Applications in Chronic Wound Healing | IJN – Dove Medical Press

Introduction

The skin is the largest organ in the body, accounting for 15% of the total body weight. It is the first line of defense against physical, chemical, and biological factors.1,2 In some cases, the anatomical structure and biological function of the skin are impaired due to internal (local blood obstruction, inflammation, or underlying diseases) or external factors (mechanical injury, chemical corrosion, electric injury, or thermal injury).1,3

After damage, skin can self-heal, and this process involves four phases: hemostasis, inflammation, proliferation, and remodeling (Figure 1).4,5 In the first few minutes after skin damage, the platelets accumulate around the wound and get activated, forming a scab to preventing bleeding.6 After 23 days, the inflammatory phase starts around the wound, and the immune cells remove the dead and devitalized tissues and prevent microbial infections.4 The proliferation phase occurs after the inflammation phase, and it is characterized by the activation of keratinocytes, fibroblasts, endothelial cells, and macrophages, which contribute to wound closure, matrix formation and angiogenesis.7 In the 12 or more months after the primary repair is completed, the regenerated skin tissue is remodeled. During this phase, the processes activated after injury slow down, and the healed wound reaches it maximum mechanical strength.4,5

Figure 1 Phases of wound healing, including the hemostasis, inflammatory, proliferation, and remodeling phase.

Notes: Reprinted from: Tavakoli S, Klar AS. Advanced Hydrogels as Wound Dressings. Biomolecules. 2020;10(8):1169. doi:10.3390/biom10081169.5 2020 by the authors. Licensee MDPI, Basel, Switzerland. Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

However, in some cases, the skins self-healing property is inadequate, leading to the formation of chronic wounds. Chronic wounds are defined as wounds that remain unhealed even after 12 weeks.8 The main factors delaying wound repair include diabetes, infections, and long-term inflammation. Diabetic mellitus damages the microenvironment of skin tissue, which is involved in wound regeneration. It causes increases in reactive oxygen species (ROS) levels and poor collagen deposition.911 The hyperglycemia weakens the functions of fibroblasts, keratinocytes, endothelial cells, and stem cells or progenitor cells involved in wound healing.12 Microbial infections deplete the energy and cells required for tissue regeneration, and the bacteria can form biofilms that display antibiotic resistance, immune evasion, and wound adherence.13,14 In unhealed skin, excess inflammation also contributes to wound chronicity owing to its cytotoxic effects and the induced tissue damage, both of which delay wound healing.1517 Traditionally, the chronic wounds are treated with wound dressing made of gauze, skin grafting, or even flap transplantation. Moreover, targeted antibiotics are administered in case of infection. However, Surgery for chronic wounds can be challenging due to limited donor sites, donor damage, scar formation, and even severe functional and psycho-social disorders.1820 Moreover, antibiotic overuse can lead to drug resistance, creating new problems for infectious chronic wounds.21,22 Moreover, chronic wounds become refractory due to infections, diabetes, ischemia, over-degradation of collagen, and other factors, leading to the failure of traditional treatment methods. Thus, novel methods for treating chronic wounds need to be explored.

Skin wounds are the most common type of tissue injury, and they can be caused by trauma, surgery, burns, chronic diseases, or cancers.4,23 Under adverse conditions, wounds often turn chronic. The acceleration of wound repair and improvement of the healing process are the primary objectives of chronic wound treatment. Nanobiotechnology, which involves the use of nano-sized particles in biological systems, represents the convergence of several scientific fields, including chemistry, biology, physics, optics, mechanics, and nanoscale Science and technology. Nanobiotechnology can provide tools and technologies for examining and modulating biological systems.24,25 By applying nanotechnology in the field of bioMedicine, several novel biomaterials, biosensors, and bio-therapies have been designed and studied. It is believed that the combination of nanotechnology and biology can aid in wound management, monitoring, and repair.26,27 Initially, the application of nanobiotechnology in chronic wound treatment was focused on the provision of scaffolds for cell migration and the replacement of traditional gauze dressing.2830 However, with the development of nanotechnology and our understanding of wound healing mechanisms, various nanobiotechnology-based wound-treatments systems including drug and gene delivery platforms, antimicrobial systems, and cell-carrying systems have been developed and found to have prospective applications.3136 Nevertheless, despite these advances, wound dressings remain largely primitive and lack functions that allow wound monitoring and dynamic wound responses. Therefore, smart hydrogels or bandage systems developed using nano-sized biomaterials, which can respond to stimuli or monitor the status of chronic wounds, have been examined.37,38

This review article provides a summary of nanobiotechnology-based scaffold, delivery, antimicrobial, cell-carrying, collagen modulating, stimuli-responsive, and wound monitoring systems for chronic wound healing. Further, the prospects of nanobiotechnology to achieve better treatment outcomes for chronic wounds are discussed.

Physiologically, the wound healing process is affected by several factors, including gene expression; cell functions such as migration, proliferation, and differentiation; the skin microenvironment; infection; ischemiahypoxia; inflammation; and collagen formation and arrangement.1,3,17,3942 These factors are used as references for the design of nanobiotechnology systems that promote chronic wound repair (Figure 2) and need to be carefully considered before designing such systems.

Figure 2 Nanoplatform for chronic wound healing.

To repair tissue defects in the wound area, a platform for cell adhesion, migration, and proliferation ie, a scaffold for cells needs to be established. Such a scaffold can also serve as a platform for multi-functional modification. Given their good biocompatibility, angiogenic capacity, and biomimetic behavior to natural human skin, nano-scaffold systems are widely used in tissue engineering.4346

Tradition treatment methods for chronic wounds that show delayed Union involve local or systemic drug administration. However, the performance of these drugs is suboptimal owing to limitations such as low solubility and low bioactivity. Nanobiotechnology has thus been leveraged for the development of drug, gene, and exosome delivery systems that can help in overcoming these limitations.34,47,48

Infections, which impede tissue repair, should receive careful attention in chronic wound treatment. Silver nano-particles, a product of nanobiotechnology, have been used clinically in the treatment of microbial infection for decades. Moreover, several more recent studies have explored new nanoplatform-based anti-infection therapies, including potential anti-infection nanoparticles (NPs).4952

Cell therapy, especially stem cell therapy, is currently a focus in regenerative medicine and diabetic wound repair. In some basic medical and preclinical studies, chronic wound treatment with stem cells has shown excellent outcomes.5355 However, despite its great potential, the clinical translation of stem cell therapy for chronic wound healing is hindered by the lack of appropriate methods for cell encapsulation and transplantation. Thus, the development of nanobiotechnology-based cell-carrying systems can provide improved therapeutic effects.56,57

With the development of precision medicine, therapeutic systems that monitor wounds and respond to individual stimuli are expected to become popular. One such system is based on ferrihydrite NPs, which can respond to blue light and are effective for antimicrobial and wound healing treatments.58 More stimuli-responsive materials and monitoring systems for chronic wound healing can be generated through nanobiotechnology.

The term scaffold system generally refers to materials that can integrate with living tissues and cells and can be implanted into different tissues where they supplement natural tissue function based on specific conditions. In order to enable seed cells to proliferate and differentiate, a scaffold composed of biological materials that acts as an artificial extracellular matrix (ECM) is required. Scaffolds are critical for tissue engineering systems, including those for bone, cartilage, blood vessels, nerves, skin, and artificial organs (eg, liver, spleen, kidney, and bladder).

Nano-scaffold systems aimed at chronic wound healing need to possess certain important features.

1. Safety and good biocompatibility: Scaffolds should be safe. Furthermore, their chemical components and degradation products should cause minimal immune or inflammatory responses in the body during a predetermined period.59

2. Appropriate size, dimensions, and mechanical strength: The chemical features of the scaffold should provide suitable microenvironments and maintain the biological activity of loaded cells or tissues for a long time.

3. Appropriate pore size and distribution: Scaffolds should have a highly and well-connected porous structure with an ideal pore size to allow cells, drugs, and bioactive molecules to get evenly distributed throughout the scaffold.60

4. Excellent biological behaviors: Scaffolds and the substances present in the scaffold should promote the proliferation and migration of fibroblasts, keratinocytes, and endothelial cells, thus promoting wound healing.61,62

5. Appropriate wound healing environment: The scaffold system should be able to absorb the wound exudate and prevent wound dehydration, reducing surface necrosis on the wound.63,64

Scaffold systems can be classified as follows based on the source and function of the materials.

