Regenerative Engineering and Translational Medicine

Altered Biodistribution and Tissue Retention of Nanoparticles Targeted with P-Glycoprotein Substrates Abstract Low molecular weight substrates of the efflux transporter, P-glycoprotein, alter the biodistribution and tissue retention of nanoparticles following intravenous administration. Of particular interest is the retention of the targeted nanoparticles in the brain. Drug delivery to the brain is hindered by the restricted transport of drugs through the blood-brain barrier (BBB). Drugs that passively diffuse across the BBB also have large volumes of distribution; therefore, alteration of their biodistribution to increase their concentration in the brain may help to enhance efficacy and reduce off-target side effects. In this work, targeted nanoparticles were used to explore a new approach to target drugs to the brain—the exploitation of the P-glycoprotein efflux pump. The retention of nanoparticles containing a strong P-glycoprotein substrate, rhodamine 6G, tethered to a PLA nanoparticle through a PEG spacer was greater than twofold relative to untargeted nanoparticles and to nanoparticles tethered to a weaker P-glycoprotein substrate, rhodamine 123. In a P-glycoprotein knockout mouse model (mdr1a (-/-)), there were no significant differences in brain accumulation between rhodamine 6G-targeted particles and controls, strongly supporting the role of P-glycoprotein. This proof of concept report shows the potential applicability of low molecular weight P-gp substrates to alter nanoparticle biodistribution. Lay Summary The efficacy of medicines can be improved by diverting drugs to specific tissues. Finding new ways to target medicines to diseased tissue is an active area of research across disciplines. Drug-loaded nanoparticles, delivered to tissues of interest, are one way to accomplish this goal. The work reported in this manuscript explores the possibility of using small molecules to get nanoparticles to bind to a drug efflux pump, P-glycoprotein (P-gp), that is present in various tissues in the body. P-gp functions to remove drugs from tissues, and it is usually considered a hindrance to drug targeting. The research in this paper shows that the natural function of P-gp can be used favorably to retain nanoparticles in various tissues. Future Work The data reported in this manuscript serves to establish a proof-of-concept that low molecular weight P-gp substrates can be used to alter the biodistribution of nanoparticles. Future work includes (1) understanding the targeting mechanism(s) that lead to these results, (2) identifying FDA-approved drugs that can target nanoparticles, and (3) evaluating how nanoparticle biodistribution is altered by using P-gp substrates with different binding constants.

Increased NF-κB Activity in Osteoprogenitor-Lineage Cells Impairs the Balance of Bone Versus Fat in the Marrow of Skeletally Mature Mice Abstract "Senile osteoporosis" is defined as significant aging-associated bone loss and is accompanied by increased fat in the bone marrow. The proportion of adipocytes in the bone marrow is inversely correlated with bone formation and is associated with increased risk of fracture. NF-κB is a transcription factor that functions as a master regulator of inflammation and bone remodeling. NF-κB activity increases during aging; furthermore, constitutive activation of NF-κB significantly impairs skeletal development in neonatal mice. However, the effects of NF-κB activation using a skeletally mature animal model have not been examined. In the current study, an osteoprogenitor (OP)-specific, doxycycline-regulated NF-κB-activated transgenic mouse model (iNF-κB/OP) was generated to investigate the role of NF-κB in bone remodeling in skeletally mature mice. Reduced osteogenesis in the OP-lineage cells isolated from iNF-κB/OP mice was only observed in the absence of doxycycline in vitro. Bone mineral density in the metaphyseal regions of femurs and tibias was reduced in iNF-κB/OP mice. No significant differences in bone volume fraction and cortical bone thickness were observed. Osmium-stained bone marrow fat was increased in epiphyseal and metaphyseal areas in the tibias of iNF-κB/OP mice. These findings suggest that targeting NF-κB activity as a therapeutic strategy may improve bone healing and prevent aging-associated bone loss in aged patients. Lay Summary "Senile osteoporosis" denotes significant aging-associated bone loss from the axial and peripheral skeleton and is accompanied by increased fat in the bone marrow. This imbalance in osteogenesis and adipogenesis is associated with an increased incidence of fragility fractures of the spine, hip, knee, shoulder, and wrist. NF-κB is a key regulator of bone remodeling. Increased NF-κB activity was found in many organs during the natural aging process. Clarification of the specific effect of increased NF-κB activity on osteoprogenitors during aging will delineate novel therapeutic approaches to mitigate the adverse effects of chronic inflammation and suppressed bone formation in aging-associated osteoporosis.

