Advancements and Future Perspectives in Cell Electrospinning and Bio-Electrospraying.

In recent years, researchers have tried to include living cells into electrospun nanofibers or droplets, leading to the field of live cell electrospinning and bio-electrospraying . In live cell electrospinning and bio-electrospraying, cells are embedded in a polymer and subject to the process of mechanical and electrical stimulation of the process. The resulting nanofiber mats or droplets with embedded cells have several potential applications in tissue engineering. The nanofiber structure provides a supportive and porous environment for cells to grow and interact with their surroundings. This can be favorable for tissue regeneration, where the goal is to create functional tissues that closely mimic the extracellular matrix. However, there are also challenges associated with live cell electrospinning and electrospraying, including maintaining cell viability and uniform cell distribution within the nanofiber mat. Additionally, the electrospinning/electrospraying process can have an impact on cell behavior, phenotype, and genotype, which must be cautiously monitored and studied. Overall, the goal of this review paper is to provide a comprehensive and critical analysis of the existing literature on cell electrospinning and bio-electrospraying.

Effect of ellagic acid and retinoic acid on collagen and elastin production by human dermal fibroblasts.

BACKGROUND: Elastin is a fibrous protein key to the structure and support of skin as well as other organ tissues. Elastic fibers are located in the skin's dermal layer and make up approximately 2%-4% of the fat-free dry weight of the dermis in the skin of adults. Aging causes the progressive degradation of elastin fibers. Loss of these fibers can cause skin sagging and wrinkling, loss of healthy blood vessels and lung capacity, aneurysms, and Chronic Obstructive Pulmonary Disease (COPD). OBJECTIVE: We hypothesized that ellagic acid, a polyphenol, will increase elastin in human dermal fibroblasts (HDF) due to polyphenols' elastin binding properties. METHOD: We treated HDF's with 2 µg/ml ellagic acid for 28 days to see the elastin deposition in HDF cell cultures. To test this, we treated HDFs with polyphenols ellagic acid for 3, 7, 14 and 21 days. For comparison purposes, we included a group of ellagic acid and retinoic acid since retinoic acid is already in the market for elastin regeneration purposes. RESULTS: When ellagic acid and retinoic acid were introduced together, insoluble elastin and collagen deposition were significantly higher in HDFs compared to other groups. CONCLUSION: Polyphenols and retinoic acid can improve skin extracellular matrix production of elastin and collagen and may improve skin fine wrinkles.

Mutability of druggable kinases and pro-inflammatory cytokines by their proximity to telomeres and A+T content.

Mutations of protein kinases and cytokines are common and can cause cancer and other diseases. However, our understanding of the mutability in these genes remains rudimentary. Therefore, given previously known factors which are associated with high mutation rates, we analyzed how many genes encoding druggable kinases match (i) proximity to telomeres or (ii) high A+T content. We extracted this genomic information using the National Institute of Health Genome Data Viewer. First, among 129 druggable human kinase genes studied, 106 genes satisfied either factors (i) or (ii), resulting in an 82% match. Moreover, a similar 85% match rate was found in 73 genes encoding pro-inflammatory cytokines of multisystem inflammatory syndrome in children. Based on these promising matching rates, we further compared these two factors utilizing 20 de novo mutations of mice exposed to space-like ionizing radiation, in order to determine if these seemingly random mutations were similarly predictable with this strategy. However, only 10 of these 20 murine genetic loci met (i) or (ii), leading to only a 50% match. When compared with the mechanisms of top-selling FDA approved drugs, this data suggests that matching rate analysis on druggable targets is feasible to systematically prioritize the relative mutability-and therefore therapeutic potential-of the novel candidates.

M1 to M2 induction in macrophages using a retinoic acid-releasing mesenchymal stem cell scaffold.