When designing scaffold systems for chronic wound, an appropriate matrix source needs to be selected. Table 1 lists a few sources of nanocomposites used in wound dressing. Natural nanomaterials and their derivates have good biocompatibility and can be degraded by enzymes or water. However, their characters and quality differ from batch to batch and cannot be standardized. In contrast, synthetic biomaterials, such as polyethylene glycol (PEG) nano-scaffolds, show more stable structural properties and can be chemically modified. However, the biosafety of synthetic materials needs to be strictly examined.

Table 1 Sources of Nanocomposites

According to their functions, tissue engineering materials can be used for bones, nerves, blood vessels, skin, and other tissues (eg, tendon, ligament, cornea, liver, and kidneys).

Tissue engineering scaffolds for the skin can be of several types. These include natural polymers (chitosan, hyaluronic acid, and collagen), nanocomposite scaffolds (eg, nanobioactive glass and metal NPs), and conducting polymers (eg, polyaniline, polypyrrole, and polythiophene).7375 Taghiabadi et al synthesized an intact amniotic membrane-based scaffold for cultivating adipose-derived stromal cells (ASCs). By ASCs on an acellular human amniotic membrane (HAM), they created a neoteric skin substitute.76 Zhang et al designed a conductive and antibacterial hydrogel based on polypyrrole and functionalized Znchitosan molecules for the management of infected chronic wounds. They demonstrated the promising potential of the hydrogel in promoting the healing of the infected chronic wound after electrical stimulation. Currently, other tissue engineering scaffolds such as calcium phosphates and composite materials (eg, hydroxyapatite, -tricalcium phosphate, and whitlockite) for bone tissue engineering and amniotic membranes for corneal tissue engineering are under research.69,77

Skin tissue engineering scaffolds can be categorized as porous, fibrous, microsphere, hydrogel, composite, and acellular materials.73 Typically, natural biomaterials and their derivatives are biodegradable, absorbable, and harmless to the body, but their strength and processing performance are poor and their degradation speed cannot be controlled. Hence, in order to improve the mechanical and biological properties of scaffolds (eg, adhesion, strength, processing performance, and degradation speed) and accelerate wound healing, composite scaffolds have been developed by combining the characteristics and advantages of different materials. Depending on their constituents, these composite scaffolds can achieve specific functions. Currently, most novel scaffolds being developed use composite materials to obtain multifunctional characteristics.

Delivery systems are used to deliver drugs, cells, genes, and other neoteric bioactive molecules to the body or target area via transplantation or injection.78 Traditionally, delivery systems are broadly divided into two categories, drug delivery and cell delivery. With continuous Innovation in scientific research, new approaches, including gene delivery and the delivery of bioactive molecules such as growth factors, proteins, and peptides, are being developed.

Recently, there has been a significant increase in new biotechnology-based treatments, among which cell and gene therapies are quite sophisticated. Exosomes have shown superior therapeutic potential against various conditions, and delivery methods are being devised to maximize their therapeutic effectiveness. Moreover, exosomes are also emerging as a delivery system for other substances (eg, small molecules and miRNAs).79 NPs are essential for the delivery of these refined substances. In addition to serving as delivery vehicles, NPs can also act as diagnostic and therapeutic agents for some diseases.80 Research on nanoparticle-based drug delivery has mainly been focused on targeted drug delivery, and especially tumor-targeted drug delivery.81

A drug delivery system serves as a vehicle for therapeutic molecules. It allows drug delivery in the body, improves drug efficacy, and allows safe and controlled drug release.

The conventional routes for drug delivery80 are gastrointestinal drug delivery (eg, oral and rectal), parenteral administration (eg, subcutaneous, intramuscular, and intravenous injection) and topical administration (eg, percutaneous injection and wound dressings). Novel drug delivery systems for wound healing can be classified into the following categories: NPs, microcarriers, and tissue-engineered scaffolds.82 Skin tissue engineering scaffolds have been introduced earlier in this review, and NPs and microcarriers will be introduced in detail here (Table 2).

Table 2 Drug Delivery Systems Developed Using Nanotechnology

Drug-loaded nano-scaffolds that promote wound healing after topical administration have been developed. However, due to their poor solubility, short half-life, and other drawbacks, some drugs do not accumulate at an optimal concentration at the wound site for a long duration.83 Nano-scaffolds with varying porous structures can be used to load drugs or bioactive molecules, and the porous structure can provide a breathable environment for the wound.84 NPs carrying poorly soluble drugs are widely used to prepare controlled drug delivery systems. Nano-scaffolds typically show slow degradation, allowing long-term drug release and thereby maintaining an ideal concentration of the drug in the plasma.85 Shamloo et al developed polyvinyl alcohol (PVA)/chitosan/gelatin hydrogels to overcome the short half-life of basic fibroblast growth factor (bFGF). The biocompatibility of the hydrogel supported the continuous delivery of bFGF and significantly accelerated wound healing.86

During the treatment of chronic wounds, the drug is usually applied directly on affected region. Nanotechnology-based drug delivery systems could enable controlled drug release. Meanwhile, the degradability and stability of the drug could also be modified using nanosystems. Hence, these drug delivery systems could improve treatment compliance among patients with chronic wounds by reducing the application frequency and the cost of treatment.

It is widely acknowledged that metal ion-based biomaterials exhibit promising antimicrobial activity when applied to wounds, making them very suitable for the management of diabetic wounds, which are prone to infection. Given their reducing properties, under oxidative stress, cuprous ions provide a promising therapeutic option for diabetic wounds. Copper ions have also been reported to promote angiogenesis.115117 Equipped with infrared absorption and efficient heat generation abilities, semiconductor cuprous sulfide (Cu2S) NPs are widely employed as photothermal agents. Wang et al utilized the photothermal effect of Cu2S and the angiogenic effect of Cu ions to prepare electrospun fibers containing Cu2S NPs, achieving a combination of advantages based on the components and successfully promoting diabetic wound healing. Moreover, their biomaterial could also effectively inhibit the growth of skin tumors both in vivo and in vitro.70 This system demonstrated the effectiveness of bifunctional tissue engineering biomaterials, providing a novel method for drug delivery for the treatment of biological conditions.

Classic gene therapy generally involves the expression of exogenous genes or the silencing of target genes via viral or non-viral delivery.118,119 In general, gene delivery via viral transfection may be carcinogenic.119 Most gene therapies for diabetic wounds are based on siRNAs. Gene therapy has become a promising strategy for the treatment of various diseases, and its effects are mediated via the regulation of RNA and protein expression.120 Many unmodified gene therapy agents, such as proteins, peptides, and nucleic acids, are rapidly degraded or eliminated from systemic circulation before they can accumulate at effective concentrations at the target site. Owing to poor pharmacokinetics, repeated administration is warranted. This, in addition to the narrow range of safe doses, often leads to adverse effects during treatment.121

Several studies on wound management and especially chronic diabetic wound management have focused on gene- or RNA-based (eg, mRNA, microRNA, circRNA, and lncRNA) therapies.122 Subcutaneous local injections can be used to directly deliver RNAs or proteins to the wound site.123 However, due to the short half-life of the therapeutic agent, repeated administration is required, often leading to pain and poor treatment compliance. Drug delivery systems not only solve these problems but also protect gene-related small molecules from degradation and eliminated from the body. The greatest challenge in gene therapy is ensuring the successful transduction or transfection of target genes into host cells by crossing extracellular and intracellular barriers. Therefore, the engineering of gene delivery vehicles is complex.118 Moreover, the materials used to encapsulate gene-related small molecules are required to have low toxicity and promote a high transfection efficiency.124 Currently, the NPs that deliver siRNAs to promote wound management are composed of lipids, polymers (eg, chitosan, PEG), hyperbranched cationic polysaccharides (HCP), and silicon.125130

Shaabani et al developed layer-by-layer self-assembled siRNA-loaded gold NPs with two different outer layers Chitosan ([emailprotected]) and Poly L-arginine ([emailprotected]).126 They compared the two types of NPs, which had a similar core structure. They found that the two polymers had different escape mechanisms: the buffering capacity of chitosan resulted in endosome disruption,131 while PLA bound to the endosome lipid bilayer and promoted escaped through pore formation. Their results indicated that an outer layer of PLA allows the endosomal escape of siRNA, thus improving transfection efficiency and delivering target molecules to promote diabetic wound healing. Given that naked siRNAs are easily eliminated from the body, Li et al and Lan et al designed four HCP derivative-based vehicles128,129 for the delivery of siRNA against MMP9. This treatment led to the knockdown of MMP9, which prevents the healing of diabetic wounds, and thus promoted diabetic wound healing. Currently, nanocomposite-based gene delivery applications are focused on siRNA. However, efforts to deliver other products such as miRNA, lncRNA, or even DNA will be required in the future.