News and Views June 2019

Adipose-Derived Stem Cells Can Contribute to Vascular Network Formation in Poly(ethylene Glycol) Hydrogel Scaffolds Abstract Although adipose-derived stem cells (ADSCs) can influence wound healing, their role in neovascularization is unclear. Utilizing three-dimensional in vitro, we sought to determine whether ADSCs cultured under varying conditions could contribute to vascular network formation, functioning as either endothelial cells (ECs) or supporting pericytes.. To study this, ADSCs were encapsulated in 3D hydrogels either with human brain vascular pericytes (HBVP, to show ADSC functions as ECs) or with human umbilical vein endothelial cells (HUVEC, to show ADSC functions as pericytes). The hydrogel used provides a 3D cell-adhesive, proteolytically degradable cell culture matrix that supports formation of vascular networks by encapsulated endothelial cells and pericytes. For this study, ADSCs were cultured in either basal media (basal) or EGM-2 media with 20 ng/mL of VEGF (stimulated) for 7 days and then encapsulated with HUVECs or HBVPs. When encapsulated with HBVPs, both basal and stimulated ADSCs were capable of forming CD31+ tubule-like networks, indicating endothelial function. In co-culture with HUVECs, basal and stimulated ADSCs were capable of enhancing and stabilizing formation of CD31+ tubule-like networks by the HUVECs and localized along the outer surfaces of the endothelial tubules, indicating pericyte-like function. These studies were repeated with diabetic ADSCs to examine the influence of this phenotype on the cells' ability to influence neovascularization. Both basal and stimulated diabetic ADSCs were capable of supporting tubule formation by HUVECs, though to a lesser degree than non-diabetic ADSCs. Notably, diabetic ADSCs were not capable of forming CD31+ tubules when co-cultured with HBVPs, indicating loss of endothelial function. Lay Summary Adipose-derived stem cells (ADSCs) are known to play a role in wound healing; however, the exact role of these cells in neovascularization is unclear. Our work focuses on understanding specific cell–cell interactions between ADSCs, endothelial cells, and mesenchymal support cells within a 3D poly(ethylene glycol) hydrogel scaffold. Furthermore, we sought to understand the effect of the diabetic phenotype on this phenomenon as well.

A Review of Decellularized Extracellular Matrix Biomaterials for Regenerative Engineering Applications Abstract Biomaterials are a cornerstone technology of the biomedical device, tissue engineering, and regenerative medicine industries. While traditional biomaterials are fully defined synthetics, growing evidence supports the use of extracellular matrix-based biomaterials produced through the decellularization of organs, tissues, or cell cultures. These materials are particularly advantageous as they largely retain the structure and the biochemical nature of the original tissue, properties that are often difficult to reproduce with synthetics. Indeed, there are many FDA-approved and clinically used extracellular matrix-based materials that are generated through decellularization processes. In this review, we first describe methods of decellularization used to produce these materials and their associated advantages and limitations, discuss the current use of extracellular matrix-based materials in regenerative engineering applications, describe the areas where current research is occurring, and forecast areas where impactful research may appear. Lay Summary The regeneration of tissues often requires a scaffold material to support and guide the cells that are performing the repair. Often, these materials are manmade and lack many of the key features present in native tissue. However, a tissue can be processed to remove its cells (a process called decellularization), leaving behind a scaffold of proteins and polysaccharides known as the extracellular matrix. These decellularized matrices are attractive scaffolds for use in regenerative medicine applications, and they are the subject of this review.

Reprogramming the Stem Cell Behavior by Shear Stress and Electric Field Stimulation: Lab-on-a-Chip Based Biomicrofluidics in Regenerative Medicine Abstract The biophysical cues of endogenous origin, i.e., shear stress and electric field, are known to significantly modulate cell functionality, in vitro. While this has been relatively well investigated in conventional petri dish culture, it is important to validate such important phenomenon in physiologically simulated cellular microenvironment. In this perspective, this review critically discusses the importance of lab-on-a-chip (LOC)-based microfluidic devices to probe into this aspect to develop an insight towards the application in regenerative medicine. While reviewing several literature reports, an emphasis has been placed to unravel the intriguing aspects of shear and electric field modulated differentiation of stem cells in the biomicrofluidics devices. The potential application focusing the stem cell culture was emphasized in this article as the stem cells are the foundation of tissue regeneration. Several challenges in tissue regeneration and introduction of personalized medicine could be addressed through microfluidic technology. Culturing of organ-specific multiple cell types within lab-on-a-chip and biophysical stimulation mediated activations of intracellular signal transduction under gradient shear/electric field are highlighted in this review. Lay Summary Conceptually, regenerative medicine is considered as an emerging approach for treating traumatized, malfunctional anatomical parts of the patients with stem cells to establish normal functionality of the tissue. The regenerated tissue should preferably be the patients' autologous tissue, grown under artificially created in vivo physiological environment. Biomicrofluidic-based lab-on-a-chip technology enables to perform in vitro cell/tissue engineering under endogenous cues, like shear and electric field. Therefore, this review discusses two aspects of regenerative medicine in terms of autologous transplantation of cells/tissues to improvise personalized regenerative medicine and to recreate an organ-specific tissue under the influence of biophysical stimulation in an attempt to improve the physiological functionality. Graphical abstract Biomicrofluidics based Lab-on-a-Chip devices have improvised the tissue regenerative approaches towards into the direction of personalised medicine.