BACKGROUND: Modulation of macrophage polarization is required for effective tissue repair and regenerative therapies. Therapeutic modulation of macrophages from an inflammatory M1 to a fibrotic M2 phenotype could help in diseases, such as chronic wounds, which are stalled in a prolonged and heightened inflammatory stage within the wound healing process. OBJECTIVE: This study evaluates the efficiency of a pullulan/gelatin nanofiber scaffold loaded with retinoic acid (RA) and adipose-derived mesenchymal stem cells (ASCs) to modulate M1 to M2 anti-inflammatory transition. METHODS: Scaffolds were fabricated by electrospinning, and crosslinked using ethylene glycol diglycidyl ether (EGDE). Exposure of RA and/or ASCs to cultured macrophages have been shown to promote M1 to M2 transition. Pullulan was chosen as a scaffold material due to its ability to quench reactive oxygen species, key signaling molecules that play an important role in the progression of inflammation, as well as for its excellent mechanical properties. Gelatin was chosen as an additional scaffold component due to the presence of cell-binding motifs and its biocompatibility. Scaffold compositions examined were 75:25 and 50:50, pullulan:gelatin. The scaffolds were crosslinked in 1:70 and 1:50 EGDE:EtOH. The scaffold composition was determined via FTIR. For the present study, the 75:25 pullulan:gelatin crosslinked with 1:70 EGDE:EtOH, forming nanofibers 328 ± 47.9 nm (mean ± SD) in diameter, was chosen as the scaffold composition due to its lower degradation and release rate, which allows a sustained delivery of RA. RESULTS: The scaffold composition degraded to approximately 80% after 14 days, with approximately 38% of the drug released after 7 days. THP-1 monocytic cells were induced into a M1 macrophage phenotype through stimulation with lipopolysaccharide (LPS) and gamma interferon (IFN-γ). These M1 macrophages were the exposed to scaffolds loaded with RA and ASCs, to induce differentiation to an M2 phenotype. CONCLUSION: Gene expression quantitation by qPCR showed a reduction of M1 biomarkers, tumor necrosis factor alpha (TNFα) and interleukin 1ß (IL1ß), and an increase of M2 biomarker CCL22 after 2 days of exposure, suggesting successful M1 to M2 transition.

Differentiation of adipose-derived stem cells to chondrocytes using electrospraying.

An important challenge in the fabrication of tissue engineered constructs for regenerative medical applications is the development of processes capable of delivering cells and biomaterials to specific locations in a consistent manner. Electrospraying live cells has been introduced in recent years as a cell seeding method, but its effect on phenotype nor genotype has not been explored. A promising candidate for the cellular component of these constructs are human adipose-derived stem cells (hASCs), which are multipotent stem cells that can be differentiated into fat, bone, and cartilage cells. They can be easily and safely obtained from adipose tissue, regardless of the age and sex of the donor. Moreover, these cells can be maintained and expanded in culture for long periods of time without losing their differentiation capacity. In this study, hASCs directly incorporated into a polymer solution were electrosprayed, inducing differentiation into chondrocytes, without the addition of any exogenous factors. Multiple studies have demonstrated the effects of exposing hASCs to biomolecules-such as soluble growth factors, chemokines, and morphogens-to induce chondrogenesis. Transforming growth factors (e.g., TGF-ß) and bone morphogenetic proteins are particularly known to play essential roles in the induction of chondrogenesis. Although growth factors have great therapeutic potential for cell-based cartilage regeneration, these growth factor-based therapies have presented several clinical complications, including high dose requirements, low half-life, protein instability, higher costs, and adverse effects in vivo. The present data suggests that electrospraying has great potential as hASCs-based therapy for cartilage regeneration.

Systemic delivery of targeted nanotherapeutic reverses angiotensin II-induced abdominal aortic aneurysms in mice.

Abdominal aortic aneurysm (AAA) disease causes dilation of the aorta, leading to aortic rupture and death if not treated early. It is the 14th leading cause of death in the U.S. and 10th leading cause of death in men over age 55, affecting thousands of patients. Despite the prevalence of AAA, no safe and efficient pharmacotherapies exist for patients. The deterioration of the elastic lamina in the aneurysmal wall is a consistent feature of AAAs, making it an ideal target for delivering drugs to the AAA site. In this research, we conjugated nanoparticles with an elastin antibody that only targets degraded elastin while sparing healthy elastin. After induction of aneurysm by 4-week infusion of angiotensin II (Ang II), two biweekly intravenous injections of pentagalloyl glucose (PGG)-loaded nanoparticles conjugated with elastin antibody delivered the drug to the aneurysm site. We show that targeted delivery of PGG could reverse the aortic dilation, ameliorate the inflammation, restore the elastic lamina, and improve the mechanical properties of the aorta at the AAA site. Therefore, simple iv therapy of PGG loaded nanoparticles can be an effective treatment option for early to middle stage aneurysms to reverse disease progression and return the aorta to normal homeostasis.