Exosomes are endosome-derived vesicles (30 to 150 nm in size) secreted by a variety of cells, including adipose stem cells (ADSCs), bone marrow stem cells (BMSCs), and mesenchymal stem cells (MSCs).132,133 Different types of cells secrete exosomes with different specific markers, which account for their specific functions. Despite their different origins, exosomes have a similar appearance and size and often have a common composition. Once they are isolated from an extracellular medium or from biological fluids, the source of exosomes cannot be ascertained of.134 Exosomes can be employed as small molecules for wound treatment. The combination of exosomes with porous NPs can increase therapeutic effects while maintaining the advantages of a scaffold. Importantly, exosomes can also be used as nanocarriers for drug delivery and targeted therapy, and these are called engineered exosomes.133,135

Exosomes can effectively promote diabetic wound healing.136,137 Shiekh et al embedded ADSC-derived exosomes (ADSC-exo) into antioxidant polyurethane scaffolds to achieve sustained exosome release. Their nanosystem leveraged the advantages of the scaffold, including antioxidant and antibacterial effects, to accelerate diabetic wounds healing both in vivo and in vitro.71 To prolong the half-life and lower the clearance rate of exosomes, Lei et al designed an ultraviolet-shielding nano-dressing based on polysaccharides that allowed exosome delivery and had self-healing, anti-infection and thermo-sensitive properties.61 These findings indicate that exosomes can be stabilized and well-delivered to target cells by combining them with porous NPs or nanocarriers and can be applied for treating chronic wounds.

It is widely accepted that infection is an important factor to monitor during the wound healing process as it can lead to progression of the chronic wound or even sepsis.138140 Conventional prevention and treatment approaches for wound infection involve local or systemic antibiotic administration, which can lead to failed anti-infection treatment or even antibiotic resistance.141,142 Several nano-formulations that have antimicrobial ability have been developed and used in anti-infectious wound therapy, playing a critical role in infection management. Table 3 lists some antimicrobial nanobiotechnology-based systems used in wound healing.

Table 3 Nanomaterials Used in Anti-Microbial Wound Dressing

Metals have been used as inorganic antimicrobial agents for thousands of years and were even used as anti-infection agents in ancient Persia.162 Metal NPs, such as AgNPs, AuNPs, and CuNPs, have attracted great attention due to their anti-infection properties and low toxicity.163 Given that metal NPs do not cause antimicrobial resistance and release metal ions or produce ROS which can kill microorganisms they appear to be suitable alternatives to antibiotics as.164,165

AgNPs, which are the more well-known metal NPs, have been used widely in clinical practice and basic medical research. Wound treatment products containing AgNPs have been commercially available for decades.166 AgNPs can continuously generate Ag+, which reacts with proteins and nucleic acids, causing molecular defects and killing bacteria and viruses.167170 Several studies have shown that AgNPs have good potential as antiseptics. Luna-Hernndez et al found that a combination of functional chitosan and silver nanocomposites showed antibacterial effects against S. aureus and P. aeruginosa in burn wounds.152 Moreover, in mice treated with the composite dressing, silver accumulation was found to be far lower than that in mice treated with the clinically used AcasinTM nanosilver dressing. Zlatko et al demonstrated that the AgNPs hydrogel serves as a versatile platform, with features such as antibacterial efficacy, exudate absorbance, low cost, biocompatibility, hemocompatibility, and improved healing for chronic wounds.171 Huang et al constructed an organic framework-based microneedle patch containing AgNPs. The product showed transdermal delivery and could prevent S. aureus, E. coli, and P. aeruginosa infections in diabetic wounds.172 In addition, several commercialized products containing AgNPs have been developed for clinical treatment. These include Acticoat, Allevyn Ag, Aquacel Ag Surgical, Atrauman Ag, Biatain Silicone Ag, Flaminal, Mepilex Transfer Ag, SILVERCEL, and Urgo Clean Ag.

Nano-sized gold is also useful as an anti-infection agent. It has been confirmed that AuNPs bind to bacterial DNA and show bactericidal and bacteriostatic properties.173,174 Some studies show that Au nanocomposites can kill MRSA and P. aeruginosa through photothermal effects and could promote wound closure.150,175

Compared with gold and silver, copper is less expensive and more easily available. CuNPs are considered the best candidates for developing future technologies for the management of infectious and communicable diseases.49 Cai et al developed a CuNP-embedded hydrogel that accelerated wound healing and showed effective antibacterial capacity against both gram-positive and gram-negative bacteria as well as great photothermal properties.176

Inorganic non-metal nano-materials have been also considered potential antimicrobial agents owing to their intrinsic anti-infection effects.177 Based on the unique structural and physio-chemical properties of carbon nanomaterials, a research team prepared a carbon nanofiber platform that inhibits the growth of E. coli and MRSA.178 In this study, CuNPs and ZnNPs were asymmetrically distributed in carbon NFs grown on an activated carbon fiber substrate using chemical vapor deposition (CVD). The carbon NFs platform inhibited the growth of gram-positive and gram-negative bacterial strains with superior efficiency than simple metal NPs. Another study showed that carbon nanotubes can be used to prepare wound-repairing bandages with infection-preventing properties.179

The natural organic biomaterial chitosan and its derivatives are popular in biomedicine. Chitosan possesses good biocompatibility, antimicrobial properties, and low immunogenicity.180 Using nanobiotechnology, Ganji et al fabricated a nanofiber with chitosan-encapsulated nanoparticles loaded with curcumin for wound dressing. The electrospun chitosan-based nanofiber inhibited the growth of E. coli and MRSA by 98.9% and 99.3% in infected wounds in mice.50 Another type of chitosan nanofiber also showed potential in wound care owing to its antibacterial and re-epithelialization-promoting effects.181 Antibiotic-loaded chitosan nanofibers have also been used for local drug delivery and wound treatment.182 Other metalorganic framework nanorods have also shown bacterial inhibition in infectious wounds.183 Dias et al developed a series of soluble potato starch nanofibers sized 70264 nm. They incorporated carvacrol during the synthesis of the potato starch nanofibers, and the obtained nanocomposites showed great anti-pathogenic activity against S. aureus, E. coli, L. monocytogenes, and S. typhimurium, highlighting their potential as agents for wound dressing.184

With respect to organic nano-materials, anti-infection approaches focus on natural antibacterial compounds such as chitosan and its derivatives. Further, owing to the bactericidal effects of metals, metal-organic frameworks are also used. Given that metal NPs are associated with the potential risks of metal deposition, organic nano-antimicrobial materials, especially natural macromolecules with antibacterial properties, may become useful for wound dressing.

Biofilm, which are made up of surface-attached groups of microbes, are considered to be the primary cause of chronic wounds owing to their role in antibiotic resistance.141,185187 Most biofilms are formed on the surface of wounds. However, some special biofilms can get implanted into the deep layers of skin tissue, making traditional diagnose and treatment challenging.188 The clinical treatment of biofilms in wounds involves wound cleansing with polyvinylpyrrolidone or hydrogen peroxide, debridement, refashioning of wound edges, dressing, and the topical or general administration of antibiotics.189 With further insights into the mechanisms of biofilm formation and developments in nanobiotechnology, nanomaterials effective for biofilm therapy have been developed.

