts for approximately one-third of these cases, precipitating ~200,000 surgical procedures annually. Continence is maintained through the interplay of sub-urethral support and urethral sphincter coaptation, particularly during activities that increase intra-abdominal pressure. Currently, surgical correction of SUI focuses on the re-establishment of sub-urethral support. However, mesh-based repairs are associated with foreign body reactions and poor localized tissue healing, which leads to mesh exposure, prompting the pursuit of technologies that restore external urethral sphincter function and limit surgical risk. The present work utilizes a human platelet-derived CD41a and CD9 expressing extracellular vesicle product (PEP) enriched for NF-κB and PD-L1 and derived to ensure the preservation of lipid bilayer for enhanced stability and compatibility with hydrogel-based sustained delivery approaches. In vitro, the application of PEP to skeletal muscle satellite cells in vitro drove proliferation and differentiation in an NF-κB-dependent fashion, with full inhibition of impact on exposure to resveratrol. PEP biopotentiation of collagen-1 and fibrin glue hydrogel achieved sustained exosome release at 37 °C, creating an ultrastructural "bead on a string" pattern on scanning electron microscopy. Initial testing in a rodent model of latissimus dorsi injury documented activation of skeletal muscle proliferation of healing. In a porcine model of stress urinary incontinence, delivery of PEP-biopotentiated collagen-1 induced functional restoration of the external urethral sphincter. The histological evaluation found that sustained PEP release was associated with new skeletal muscle formation and polarization of local macrophages towards the regenerative M2 phenotype. The results provided herein serve as the first description of PEP-based biopotentiation of hydrogels implemented to restore skeletal muscle function and may serve as a promising approach for the nonsurgical management of SUI.

MicroRNA-101a enhances trabecular bone accrual in male mice.

High-throughput microRNA sequencing was performed during differentiation of MC3T3-E1 osteoblasts to develop working hypotheses for specific microRNAs that control osteogenesis. The expression data show that miR-101a, which targets the mRNAs for the epigenetic enzyme Ezh2 and many other proteins, is highly upregulated during osteoblast differentiation and robustly expressed in mouse calvaria. Transient elevation of miR-101a suppresses Ezh2 levels, reduces tri-methylation of lysine 27 in histone 3 (H3K27me3; a heterochromatic mark catalyzed by Ezh2), and accelerates mineralization of MC3T3-E1 osteoblasts. We also examined skeletal phenotypes of an inducible miR-101a transgene under direct control of doxycycline administration. Experimental controls and mir-101a over-expressing mice were exposed to doxycycline in utero and postnatally (up to 8 weeks of age) to maximize penetrance of skeletal phenotypes. Male mice that over-express miR-101a have increased total body weight and longer femora. MicroCT analysis indicate that these mice have increased trabecular bone volume fraction, trabecular number and trabecular thickness with reduced trabecular spacing as compared to controls. Histomorphometric analysis demonstrates a significant reduction in osteoid volume to bone volume and osteoid surface to bone surface. Remarkably, while female mice also exhibit a significant increase in bone length, no significant changes were noted by microCT (trabecular bone parameters) and histomorphometry (osteoid parameters). Hence, miR-101a upregulation during osteoblast maturation and the concomitant reduction in Ezh2 mediated H3K27me3 levels may contribute to the enhanced trabecular bone parameters in male mice. However, the sex-specific effect of miR-101a indicates that more intricate epigenetic mechanisms mediate physiological control of bone formation and homeostasis.

Efficacy and Tolerability of Topical Platelet Exosomes for Skin Rejuvenation: Six-Week Results.

BACKGROUND: Exosomes are regenerative mediators for skin rejuvenation. Human platelet extract (HPE) is an allogeneic exosome product derived from US-sourced, leukocyte-reduced apheresed platelets with consistent purity and potency. OBJECTIVES: The authors sought to better characterize the safety and tolerability of novel HPE (plated) Intensive Repair Serum (Rion Aesthetics, Rochester, MN) and its maximal effects on skin rejuvenation at 6 weeks. METHODS: This prospective, single-arm, non-randomized, longitudinal study investigated the safety and efficacy of HPE. Structured sub-analysis evaluated multifactorial improvement in skin health following standardized skin care regimen to determine the maximal effect. Evaluation at baseline and 6 weeks included participant questionnaires and photo documentation with VISIA-CR Generation 5 3D PRIMOS (Canfield Scientific Inc, Fairfield, NJ). RESULTS: VISIA-CR imaging yielded quantifiable and statistically significant improvements in overall skin health (skin health score). A greater score correlated to greater overall skin health, and there was a statistically significant mean delta improvement of 224.2 ± 112.8 (mean ± standard deviation, P ≤ 0.0001) in skin health score at 6 weeks compared with baseline. This correlated to reduction in redness, wrinkles, and melanin production across all cosmetic units (P = 0.005, P = 0.0023, P ≤ 0.0001, respectively) and significant improvements in luminosity and color evenness (P ≤ 0.001). CONCLUSIONS: A topically applied platelet-derived exosome product, HPE, induced normalization to skin health at 4 to 6 weeks with improved various clinical measures of facial photodamage and cutaneous aging. It is safe, well-tolerated, and well-liked by participants.