Effect of all-trans retinoic acid and pentagalloyl glucose on smooth muscle cell elastogenesis.

BACKGROUND: The main objective of tissue engineering is to fabricate a tissue construct that mimics native tissue both biologically and mechanically. A recurring problem for tissue-engineered blood vessels (TEBV) is deficient elastogenesis from seeded smooth muscle cells. Elastin is an integral mechanical component in blood vessels, allowing elastic deformation and retraction in response to the shear and pulsatile forces of the cardiac system. OBJECTIVE: The goal of this research is to assess the effect of the vitamin A derivative all-trans retinoic acid (RA) and polyphenol pentagalloyl glucose (PGG) on the expression of elastin in human aortic smooth muscle cells (hASMC). METHODS: A polycaprolactone (PCL) and the gelatin polymer composite was electrospun and doped with RA and PGG. The scaffolds were subsequently seeded with hASMCs and incubated for five weeks. The resulting tissue-engineered constructs were evaluated using qPCR and Fastin assay for their elastin expression and deposition. RESULTS: All treatments showed an increased elastin expression compared to the control, with PGG treatments showing a significant increase in gene expression and elastin deposition.

Polyphenol treatments increase elastin and collagen deposition by human dermal fibroblasts; Implications to improve skin health.

BACKGROUND: Skin aging is marked by progressive loss in elastin and collagen that causes wrinkling and sagging of skin. Tropoelastin (TE) is the precursor monomer of elastin secreted by cells that cross-links extracellularly to create functional elastic fibers. Cells maintain the capacity to make TE during the aging process. However, the process of extracellular tropoelastin cross-linking diminishes with age. Others have shown that TE production by cells increases with UV exposure. OBJECTIVE: We hypothesize that polyphenols may help coacervate cell secreted TE due to its elastin binding property and increase insoluble elastin in human dermal fibroblasts (HDFs). Increase in TE production by short term UV exposure may further improve elastin deposition by polyphenols. METHODS: We treated HDFs with polyphenols viz epigallocatechin gallate (EGCG) and pentagalloyl glucose (PGG) either with or without intermittent (UVA, 12 min three times a week) exposure for 3, 7, and 14 days. RESULTS: Polyphenols increased insoluble elastin deposition several folds as compared to control untreated cells. Furthermore, short UVA light exposure led to several-fold increased TE production in HDFs. Still, UVA exposure alone was unable to increase insoluble elastic fibers. When polyphenols were introduced with UVA exposure, insoluble elastin deposition was further enhanced in HDFs (30-45-fold increase). Polyphenol treatments with UVA exposure also led to increased collagen deposition in cell cultures. Polyphenols also prevented cell oxidation during UVA exposure. CONCLUSIONS: Polyphenols in combination with short exposure to UVA light increase extracellular matrix deposition of elastin and collagen and may improve skin properties.

Electrospinning Live Cells Using Gelatin and Pullulan.

Electrospinning is a scaffold production method that utilizes electric force to draw a polymer solution into nanometer-sized fibers. By optimizing the polymer and electrospinning parameters, a scaffold is created with the desired thickness, alignment, and pore size. Traditionally, cells and biological constitutes are implanted into the matrix of the three-dimensional scaffold following electrospinning. Our design simultaneously introduces cells into the scaffold during the electrospinning process at 8 kV. In this study, we achieved 90% viability of adipose tissue-derived stem cells through electrospinning.

Effect of Pulsed Electromagnetic Fields on Human Mesenchymal Stem Cells Using 3D Magnetic Scaffolds.