Nanomaterials based on metals or metal oxides are widely used against wound biofilms, including silver, copper, gold, titanium, zinc oxide, magnesium oxide, copper oxide, and iron oxide.190,191 Owing to the small size of these particles, metal or metal oxide NPs can move across bacterial membranes and rupture them. They can destroy enzyme activity and the respiratory chain in bacteria. It has been demonstrated that Ag NPs and silver oxide NPs are the most effective against microbial biofilms.192,193 Abdalla et al functionalized nano-silver with lactoferrin and incorporated them in a gelatin hydrogel, generating a dual-antimicrobial action dressing for infectious wounds and maximizing the anti-biofilm property of silver.194

Chitosan, bacterial cellulose (BC) and other natural antimicrobials have been modified using nanotechnology to treat wound biofilms. Owing to the positive charge on the polymeric chain of chitosan, chitosan NPs easily adhere to the negatively charged microbial membrane, triggering changes in permeability and preventing biofilm formation.195 Zemjkoski et al obtained chitosan NPs through gamma irradiation and encapsuled them into BC to form BC-nChiD hydrogels with excellent anti-biofilm potential. These hydrogels could provide a 90% reduction in viable biofilms and a 65% reduction in biofilm height.196 Mahtab reduced the amount of bacteria in a planktonic condition by treating bacterial biofilms with photodynamic therapy using curcumin encapsulated into silica NPs. After exposure to blue light, ROS was produced owing to the photodynamic properties of silica NPs. The ROS damaged biofilms, and the curcumin released prevented bacterial growth.197

The size of nanoparticles can be controlled, and they have a large specific area, can penetrate bacterial membranes, and show bactericidal properties. Hence, nanotechnology has great potential in destroying biofilms and treating infectious chronic wounds. In addition to providing nanoparticles with anti-infection properties, nanotechnology could also be used to provide a platform for antibiotics, enhance their solubility, prolong their half-life, and reduce the required treatment dose.

Due to its superiority with respect to tissue engineering, cell-based therapy is extensively used for chronic wound treatment.198201 Stem cells derived from bone marrow, the umbilical cord, and adipose and cutaneous tissue can differentiate into various tissue types and modulate cell migration, collagen deposition, re-epithelialization, and tissue remodeling.198,202205 Nanofibers prepared using electrostatic spinning are widely used for scaffolding. Mao et al prepared polycaprolactone nanofibrous scaffolds and combined collagen with bioactive glass NPs (CPB nanofibrous scaffold). The CPB nanofibrous scaffold exerted positive effects as a cell-carrying system containing epithelial progenitor cells (EPCs). The EPC-carrying CPB bioactive complex promoted wound healing by enhancing cell proliferation, granulation tissue formation, re-epithelialization, and cell adhesion (Figure 3).206 Khojasteh et al found that curcumin-carrying chitosan/poly(vinyl alcohol) nanofibers can carry pad-derived mesenchymal stem cells and show excellent curcumin release and improve cell adhesion and proliferation, indicating that they could be useful in wound dressings.207 Kaplan et al produced an injectable silk nanofiber hydrogel embedded with BMSCs. The nanofiber hydrogel maintained the stemness of the BMSCs, successfully carrying them to the target site and promoting wound healing through increased angiogenesis and collagen deposition.57

Figure 3 Schematic of a CPB/EPC construct that promotes wound healing. CPB enhances cell proliferation, collagen deposition, and EPC differentiation via the Hif-1/VEGF/SDF-1 pathway. This results in the rapid vascularization and healing of full-thickness wounds.

Notes: Reprinted from: Wang C, Wang Q, Gao W et al. Highly efficient local delivery of endothelial progenitor cells significantly potentiates angiogenesis and full-thickness wound healing. Acta Biomaterialia. 2018;69:156169. doi:10.1016/j.actbio.2018.01.019.206 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. With permission from Elsevier. Available from: https://www.sciencedirect.com/science/article/abs/pii/S1742706118300308#f0060.

Usually, cell therapy in wound care is performed using micrometer-scale carriers as cell sizes fall in the range of microns. With the development of nanotechnology, an increasing number of nanofibers and NPs are being developed for cell therapy aimed at treating chronic wound given the excellent pro-differentiation, stemness-holding, and immunoregulation properties of the nanocomposites.

As an important component of the extracellular matrix, collagen mediates communication between cells, provides a scaffold for cell migration and adhesion, and plays a role in chronic wound healing.4 Some nanobiotechnology-based platforms have been used for collagen modulation. Sun et al loaded N-acetyl cysteine onto graphene oxide (GO) NPs to enable scarless wound healing (Figure 4).208 In their study, GO NPs decreased collagen metabolism and improved the balance between collagen formation and degradation, thus allowing the wound to heal without scarring. In another study by the same group, a polyamide nanofiber-based multi-layered scaffold was found to promote wound healing by encouraging the uniform arrangement of collagen.209 Krian et al synthesized a 3-D biomatrix with nanotized praseodymium that promotes collagen function via the stabilization of native collagen. Their rare-earth metal nanoparticles thus showed potential applications in wound care.210

Figure 4 Wound healing effect of a scaffold based on GO NPs.

Notes: Adapted from: Li J, Zhou C, Luo C et al. N-acetyl cysteine-loaded graphene oxide-collagen hybrid membrane for scarless wound healing. Theranostics. 2019;9(20):58395853. doi:10.7150/thno.34480.208 The author(s). Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/). See http://ivyspring.com/terms for full terms and conditions.

In chronic wound treatment, deposited collagen acts as a natural scaffold for cells, and therefore, modulating collagens is synonymous with re-establishing tissue structure in the wound area. As a result, collagen-modulating nano-systems have mainly been used for accelerating tissue repair. However, the studies by Suns group are inspirational and demonstrate that this approach should also be utilized for developing chronic wound treatments that decrease scarring.

Despite the availability of dozens of commercial wound-care products, bionic systems have not yet been adopted for wound healing. There is an urgent need for smart wound-healing systems that can respond to the stimuli (temperature, pH, glucose, enzyme, etc.) at the site of the chronic wound area.211,212 Through developments in nanobiotechnology, NPs with stimuli-response characteristics have received great attention. Gong et al synthesized a nanozyme consisting of poly(acrylic acid)-coated Fe3O4 NPs (pFe3O4) and then combined them with GO to produce pFe3O4@GO NCs. The pFe3O4@GO NCs could react with glucose and function as a self-supplying H2O2 nanogenerator at the wound site, allowing the chemodynamic treatment of wound infections.157 Some researchers developed photoactive electrospun nanofibers using cellulose acetate, polyethylene oxide, methylene blue, and three-layered cellulose acetate/polyethylene oxide/silk fibroin/ciprofloxacin. The nanofibers could produce ROS after light irradiation at 635 nm, accelerating the healing of infectious wounds by inhibiting S. aureus, K. pneumoniae, and P. aeruginosa biofilms.213 Zhang et al developed a hybrid hydrogel with MnO2 nanosheets. The injectable MnO2 nanosheet hydrogel could perform thermogenesis under 808-nm laser irradiation, eliminating ROS and inflammation and promoting wound repair.214 Overall, nano-structures functionalized using stimuli-response properties could simulate the biological, chemical, and physical characteristics of natural skin, enabling tissue regeneration in refractory wounds.

Given the elucidation of mechanisms and physiological changes associated with wound healing, sensors that allow real-time monitoring of wound repair have been developed.215217 A complex smart wound-monitoring wound dressing has also been invented.218 This dressing contains a nanofiber membrane made of chitosan/collagen, and promotes proliferation and regeneration by upregulating extracellular matrix secretion and promoting integrin/FAK signaling. Olivo et al added AgNPs to a fiber-based membrane monitor to increase the active surface area in the sensor, improving the detection sensitivity for biomarkers in the wound area.219 In order to avoid secondary wound damage caused by dressing changes, Jiang et al created bacterial cellulose-based membranes with aminobenzeneboronic acid-modified gold nanoclusters (A-GNCs), which could be used for treating wounds infected with multidrug-resistant bacteria.220 A-GNCs emit bright orange fluorescence under UV light, and the intensity of this fluorescence decreases with the release of A-GNCs. This allows healthcare professionals to determine when the dressing needs to be replaced. In the past few years, dressings that can monitor the status of chronic wounds in real-time have been tested. However, this field is relatively new, and current research on nanotech-based systems for monitoring chronic wounds is scarce.

Along with advances in nanobiotechnology research, several new nanosystems have advanced from the laboratory investigation stage to the clinical trial stage. Table 4 lists some clinical trials that have tested nano-therapies for wound healing. As early as 2014, Lopes et al investigated the cost-effectiveness of using nanocrystalline silver for treating burns. Their study showed that AgNPs provided faster wound healing than traditional silver sulfadiazine, requiring fewer dressing changes and reducing the human resource burden.221 Meanwhile, some clinical trials tested the use of nano-products for treating chronic wounds (Table 4). Although metal NPs were typically used for antimicrobial therapy, one clinical trial studied the efficacy and safety of autologous nano-fat combined with platelet-rich fibrin for treating refractory diabetic foot wounds. However, overall, there were few clinical trials examining the applications of nanoplatforms in chronic wound care, likely owing to inadequate previous research on biocompatibility. Moreover, few doctors participated in research on nanotechnology-based chronic-wound treatment, and hence, several clinical requirements were ignored or misunderstood.