In vitro Release and Cytotoxic Studies of Novel Alginate Nanocarrier for the Antitumor Drug: Sunitinib Abstract The present study involves the preparation, characterization, and evaluation of novel alginate nanocarrier for sunitinib (STB), an anticancer drug. Sunitinib (STB) is a weakly soluble drug in water due to its poor dissolution rate and oral bioavailability. Alginate biopolymer is one of the promising candidates for a delivery matrix. A controlled gelification method using calcium chloride as the crosslinker was done for the formulation of STB using nanoalginate. The objective of this work was to evaluate the physicochemical/biological properties of the release system via in vitro dissolution studies and cytotoxicity studies. The bare alginate nanoparticles and STB-loaded alginate nanoparticles were characterized for its physicochemical properties using FT-IR, SEM, and TEM studies. The formulation and loading chemistry were well exhibited by FTIR analysis results. SEM and TEM results revealed the spherical and rod-like shape morphology with rough surface where the drug molecules were adhered. The entrapment efficiency and dissolution studies of the drug were done through UV VIS instrument. In vitro cytotoxic studies were performed for loaded sunitinib alginate biopolymer (STB-AL-NPs). The cytotoxicity results reveal that the cytotoxicity is mainly due to the loaded anticancer drug sunitinib. Lay Summary In this study, sunitinib drug was encapsulated in alginate nanoparticles effectively. The interaction between polymer, crosslinker, and drug was well determined using FTIR, XRD, SEM, TEM, and Zeta potential. The results are compared with bare alginate nanoparticles. The expected entrapment efficiency of the drug carrier was around 99% via UV-Vis spectroscopy, proving good drug-polymer interaction. In vitro drug release studied was done by dissolution method using 900 mL of 0.2 mM PBS, to determine the sustained release of the drug from the excipient. The results proved that the drug was released to the medium in the sustained manner. Future Scope Sunitinib-encapsulated alginate nanoparticles not only offer several advantages over conventional drug therapies but also expected to overcome the side effect regarding to dosing and toxicity while administration. However, further optimization studies includes stabilization and targeting should be performed for both in vitro and in vivo.

Localized Osteoarthritis Disease-Modifying Changes due to Intra-articular Injection of Micronized Dehydrated Human Amnion/Chorion Membrane Abstract Osteoarthritis (OA) is the leading cause of joint disability, and there are no FDA-approved disease-modifying drugs. The intra-articular delivery of micronized dehydrated human amnion/chorion membrane (AmnioFix, MiMedx, GA) has been shown to have a chondro-protective effect on articular cartilage in the medial meniscus transection (MMT) pre-clinical model of OA and has entered human clinical trials. AmnioFix is a well-characterized extracellular matrix (ECM)-derived therapeutic that contains hundreds of bioactive molecules, but little is known about its therapeutic mechanism in OA. The objective of this study was to elucidate, via local gene expression analysis, the molecular mechanisms of action of AmnioFix during OA development and progression. Lewis rats underwent MMT surgery, and AmnioFix or saline was injected intra-articularly 24 h post-surgery. At 5, 7, and 21 days post-surgery, articular cartilage, synovial membrane, and osteophyte tissues from multiple regions were collected and analyzed by microarray RT-PCR. Results demonstrated regional variation in the effects of amnion treatment on gene expression. Although gene expression was unaltered in articular cartilage and osteophyte tissue, pro- and anti-inflammatory markers were more highly expressed in the medial synovial membrane for the AmnioFix treatment group compared to the saline and naïve control groups. These data suggest that the previously observed chondro-protective effects of AmnioFix treatment may be mediated via alteration of the synovial membrane microenvironment. This work is the first to provide insight into the mechanisms of action of AmnioFix within the articular joint space. Lay Summary AmnioFix (MiMedx, Marietta, GA), obtained from placenta, is a promising candidate for treatment of osteoarthritis (OA). AmnioFix has previously demonstrated protection of the cartilage on joint surfaces in an animal model of OA. However, this is the first study to elucidate the mechanisms of action of AmnioFix on multiple tissues within the joint space. Our results demonstrated that AmnioFix acts primarily through the synovial membrane, where it induces the expression of immunomodulatory markers. We suggest that the immunomodulatory properties of AmnioFix influence the microenvironment of the synovial membrane, thereby affecting joint homeostasis and inducing a chondro-protective effect on the articular cartilage. Future work should investigate the phenotype/s of cells recruited to the synovium following AmnioFix treatment, as well as the interaction of AmnioFix with synovial tissue-resident cells. Furthermore, the effects of AmnioFix on other joint disorders such as rheumatoid arthritis, a disease involving inflammation of the synovium, warrants further investigation.