Alternative bone regeneration strategies that do not rely on harvested tissue or exogenous growth factors are needed. One of the major challenges in tissue reconstruction is recreating the bone tissue microenvironment using the appropriate combination of cells, scaffold, and stimulation to direct differentiation. This study presents a bone regeneration formulation that involves the use of human adipose-derived mesenchymal stem cells (hASCs) and a three-dimensional (3D) hydrogel scaffold based on self-assembled RADA16 peptides containing superparamagnetic iron oxide nanoparticles (NPs). Although superparamagnetic NPs could be used as stimulus to manipulate the cell proliferation and differentiation, in this paper their use is explored for assisting osteogenic differentiation of hASCs in conjunction with direct stimulation by extremely low-frequency pulsed electromagnetic fields (pEMFs). Cellular morphology, proliferation, and viability, as well as alkaline phosphatase activity, calcium deposition, and osteogenic capacity were monitored for cells cultured up to 21 days in the 3D construct. The results show that the pEMFs and NPs do not have any negative effect on cell viability, but instead distinctly induced early differentiation of hASCs to an osteoblastic phenotype, when compared with cells without biophysical stimulation. This effect is attributed to synergy between the pEMFs and NPs, which may have stimulated mechanotransduction pathways, which, in turn activated biochemical signals between cells to differentiate or proliferate. This approach may offer a safe and effective option for the treatment of non-union bone fractures. Bioelectromagnetics. © 2020 The Authors. Bioelectromagnetics published by Wiley Periodicals, Inc.

Bisphosphosphonate-calcium phosphate cement composite and its properties.

Calcium phosphate cement (CPC) has been studied extensively due to its bioactivity and biodegradability. CPC is typically made by a combination of multiple calcium phosphates that form a paste that sets and hardens in the body after being combined with either water or an aqueous solution. It is highly moldable and easily manipulated, and CPCs possess osteoconductive properties. Due to these characteristics, CPCs offer great promise in bone grafting applications. CPC combined with drugs has a great potential as drug delivery system and has been studied extensively. In this review we have focused on Bisphosphonate-CPC drug delivery system. In addition, we introduce and discuss the potential of studying other bisphosphonates.

Site-specific chelation therapy with EDTA-loaded albumin nanoparticles reverses arterial calcification in a rat model of chronic kidney disease.

Medial arterial calcification (MAC) is a common outcome in diabetes and chronic kidney disease (CKD). It occurs as linear mineral deposits along the degraded elastin lamellae and is responsible for increased aortic stiffness and subsequent cardiovascular events. Current treatments for calcification, particularly in CKD, are predominantly focused on regulating the mineral disturbance and other risk factors. Ethylene diamine tetraacetic acid (EDTA), a chelating agent, can resorb mineral deposits, but the systemic delivery of EDTA may cause side effects such as hypocalcemia and bone resorption. We have developed elastin antibody conjugated albumin nanoparticles that target only degraded elastin in vasculature while sparing healthy tissues. In this study, we tested a targeted nanoparticle-based EDTA chelation therapy to reverse CKD-associated MAC. Renal failure was induced in Sprague-Dawley rats by a high adenine diet supplemented by high P and Ca for 28 days that led to MAC. Intravenous delivery of DiR dye-loaded nanoparticles confirmed targeting to vascular degraded elastin and calcification sites within 24 hours. Next, EDTA-loaded albumin nanoparticles conjugated with an anti-elastin antibody were intravenously injected twice a week for two weeks. The targeted nanoparticles delivered EDTA at the site of vascular calcification and reversed mineral deposits without any untoward effects. Systemic EDTA injections or blank nanoparticles were ineffective in reversing MAC. Reversal of calcification seems to be stable as it did not return after the treatment was stopped for an additional four weeks. Targeted EDTA chelation therapy successfully reversed calcification in this adenine rat model of CKD. We consider that targeted NP therapy will provide an attractive option to reverse calcification and has a high potential for clinical translation.

Gene Expression Profiling of Human Adipose Tissue Stem Cells during 2D versus 3D Adipogenesis.