Table 4 List of Clinical Trials for Nanobiotechnology-Based Wound Treatment

As nanobiotechnology has developed, nano-sized biomaterials have been widely applied for treating chronic wounds. This review article highlights that the application of nanotechnology in chronic wound treatment has, so far, largely focused on scaffold construction, anti-infection treatment, and substance delivery.34,45,47,130,147

In scaffold systems, nanobiotechnology provides both materials and techniques for managing chronic wounds. Electrospinning, a nanotechnique, allows the production of biomimetic structures that mimic the natural skin and help in healing refractory wounds.50 Furthermore, some nano-scaffolds promote cell adhesion and migration by mimicking the construction of natural tissues, thus promoting chronic wound healing. Nevertheless, there is further scope to improve the quality of natural nano-biomaterials and the biocompatibility of synthetic nano-biomaterials to increase their application.

Dozens of metal NPs, and especially AgNPs, have been used in antimicrobial therapy for chronic wounds.163 However, metal deposition can cause DNA and cell damage. Hence, nanomaterials that prevent infection without causing toxicity are required. Further effort should be made to decrease the accumulation of heavy metals. Alternatively, nanocomposites without metal elements should be adopted more often in the future.

To overcome the ever-changing environment of the skin during chronic wound healing, several wound-monitoring and stimuli-responsive biomaterials have been developed.58,157,218 By leveraging specific characteristics, such as the photothermal effect, chemo-dynamic effect, fluorescence, and thermo-sensitivity, more nano-biomaterials that can be used in stimuli-responsive and dynamic monitoring systems for wound care should be developed. Most studies on wound healing have focused on migration-promoting effects, antimicrobial activity, and substance delivery. However, few nanotech-based multifunctional smart systems, such as smart dressings that show specific responses to stimuli, have been developed. Researchers in this field should work towards developing smart systems based on the mechanisms of disunion in chronic wounds, which could effectively demonstrate the potential of nanobiotechnology in promoting chronic wound repair.

Despite the decades-long history of nanotechnology research, few products and therapies based on nanobiotechnology have become available commercially or entered the clinical trial phase. One reason for this is that most basic nanotech research on chronic wound healing is performed in rodent models, such as C57BL/6 mice or SpragueDawley rats, even though the skin structure and chronic wound healing processes differ between rodents and humans.222 The wound healing effects observed in primates, such as humans, may not be as good as those in rats and mice. Meanwhile, the cost of nano-materials and processing platforms required for large-scale preparation also hinder the clinical translation of nanotechnologies.

During the past few years, numerous nano-materials and techniques have been used to repair chronic wounds. This review summarizes some nanobiotechnology-based systems and nanoplatform designs that can be used for treating chronic wounds. It highlights that a smart dressing for chronic wounds that allows real-time monitoring and has stimuli-responsive abilities is one possible direction for the future of nano-wound-repairing systems. We hope this review motivates the development of more sophisticated wound management systems based on nanobiotechnology in the future.

The authors acknowledge the support from the National Natural Science Foundation of China (81974289, 81772094), the Key Research and Development Program of Hubei Province (grant number 2020BCB031), the Guangdong Basic and Applied Basic Research Foundation (2019B1515120043), the international cooperation research project of Shenzhen, the international cooperation research project of Shenzhen (GJHZ20190822091601691), and the Key Project of Basic Research of Shenzhen (JCYJ20200109113603854).

The authors report no conflicts of interest in relation to this work.

1. Diegelmann RF, Evans MC. Wound healing: an overview of acute, fibrotic and delayed healing. Front Biosci-Landmrk. 2004;9:283289. doi:10.2741/1184

2. Vig K, Chaudhari A, Tripathi S, et al. Advances in skin regeneration using tissue engineering. Int J Mol Sci. 2017;18(4):789.

3. Stojadinovic A, Carlson JW, Schultz GS, Davis TA, Elster EA. Topical advances in wound care. Gynecol Oncol. 2008;111(2):S70S80.

4. Gurtner GC, Werner S, Barrandon Y, Longaker MT. Wound repair and regeneration. Nature. 2008;453(7193):314321.

5. Tavakoli S, Klar AS. Advanced hydrogels as wound dressings. Biomolecules. 2020;10(8):1169.

6. Versteeg HH, Heemskerk JW, Levi M, Reitsma PH. New fundamentals in hemostasis. Physiol Rev. 2013;93(1):327358.

7. Wilkinson HN, Hardman MJ. Wound healing: cellular mechanisms and pathological outcomes. Open Biol. 2020;10(9):200223.

8. Olsson M, Jarbrink K, Divakar U, et al. The humanistic and economic burden of chronic wounds: a systematic review. Wound Repair Regen. 2019;27(1):114125.

9. Stadelmann WK, Digenis AG, Tobin GR. Physiology and healing dynamics of chronic cutaneous wounds. Am J Surg. 1998;176(2ASuppl):26S38S.

10. Lerman OZ, Galiano RD, Armour M, Levine JP, Gurtner GC. Cellular dysfunction in the diabetic fibroblast: impairment in migration, vascular endothelial growth factor production, and response to hypoxia. Am J Pathol. 2003;162(1):303312. doi:10.1016/S0002-9440(10)63821-7

11. Duscher D, Januszyk M, Maan ZN, et al. Comparison of the hydroxylase inhibitor dimethyloxalylglycine and the iron chelator deferoxamine in diabetic and aged wound healing. Plast Reconstr Surg. 2017;139(3):695e706e. doi:10.1097/PRS.0000000000003072

12. Rodrigues M, Wong VW, Rennert RC, Davis CR, Longaker MT, Gurtner GC. Progenitor cell dysfunctions underlie some diabetic complications. Am J Pathol. 2015;185(10):26072618. doi:10.1016/j.ajpath.2015.05.003

13. Costerton JW, Stewart PS, Greenberg EP. Bacterial biofilms: a common cause of persistent infections. Science. 1999;284(5418):13181322. doi:10.1126/science.284.5418.1318

14. Versey Z, da Cruz Nizer WS, Russell E, et al. Biofilm-innate immune interface: contribution to chronic wound formation. Front Immunol. 2021;12:648554.

15. Wang X, Coradin T, Helary C. Modulating inflammation in a cutaneous chronic wound model by IL-10 released from collagen-silica nanocomposites via gene delivery. Biomater Sci. 2018;6(2):398406.

16. Xu Z, Liang B, Tian J, Wu J. Anti-inflammation biomaterial platforms for chronic wound healing. Biomater Sci. 2021;9(12):43884409.

17. Zhao R, Liang H, Clarke E, Jackson C, Xue M. Inflammation in chronic wounds. Int J Mol Sci. 2016;17(12):2085.

18. Boateng JS, Matthews KH, Stevens HNE, Eccleston GM. Wound healing dressings and drug delivery systems: a review. J Pharm Sci-Us. 2008;97(8):28922923.

19. Schiestl C, Stiefel D, Meuli M. Giant naevus, giant excision, eleg(i)ant closure? Reconstructive surgery with Integra Artificial Skin to treat giant congenital melanocytic naevi in children. J Plast Reconstr Aesthet Surg. 2010;63(4):610615.

20. Schiestl C, Neuhaus K, Biedermann T, Bottcher-Haberzeth S, Reichmann E, Meuli M. Novel treatment for massive lower extremity avulsion injuries in children: slow, but effective with good cosmesis. Eur J Pediatr Surg. 2011;21(2):106110.

21. Eming SA, Wynn TA, Martin P. Inflammation and metabolism in tissue repair and regeneration. Science. 2017;356(6342):10261030.

22. Mookherjee N, Anderson MA, Haagsman HP, Davidson DJ. Antimicrobial host defence peptides: functions and clinical potential. Nat Rev Drug Discov. 2020;19(5):311332.

23. Eming SA, Martin P, Tomic-Canic M. Wound repair and regeneration: mechanisms, signaling, and translation. Sci Transl Med. 2014;6(265):265sr6.

24. Paradise J, Wolf SM, Kuzma J, RamacHandran G, Kokkoli E. Introduction: the challenge of developing oversight approaches to nanobiotechnology. J Law Med Ethics. 2009;37(4):543545.

25. Roco MC. Nanotechnology: convergence with modern biology and medicine. Curr Opin Biotechnol. 2003;14(3):337346.

26. Wang B, Kostarelos K, Nelson BJ, Zhang L. Trends in micro-/nanorobotics: materials development, actuation, localization, and system integration for biomedical applications. Adv Mater. 2021;33(4):2002047.