Brd4 is required for chondrocyte differentiation and endochondral ossification.

Differentiation of multi-potent mesenchymal stromal cells (MSCs) is directed by the activities of lineage-specific transcription factors and co-factors. A subset of these proteins controls the accessibility of chromatin by recruiting histone acetyl transferases or deacetylases that regulate acetylation of the N-termini of H3 and H4 histone proteins. Bromodomain (BRD) proteins recognize these acetylation marks and recruit the RNA pol II containing transcriptional machinery. Our previous studies have shown that Brd4 is required for osteoblast differentiation in vitro. Here, we investigated the role of Brd4 on endochondral ossification in C57BL/6 mice and chondrogenic differentiation in cell culture models. Conditional loss of Brd4 in the mesenchyme (Brd4 cKO, Brd4fl/fl: Prrx1-Cre) yields smaller mice that exhibit alteration in endochondral ossification. Importantly, abnormal growth plate morphology and delayed long bone formation is observed in juvenile Brd4 cKO mice. One week old Brd4 cKO mice have reduced proliferative and hypertrophic zones within the physis and exhibit a delay in the formation of the secondary ossification center. At the cellular level, Brd4 function is required for chondrogenic differentiation and maturation of both ATDC5 cells and immature mouse articular chondrocytes. Mechanistically, Brd4 loss suppresses Sox9 levels and reduces expression of Sox9 and Runx2 responsive endochondral genes (e.g., Col2a1, Acan, Mmp13 and Sp7/Osx). Collectively, our results indicate that Brd4 is a key epigenetic regulator required for normal chondrogenesis and endochondral ossification.

Brd4 Inactivation Increases Adenoviral Delivery of BMP2 for Paracrine Stimulation of Osteogenic Differentiation as a Gene Therapeutic Concept to Enhance Bone Healing.

Bromodomain (BRD) proteins are histone code interpreters that recognize acetylated lysines and link the dynamic state of chromatin with the transcriptional machinery. Here, we demonstrate that ablation of the Brd4 gene in primary mouse bone marrow-derived mesenchymal stem cells via a conditional Brd4fl/fl allele suppresses osteogenic lineage commitment. Remarkably, loss of Brd4 function also enhances expression of genes in engineered adenoviral vectors, including Cre recombinase and green fluorescent protein (GFP). Similarly, vector-based expression of BMP2 mRNA and protein levels are enhanced upon Brd4 depletion in cells transduced with an adenoviral vector that expresses BMP2 (Ad-BMP2). Importantly, Brd4 depletion in MC3T3-E1 and human adipose-derived mesenchymal stem cells (AMSCs) transduced with Ad-BMP2 enhances osteogenic differentiation of naïve MC3T3-E1 cells via paracrine mechanisms based on transwell and conditioned medium studies. Our studies indicate that Brd4 depletion enhances adenoviral transgene expression in mammalian cells, which can be leveraged as a therapeutic strategy to improve viral vector-based gene therapies. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Multiple pharmacological inhibitors targeting the epigenetic suppressor enhancer of zeste homolog 2 (Ezh2) accelerate osteoblast differentiation.

Skeletal development and bone formation are regulated by epigenetic mechanisms that either repress or enhance osteogenic commitment of mesenchymal stromal/stem cells and osteoblasts. The transcriptional suppressive trimethylation of histone 3 lysine 27 (H3K27me3) hinders differentiation of pre-committed osteoblasts. Osteoblast maturation can be stimulated by genetic loss of the H3K27 methyltransferase Ezh2 which can also be mimicked pharmacologically using the classical Ezh2 inhibitor GSK126. Identification of other Ezh2 inhibitors (iEzh2) that enhance osteogenic potential would increase chemical options for developing new bone stimulatory compounds. In this study, we examined a panel of iEzh2s and show that all eight inhibitors we tested are capable of accelerating osteoblast differentiation to different degrees at concentrations that are well below cytotoxic concentrations. Inhibition of Ezh2 is commensurate with loss of cellular H3K27me3 levels while forced expression of Ezh2 reverses the effect of Ezh2 suppression. Reduced Ezh2 function by siRNA depletion of Ezh2 mRNA and protein levels also stimulates osteoblastogenesis, consistent with the specificity of iEzh2 to target the active site of Ezh2. Diminished Ezh2 levels preempt the effects of iEzh2s on H3K27me3. GSK126, EPZ-6438 and siRNA depletion of Ezh2 each are effective in reducing H3K27me3 levels. However, EPZ-6438 is more potent than GSK126 in stimulating osteoblastogenesis, as reflected by increased extracellular matrix mineralization. Collectively, our data indicate that Ezh2 inhibitors properly target Ezh2 consistent with their biochemical affinities. The range of compounds capable of promoting osteogenesis presented in this study offers the opportunity to develop diverse bone anabolic strategies for distinct clinical scenarios, including spine fusion, non-union of bone and dental implant enhancement.