Much of the current understanding on molecular and cellular events of adipose developmental biology comes from monolayer cell culture models using preadipocyte cell lines, although in vivo adipose tissue consists of a much more complex three-dimensional microenvironment of diverse cell types, extracellular network, and tissue-specific morphological and functional features. Added to this fact, the preadipocytes, on which the adipogenesis mechanisms are mostly explored, possess some serious limitations (e.g., time of initial subculture and adipogenic differentiation time), which, perhaps, can efficiently be replaced with progenitor cells such as adipose tissue-derived stem cells (ASCs). With the objective of developing a better in vitro model for adipose developmental biology, this project involves gene expression profiling of human ASCs (hASCs) during their differentiation to adipocytes in a 2D versus 3D culture model. This transcriptional-level analysis revealed that gene expression patterns of adipogenesis-induced hASCs in a 3D self-assembled polypeptide hydrogel are relatively different from the 2D monolayered cells on plastic hard substrate. Moreover, analysis of adipogenic lineage progression 9 days after adipogenic induction shows earlier differentiation of hASCs in 2D over their 3D counterparts. However, differentiation in 2D shows some unexpected behavior in terms of gene expression, which does not seem to be related to adipogenic lineage specification. Since hASCs are already being used in clinical trials due to their therapeutic potential, it is important to have a clear understanding of the molecular mechanisms in an in vivo model microenvironment like the one presented here.

Pentagalloyl glucose increases elastin deposition, decreases reactive oxygen species and matrix metalloproteinase activity in pulmonary fibroblasts under inflammatory conditions.

Emphysema is characterized by degradation of lung alveoli that leads to poor airflow in lungs. Irreversible elastic fiber degradation by matrix metalloproteinases (MMPs) and reactive oxygen species (ROS) activity leads to loss of elasticity and drives the progression of this disease. We investigated if a polyphenol, pentagalloyl glucose (PGG) can increase elastin production in pulmonary fibroblasts. We also studied the effect of PGG treatment in reducing MMP activity and ROS levels in cells. We exposed rat pulmonary fibroblasts to two different types of inflammatory environments i.e., tumor necrosis factor-α (TNF-α) and cigarette smoke extract (CSE) to mimic the disease. Parameters like lysyl oxidase (LOX) and elastin gene expression, MMP-9 activity in the medium, lysyl oxidase (LOX) activity and ROS levels were studied to assess the effect of PGG on pulmonary fibroblasts. CSE inhibited lysyl oxidase (LOX) enzyme activity that resulted in a decreased elastin formation. Similarly, TNF-α treated cells showed less elastin in the cell layers. Both these agents caused increase in MMP activity and ROS levels in cells. However, when supplemented with PGG treatment along with these two inflammatory agents, we saw a significant increase in elastin deposition, reduction in both MMP activity and ROS levels. Thus PGG, which has anti-inflammatory, anti-oxidant properties coupled with its ability to aid in elastic fiber formation, can be a multifunctional drug to potentially arrest the progression of emphysema.

Reversal of Vascular Calcification and Aneurysms in a Rat Model Using Dual Targeted Therapy with EDTA- and PGG-Loaded Nanoparticles.

Degeneration of elastic lamina and vascular calcification are common features of vascular pathology such as aortic aneurysms. We tested whether dual therapy with targeted nanoparticles (NPs) can remove mineral deposits (by delivery of a chelating agent, ethylene diamine tetraacetic acid (EDTA)) and restore elastic lamina (by delivery of a polyphenol, pentagalloyl glucose (PGG)) to reverse moderate aneurysm development. EDTA followed by PGG NP delivery led to reduction in macrophage recruitment, matrix metalloproteinase (MMP) activity, elastin degradation and calcification in the aorta as compared to delivery of control blank NPs. Such dual therapy restored vascular elastic lamina and improved vascular function as observed by improvement in circumferential strain. Therefore, dual targeted therapy may be an attractive option to remove mineral deposits and restore healthy arterial structures in moderately developed aneurysms.

Systemic Delivery of Nanoparticles Loaded with Pentagalloyl Glucose Protects Elastic Lamina and Prevents Abdominal Aortic Aneurysm in Rats.