View post:
Applications in Chronic Wound Healing | IJN - Dove Medical Press

Blood, sweat, and many miles for cellular therapy – Martha’s Vineyard Times

From the left, Theresa Janeczek (Edgartown), Cathy Mayone (Edgartown), Lisa Conroy Murphy (Edgartown), Jacki Reich, Bob Falkenberg, Chuck Klaniecki, and Matt Berg (right) at a rest stop. Stefanie Cronin

The team met at the Steamship Authority in Vineyard Haven in the morning to get ready to ride. Natalie Aymond

The team of bikers who met in the morning (left to right): Bruce Rayvid (Cycling Club of MV), Bob Falkenberg, Elle Crofton, Chuck Klaniecki, Jacki Reich, Matt Berg, Cathy Mayone, Harry Crofton, Roger Moffat (Cycling Club of MV), and Lisa Conroy Murphy (Cycling Club of MV). Natalie Aymond

Harry Crofton (left) and Bob Falkenberg arriving at the Edgartown Memorial Wharf. Natalie Aymond

Roger Moffat (left), Harry Crofton, Elle Crofton, and Bob Falkenberg cheering on the rest of the team as they arrive. Natalie Aymond

Matt Berg (left), Bob Falkenberg, Elle Crofton, Harry Crofton, Cathy Mayone (right), and Theresa Janeczek (below) at the Edgartown Memorial Wharf. Natalie Aymond

Bob Falkenberg receiving his transplant through Be The Match. Be The Match

Bob Falkenberg, a 13-year leukemia survivor, is just one example of a transplant recipient that Be The Match (BTM) has been able to treat and save by connecting him with a blood marrow donor. But while Falkenberg is a face of success in matching patients with a donor, the 12,000 patients diagnosed with life-threatening blood cancers or other blood cell diseases each year are not always as easily treated, especially with financial barriers and a disparity in match rates for non-white patients.

Just two years after his transplant, and with a push from a 100-mile bike ride challenge from his friend, Falkenberg has been biking to raise money and awareness for the need for transplant donors for the past 11 years. He and his team of nine riders took to the Island July 20 to continue this support for Be The Match, part of their month-long East Coast ride event, Tour De TC. This annual bike ride raises critical funds for Be The Match, with this years ride aimed at raising enough funds to financially support 25 families in need of treatment.

In his past years of biking for Be The Match, Falkenberg has embarked on rides from Boston to Key West, Vancouver to San Francisco, and Vancouver to Florida. His hope is to hit all 50 states with future rides. For the second half of this years July tour, the crew has already gone from Boston to the Cape and Marthas Vineyard, but will continue on via ferry to Rhode Island and Connecticut. From there, the crew will ferry to the east end of Long Island and into New York City to meet people from the transplant center there. Finally, the riders will head to the childrens hospital in Philadelphia.

In past years, the rides have been more family and friends oriented, according to Falkenberg. But this year, he said that the rides have been more open to participation. From this, he added, they have gotten a larger response and have already raised $100,000 for this year, three times as much as last years raised funds. This is just the start, as he expects they will double all raised funds from this year next year.

Funds raised go toward adding more donors to the Be The Match registry, research to ease the safety of transplant procedures, as well as financial assistance for families and patients in need of transplants and in post-transplant recovery. For adding donors, there is a cost to do the human leukocyte antigen (HLA) tests in order to add donors to the registry and match them to patients. Funds for researching the transplant procedures include identifying and preventing issues that can impact chance of survival, which has increased from 30% to closer to 50% in the past 13 years, according to Falkenberg. For many people, the journey of treatment and recovery can even be such a financial toll it can prevent patients from moving forward with potentially life-saving procedures if careers are put on hold, decreasing household income, says BTM in an information sheet.

Leukemia is the number one childhood cancer, so a lot of this is for kids. When the parents travel, they have to stay there for a long time sometimes, Falkenberg says, which takes time away from work and puts financial strain on family support. There are also financial grants through BTM that increase this family and patient support while covering costs that insurance will not.

Money is not the only thing that decreases the success of patient survival, as there has grown to be a disparity in the diversity of donors. According to BTM, out of almost 300,000 potential U.S. donors added to the registry last year, only 31% were ethnically diverse. Falkenberg commented on this issue saying, Theres about 20 million people on the registry, but if youre Black you only have about a 29% chance right now of finding a single donor on the registry because there just arent enough Black donors and its tied to your DNA and ethnicity. Falkenberg also said that there is a similar struggle for Asian or Pacific Islander patients, Hispanic or Latino patients, and Native American patients, though not quite as bad as the odds for Black or African American patients.

Elle Crofton, a first year rider diagnosed with a blood cancer nine years ago, works as an advocate for BTM alongside Falkenberg and spoke to The Times about this issue of ethnic disparity in donors. Like Falkenberg, Crofton was able to find multiple full matches on the BTM registry that allowed her to get a transplant seven years ago, but said, For people who are not white, they have a lot less likelihood of finding a match. She added that the team is trying to get the word out to get more people of color on the registry saying, We hope we can make the need for everyone to have that equal ability to find a match smaller.

Beyond volunteering and riding, Falkenberg and Crofton have begun legislative advocacy work, lobbying congress to provide legal support to donors. The two reached out to Joe Neguse, the U.S. representative for Colorados 2nd congressional district for support. Falkenberg and Crofton had a virtual meeting surrounding their current work on a Life Saving Leave Act, to which Neguse co-sponsored the next day. The act would work to allow 40 hours of non-consecutive time off of work and protect workers from being fired while undergoing the donation process. This process includes a physical exam and an injection to increase stem cell production, for which the travel and donation can take up to two days to complete. So while not a paid leave, the act would mitigate the fear for potential donors to lose their job.

Alongside the act, Be The Match reimburses for associated costs with the donation process. Its common sense stuff, just not the law right now, Falkenberg said and added, Unfortunately, the people that are more likely to say Im worried about losing my job also line up with the groups underrepresented on the registry. So, every donor that donates matters.

To donate, become a donor and join the registry, or find out other ways to support the organization visit BeTheMatch.org. Eligibility to become a donor is met if you meet the health guidelines and are between 18 and 40 years old. For registration, completion of a health history form and a swab of cheek cells is needed. The swab kit is mailed to the registrants home.

Original post:
Blood, sweat, and many miles for cellular therapy - Martha's Vineyard Times

Inhibition of pancreatic EZH2 restores progenitor insulin in T1D donor | Signal Transduction and Targeted Therapy – Nature.com

Human samples

Rapid harvesting of cadaveric pancreatic tissues was obtained with informed consent from next of kin, from heart-beating, brain-dead donors, with research approval from the Human Research Ethics Committee at St Vincents Hospital, Melbourne. Pancreas from individuals without and with diabetes, islet, acinar and ductal samples were obtained as part of the research consented tissues through the National Islet Transplantation Programme (at Westmead Hospital, Sydney and the St Vincents Institute, Melbourne, Australia), HREC Protocol number: 011/04. The donor characteristics of islet cell donor isolations are presented in Table 1.

Islets were purified by intraductal perfusion and digestion of the pancreases with collagenase AF-1.24 (SERVA/Nordmark, Germany) followed by purification using Ficoll density gradients.25 Purified islets, from low-density gradient fractions and acinar/ductal tissue, from high-density fractions, were cultured in Miami Media 1A (Mediatech/Corning 98021, USA) supplemented with 2.5% human serum albumin (Australian Red Cross, Melbourne, VIC, Australia), in a 37C, 5% CO2 incubator.

Total RNA from human ex vivo pancreatic cells was isolated using TRIzol (Invitrogen) and RNeasy Kit (QIAGEN) including a DNase treatment. First-strand cDNA synthesis was performed using a high-capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturers instructions. cDNA primers were designed using oligoperfect designer (Thermo Fisher Scientific), as shown in Table 2. Briefly, quantitative RT-PCR analyses were undertaken using the PrecisionFast 2 qPCR Master Mix (Primerdesign) and primers using Applied Biosystems 7500 Fast Real-Time PCR System. Each qPCR reaction contained: 6.5l qPCR Master Mix, 0.5l of forward and reverse primers, 3.5l H2O and 2l of previously synthesised cDNA, diluted 1/20. Expression levels of specific genes were tested and normalised to 18s ribosomal RNA housekeeping gene.