Bioactive Poly(ethylene Glycol) Acrylate Hydrogels for Regenerative Engineering Abstract Poly(ethylene glycol) (PEG)-based hydrogels have been used in regenerative engineering applications due to attributes such as the ability to encapsulate cells, control the presentation of bioactive ligands, and manipulate the mechanical properties of the hydrogels. PEG chains are highly hydrophilic, uncharged and possess high chain mobility. This allows resistance to protein adsorption, making PEG very bioinert. Certain derivatives, such as PEG diacrylate (PEGDA), can be crosslinked to form hydrogels under conditions mild enough to allow cell encapsulations. PEGDA hydrogels can also be manipulated to span a range of stiffnesses relevant to soft tissues. Additionally, PEG chains can easily be covalently modified with peptides and proteins to allow cell adhesion or provide intrinsic cues to cells within the PEG hydrogel. Among the extensive uses of PEG acrylate-based hydrogels for regenerative engineering purposes, this review will first focus on the formation of bioactive PEG-acrylate hydrogels and then highlight tissue engineering applications of PEGDA-based hydrogels, with specific examples for cartilage tissue engineering, bone tissue engineering, vasculogenesis, liver tissue engineering, cardiac tissue engineering and the development of tumor models. Lay Summary Regenerative engineering seeks to combine materials with cells to generate new tissues outside of the body. In order to interact with cells and support the formation of tissues, the materials must be rendered biologically active and adopt certain characteristics of the native tissue environment. This review focuses on poly(ethylene glycol) (PEG) acrylate materials for regenerative engineering purposes. PEG acrylate-based materials are easily modified to be biologically active and are capable of mimicking a range of characteristics of the native tissue environment. These PEG materials have supported the formation cartilage tissues, bone tissues, blood vessels, liver tissues, cardiac tissues and tumor models. Future work will apply these results to the continued modifications of PEG-acrylate materials to generate more complex tissues. Specifically, the ability to mimic transient characteristics of native tissue microenvironments and the relevance of cell types in each tissue generated will need to be investagted within the PEG acylate materials.

Functional Graphenic Materials, Graphene Oxide, and Graphene as Scaffolds for Bone Regeneration Abstract Insufficient bone regeneration is a complex problem affecting millions, and treatment would benefit from a complex material that recapitulates the properties of native bone. No current tissue-engineered scaffold can capture all of the properties of healthy bone. Graphene, graphene oxide (GO), and functional graphenic materials (FGMs) have a variety of interesting properties that make them promising foundations on which to craft sophisticated, biomimetic, osteoinductive, synthetic scaffolds for bone regeneration. GO has shown promise in the osteoinduction of stem cells, especially when coupled with growth factors. Additionally, FGMs have tunable surface chemistry and mechanical properties, as well as periodic long-range order, making this class of materials a promising candidate for regeneration of hard tissues. Strategies for controlling and modifying the surface chemistry of graphenic materials have become increasingly sophisticated in recent years, providing access to new FGMs with distinct implications in biomaterials and medicine. In this review, we discuss promising emerging strategies for the use of graphenic materials in bone regeneration, with a focus on biomimetic and bioinstructive FGMs. Lay Summary The regeneration of bone is an interdisciplinary medical, scientific, and engineering challenge. While small fractures heal spontaneously into adulthood, larger injuries and deformities require surgical correction. Currently, grafts or metallic prosthetics are the standard of care, but both suffer limitations. Graphene, graphene oxide, and functional graphenic materials comprise a class of materials that can be derived from graphite (pencil lead) and offer a plethora of promising properties including long-range order, mechanical stability, and autodegradability that make them promising scaffold materials for bone regeneration. Graphical Abstract ᅟ

Alexandros Sfakianakis
Anapafseos 5 . Agios Nikolaos
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6948891480

alsfakia@gmail.com