Degeneration of elastin plays a vital role in the pathology and progression of abdominal aortic aneurysm (AAA). Our previous study showed that pentagalloyl glucose (PGG), a core derivative of tannic acid, hinders the development of AAAs in a clinically relevant animal model when applied locally. In this study, we tested whether targeted nanoparticles (NPs) can deliver PGG to the site of an aneurysm and prevent aneurysmal growth by protecting elastin. PGG-loaded albumin NPs with a surface-conjugated elastin-specific antibody were prepared. Aneurysms were induced by calcium chloride-mediated injury to the abdominal aorta in rats. NPs were injected into the tail vein after 10 days of CaCl2 injury. Rats were euthanized after 38 days. PGG delivery led to reduction in macrophage recruitment, matrix metalloproteinase (MMP) activity, elastin degradation, calcification, and development of aortic aneurysm. Such NP delivery offers the potential for the development of effective and safe therapies for AAA.

Prevention of abdominal aortic aneurysm progression by targeted inhibition of matrix metalloproteinase activity with batimastat-loaded nanoparticles.

RATIONALE: Matrix metalloproteinases (MMPs)-mediated extracellular matrix destruction is the major cause of development and progression of abdominal aortic aneurysms. Systemic treatments of MMP inhibitors have shown effectiveness in animal models, but it did not translate to clinical success either because of low doses used or systemic side effects of MMP inhibitors. We propose a targeted nanoparticle (NP)-based delivery of MMP inhibitor at low doses to the abdominal aortic aneurysms site. Such therapy will be an attractive option for preventing expansion of aneurysms in patients without systemic side effects. OBJECTIVE: Our previous study showed that poly(d,l-lactide) NPs conjugated with an antielastin antibody could be targeted to the site of an aneurysm in a rat model of abdominal aortic aneurysms. In the study reported here, we tested whether such targeted NPs could deliver the MMP inhibitor batimastat (BB-94) to the site of an aneurysm and prevent aneurysmal growth. METHODS AND RESULTS: Poly(d,l-lactide) NPs were loaded with BB-94 and conjugated with an elastin antibody. Intravenous injections of elastin antibody-conjugated BB-94-loaded NPs targeted the site of aneurysms and delivered BB-94 in a calcium chloride injury-induced abdominal aortic aneurysms in rats. Such targeted delivery inhibited MMP activity, elastin degradation, calcification, and aneurysmal development in the aorta (269% expansion in control versus 40% elastin antibody-conjugated BB-94-loaded NPs) at a low dose of BB-94. The systemic administration of BB-94 alone at the same dose was ineffective in producing MMP inhibition. CONCLUSIONS: Targeted delivery of MMP inhibitors using NPs may be an attractive strategy to inhibit aneurysmal progression.

Targeted chelation therapy with EDTA-loaded albumin nanoparticles regresses arterial calcification without causing systemic side effects.

BACKGROUND AND AIMS: Elastin-specific medial arterial calcification (MAC) is an arterial disease commonly referred as Monckeberg's sclerosis. It causes significant arterial stiffness, and as yet, no clinical therapy exists to prevent or reverse it. We developed albumin nanoparticles (NPs) loaded with disodium ethylene diaminetetraacetic acid (EDTA) that were designed to target calcified elastic lamina when administrated by intravenous injection. METHODS AND RESULTS: We optimized NP size, charge, and EDTA-loading efficiency (150-200 nm, zeta potential of -22.89--31.72 mV, loading efficiency for EDTA~20%) for in vivo targeting in rats. These NPs released EDTA slowly for up to 5 days. In both ex-vivo study and in vivo study with injury-induced local abdominal aortic calcification, we showed that elastin antibody-coated and EDTA-loaded albumin NPs targeted the damaged elastic lamina while sparing healthy artery. Intravenous NP injections reversed elastin-specific MAC in rats after four injections over a 2-week period. EDTA-loaded albumin NPs did not cause the side effects observed in EDTA injection alone, such as decrease in serum calcium (Ca), increase in urine Ca, or toxicity to kidney. There was no bone loss in any treated groups. CONCLUSION: We demonstrate that elastin antibody-coated and EDTA-loaded albumin NPs might be a promising nanoparticle therapy to reverse elastin-specific MAC and circumvent side effects associated with systemic EDTA chelation therapy.