Modification of Histone H3 and histone-associated Ezh2 protein signals were quantified in human pancreatic ductal epithelial cells (AddexBio) by the LI-COR Odyssey assay. The cells were treated with 5 or 10M of GSK 126 (S7061, Selleckchem) for 48h. Histones and their associated proteins were examined using an acid extraction and immunoblotting as described previously.18 Protein concentrations were determined using Coomassie Reagent (Sigma) with BSA as a standard. Equal amounts (3g) of acid extract were separated by Nu-PAGE (Invitrogen), transferred to a PVDF membrane (Immobilon-FL; Millipore) and then probed with antibodies against H3K27me3 (07449, Millipore), H3K27ac (ab4729, Abcam), H3K9me3 (ab8898, Abcam), H3K9me2 (ab1220, Abcam), H3K4me3 (39159, Active Motif), Ezh2 (#4905, Cell Signaling Technology), and total histone H3 (#14269, Cell Signaling Technology). Protein blotting signals were quantified by an infra-red imaging system (Odyssey; LI-COR). Modification of Histone H3 and histone-associated Ezh2 signals were quantified using total histone H3 signal as a loading control.

Chromatin immunoprecipitation assays in human exocrine cells were performed previously described.26,27 Cells were fixed for 10min with 1% formaldehyde and quenched for 10min with glycine (0.125M) solution. Fixed cells were resuspended in sodium dodecyl (lauryl) sulfate (SDS) lysis buffer (1% SDS, 10mM EDTA, 50mM Tris-HCl pH 8.1) including a protease inhibitor cocktail (Roche Diagnostics GmBH, Mannheim, Germany) and homogenised followed by incubation on ice for 5min. Soluble samples were sonicated to 200600bp and chromatin was resuspended in ChIP Dilution Buffer (0.01% SDS, 1.1% Triton X-100, 1.2mM EDTA, 16.7mM Tris-HCl pH 8.0, and 167mM NaCl) and 20l of Dynabeads Protein A (Invitrogen, Carlsbad, CA, USA) was added and pre-cleared. H3K27me3 antibody was used for immunoprecipitation of chromatin and incubated overnight at 4C as previously described.28 Immunoprecipitated DNA were collected by magnetic isolation, washed low salt followed by high salt buffers and eluted with 0.1M NaHCO3 with 1% SDS. Protein-DNA cross-links were reversed by adding Proteinase K (Sigma, St. Louis, MO, USA) and incubation at 62C for 2h. DNA was recovered using a Qiagen MinElute column (Qiagen Inc., Valencia, CA, USA). H3K27me3 content at the promoters of the INS, INS-IGF2, NGN3 and PDX1 genes were assessed by qPCR using primers designed from the integrative ENCODE resource.29 ChIP primers are shown in Table 3.

Insulin and glucagon localisation in human islets were assessed using paraffin sections (5m thickness) of human pancreas tissue fixed in 10% neutral-buffered formalin and stained with hematoxylin and eosin (H&E) or prepared for immunohistochemistry. Insulin and glucagon were detected using Guinea Pig anti-insulin (1/100, DAKO) or mouse anti-glucagon (1/50) mAbs (polyclonal Abs, Sigma-Aldrich).

Pharmacological inhibition of EZH2, human pancreatic exocrine cells were kept untreated or stimulated with 10M GSK-126 (S7061, Selleckchem) at a cell density of 1105 per well for 24h. After 24h of treatment, fresh Miami Media was added to the cells, which were treated again with 10 GSK-126 and cultured for a further 24h. All cell incubations were performed in Miami Media 1A (Mediatech/Corning 98-021, USA) supplemented with 2.5% human serum albumin (Australian Red Cross, Melbourne, VIC, Australia), in a cell culture incubator at 37C in an atmosphere of 5% CO2 for 48h using non-treated six-well culture plates (Corning).

Read the rest here:
Inhibition of pancreatic EZH2 restores progenitor insulin in T1D donor | Signal Transduction and Targeted Therapy - Nature.com

Bristol Myers Squibb Receives Positive CHMP Opinion Recommending Approval for LAG-3-Blocking Antibody Combination Opdualag (nivolumab and relatlimab)…

PRINCETON, N.J.--(BUSINESS WIRE)--Bristol Myers Squibb (NYSE: BMY) today announced that the Committee for Medicinal Products for Human Use (CHMP) of the European Medicines Agency (EMA) has recommended approval of the fixed-dose combination of nivolumab and relatlimab for the first-line treatment of advanced (unresectable or metastatic) melanoma in adults and adolescents 12 years of age and older with tumor cell PD-L1 expression < 1%. The European Commission (EC), which has the authority to approve medicines for the European Union (EU), will now review the CHMP opinion.

We are very proud of the role we have played in progressing the treatment of advanced melanoma over the years. As part of our mission to deliver new medicines for patients, we have continued to develop new dual immunotherapy combinations, said Paul Basciano, development lead, relatlimab, Bristol Myers Squibb. This positive CHMP opinion marks the first step toward the potential approval of the first LAG-3 blocking antibody combination and the third distinct checkpoint inhibitor for BMS for advanced melanoma patients in the EU.

The positive opinion is based upon efficacy and safety results from the Phase 2/3 RELATIVITY-047 trial. The trial showed that treatment with the fixed-dose combination of nivolumab and relatlimab more than doubled the median progression-free survival (PFS), including in patients with tumor cell PD-L1 expression < 1%, when compared to nivolumab monotherapy an established standard of care. The proposed indication for the EU is based upon an exploratory analysis of the data in patients with tumor cell PD-L1 expression < 1%. No new safety events were identified with the combination when compared to nivolumab monotherapy.

On March 18, 2022, the U.S. Food and Drug Administration (FDA) approved the fixed-dose combination of nivolumab and relatlimab as Opdualag (nivolumab and relatlimab-rmbw) for the treatment of adult and pediatric patients 12 years of age or older with unresectable or metastatic melanoma. Please see important safety information from the U.S. prescribing information below.

Bristol Myers Squibb thanks the patients and investigators involved in the RELATIVITY-047 trial.

About RELATIVITY-047

RELATIVITY-047 is a global, randomized, double-blind Phase 2/3 study evaluating the fixed-dose combination of nivolumab and relatlimab versus nivolumab alone in patients with previously untreated metastatic or unresectable melanoma. Patients were enrolled regardless of tumor cell PD-L1 expression. The trial excluded patients with active autoimmune disease, medical conditions requiring systemic treatment with moderate or high dose corticosteroids or immunosuppressive medications, uveal melanoma, and active or untreated brain or leptomeningeal metastases. The primary endpoint of the trial is progression-free survival (PFS) determined by Blinded Independent Central Review (BICR) using Response Evaluation Criteria in Solid Tumors (RECIST v1.1) in the all-comer population. The secondary endpoints are overall survival (OS) and objective response rate (ORR) in the all-comer population. A total of 714 patients were randomized 1:1 to receive a fixed-dose combination of nivolumab (480 mg) and relatlimab (160 mg) or nivolumab (480 mg) by intravenous infusion every four weeks until disease progression, unacceptable toxicity or withdrawal of consent.

About LAG-3

Lymphocyte-activation gene 3 (LAG-3) is a cell-surface molecule expressed on effector T cells and regulatory T cells (Tregs) and functions to control T-cell response, activation and growth. Preclinical studies indicate that inhibition of LAG-3 may restore effector function of exhausted T cells and potentially promote an anti-tumor response. Early research demonstrates that targeting LAG-3 in combination with other potentially complementary immune checkpoints may be a key strategy to more effectively potentiate anti-tumor immune activity.

Bristol Myers Squibb is evaluating relatlimab, its LAG-3-blocking antibody, in clinical trials in combination with other agents in a variety of tumor types.

About Melanoma

Melanoma is a form of skin cancer characterized by the uncontrolled growth of pigment-producing cells (melanocytes) located in the skin. Metastatic melanoma is the deadliest form of the disease and occurs when cancer spreads beyond the surface of the skin to other organs. The incidence of melanoma has been increasing steadily for the last 30 years. In the United States, 106,110 new diagnoses of melanoma and about 7,180 related deaths are estimated for 2021. Globally, the World Health Organization estimates that by 2035, melanoma incidence will reach 424,102, with 94,308 related deaths. Melanoma can be mostly treatable when caught in its very early stages; however, survival rates can decrease as the disease progresses.

Bristol Myers Squibb: Creating a Better Future for People with Cancer

Bristol Myers Squibb is inspired by a single vision transforming patients lives through science. The goal of the companys cancer research is to deliver medicines that offer each patient a better, healthier life and to make cure a possibility. Building on a legacy across a broad range of cancers that have changed survival expectations for many, Bristol Myers Squibb researchers are exploring new frontiers in personalized medicine, and through innovative digital platforms, are turning data into insights that sharpen their focus. Deep scientific expertise, cutting-edge capabilities and discovery platforms enable the company to look at cancer from every angle. Cancer can have a relentless grasp on many parts of a patients life, and Bristol Myers Squibb is committed to taking actions to address all aspects of care, from diagnosis to survivorship. Because as a leader in cancer care, Bristol Myers Squibb is working to empower all people with cancer to have a better future.

OPDUALAG U.S. INDICATION

Opdualag (nivolumab and relatlimab-rmbw) is indicated for the treatment of adult and pediatric patients 12 years of age or older with unresectable or metastatic melanoma.

OPDUALAG IMPORTANT SAFETY INFORMATION

Severe and Fatal Immune-Mediated Adverse Reactions

Immune-mediated adverse reactions (IMARs) listed herein may not include all possible severe and fatal immune-mediated adverse reactions.

IMARs which may be severe or fatal, can occur in any organ system or tissue. IMARs can occur at any time after starting treatment with a LAG-3 and PD-1/PD-L1 blocking antibodies. While IMARs usually manifest during treatment, they can also occur after discontinuation of Opdualag. Early identification and management of IMARs are essential to ensure safe use. Monitor patients closely for symptoms and signs that may be clinical manifestations of underlying IMARs. Evaluate clinical chemistries including liver enzymes, creatinine, and thyroid function at baseline and periodically during treatment. In cases of suspected IMARs, initiate appropriate workup to exclude alternative etiologies, including infection. Institute medical management promptly, including specialty consultation as appropriate.

Withhold or permanently discontinue Opdualag depending on severity (please see section 2 Dosage and Administration in the accompanying Full Prescribing Information). In general, if Opdualag requires interruption or discontinuation, administer systemic corticosteroid therapy (1 to 2 mg/kg/day prednisone or equivalent) until improvement to Grade 1 or less. Upon improvement to Grade 1 or less, initiate corticosteroid taper and continue to taper over at least 1 month. Consider administration of other systemic immunosuppressants in patients whose IMARs are not controlled with corticosteroid therapy. Toxicity management guidelines for adverse reactions that do not necessarily require systemic steroids (e.g., endocrinopathies and dermatologic reactions) are discussed below.

Immune-Mediated Pneumonitis

Opdualag can cause immune-mediated pneumonitis, which may be fatal. In patients treated with other PD-1/PD-L1 blocking antibodies, the incidence of pneumonitis is higher in patients who have received prior thoracic radiation. Immune-mediated pneumonitis occurred in 3.7% (13/355) of patients receiving Opdualag, including Grade 3 (0.6%), and Grade 2 (2.3%) adverse reactions. Pneumonitis led to permanent discontinuation of Opdualag in 0.8% and withholding of Opdualag in 1.4% of patients.

Immune-Mediated Colitis

Opdualag can cause immune-mediated colitis, defined as requiring use of corticosteroids and no clear alternate etiology. A common symptom included in the definition of colitis was diarrhea. Cytomegalovirus infection/reactivation has been reported in patients with corticosteroid-refractory immune-mediated colitis. In cases of corticosteroid-refractory colitis, consider repeating infectious workup to exclude alternative etiologies.

Immune-mediated diarrhea or colitis occurred in 7% (24/355) of patients receiving Opdualag, including Grade 3 (1.1%) and Grade 2 (4.5%) adverse reactions. Colitis led to permanent discontinuation of Opdualag in 2% and withholding of Opdualag in 2.8% of patients.

Immune-Mediated Hepatitis

Opdualag can cause immune-mediated hepatitis, defined as requiring the use of corticosteroids and no clear alternate etiology.

Immune-mediated hepatitis occurred in 6% (20/355) of patients receiving Opdualag, including Grade 4 (0.6%), Grade 3 (3.4%), and Grade 2 (1.4%) adverse reactions. Hepatitis led to permanent discontinuation of Opdualag in 1.7% and withholding of Opdualag in 2.3% of patients.

Immune-Mediated Endocrinopathies

Opdualag can cause primary or secondary adrenal insufficiency, hypophysitis, thyroid disorders, and Type 1 diabetes mellitus, which can be present with diabetic ketoacidosis. Withhold or permanently discontinue Opdualag depending on severity (please see section 2 Dosage and Administration in the accompanying Full Prescribing Information).

For Grade 2 or higher adrenal insufficiency, initiate symptomatic treatment, including hormone replacement as clinically indicated. In patients receiving Opdualag, adrenal insufficiency occurred in 4.2% (15/355) of patients receiving Opdualag, including Grade 3 (1.4%) and Grade 2 (2.5%) adverse reactions. Adrenal insufficiency led to permanent discontinuation of Opdualag in 1.1% and withholding of Opdualag in 0.8% of patients.

Hypophysitis can present with acute symptoms associated with mass effect such as headache, photophobia, or visual field defects. Hypophysitis can cause hypopituitarism; initiate hormone replacement as clinically indicated. Hypophysitis occurred in 2.5% (9/355) of patients receiving Opdualag, including Grade 3 (0.3%) and Grade 2 (1.4%) adverse reactions. Hypophysitis led to permanent discontinuation of Opdualag in 0.3% and withholding of Opdualag in 0.6% of patients.

Thyroiditis can present with or without endocrinopathy. Hypothyroidism can follow hyperthyroidism; initiate hormone replacement or medical management as clinically indicated. Thyroiditis occurred in 2.8% (10/355) of patients receiving Opdualag, including Grade 2 (1.1%) adverse reactions. Thyroiditis did not lead to permanent discontinuation of Opdualag. Thyroiditis led to withholding of Opdualag in 0.3% of patients. Hyperthyroidism occurred in 6% (22/355) of patients receiving Opdualag, including Grade 2 (1.4%) adverse reactions. Hyperthyroidism did not lead to permanent discontinuation of Opdualag. Hyperthyroidism led to withholding of Opdualag in 0.3% of patients. Hypothyroidism occurred in 17% (59/355) of patients receiving Opdualag, including Grade 2 (11%) adverse reactions. Hypothyroidism led to the permanent discontinuation of Opdualag in 0.3% and withholding of Opdualag in 2.5% of patients.

Monitor patients for hyperglycemia or other signs and symptoms of diabetes; initiate treatment with insulin as clinically indicated. Diabetes occurred in 0.3% (1/355) of patients receiving Opdualag, a Grade 3 (0.3%) adverse reaction, and no cases of diabetic ketoacidosis. Diabetes did not lead to the permanent discontinuation or withholding of Opdualag in any patient.

Immune-Mediated Nephritis with Renal Dysfunction

Opdualag can cause immune-mediated nephritis, which is defined as requiring use of steroids and no clear etiology. In patients receiving Opdualag, immune-mediated nephritis and renal dysfunction occurred in 2% (7/355) of patients, including Grade 3 (1.1%) and Grade 2 (0.8%) adverse reactions. Immune-mediated nephritis and renal dysfunction led to permanent discontinuation of Opdualag in 0.8% and withholding of Opdualag in 0.6% of patients.

Withhold or permanently discontinue Opdualag depending on severity (please see section 2 Dosage and Administration in the accompanying Full Prescribing Information).

Immune-Mediated Dermatologic Adverse Reactions

Opdualag can cause immune-mediated rash or dermatitis, defined as requiring use of steroids and no clear alternate etiology. Exfoliative dermatitis, including Stevens-Johnson syndrome, toxic epidermal necrolysis, and Drug Rash with eosinophilia and systemic symptoms has occurred with PD-1/L-1 blocking antibodies. Topical emollients and/or topical corticosteroids may be adequate to treat mild to moderate non-exfoliative rashes.

Withhold or permanently discontinue Opdualag depending on severity (please see section 2 Dosage and Administration in the accompanying Full Prescribing Information).

Immune-mediated rash occurred in 9% (33/355) of patients, including Grade 3 (0.6%) and Grade 2 (3.4%) adverse reactions. Immune-mediated rash did not lead to permanent discontinuation of Opdualag. Immune-mediated rash led to withholding of Opdualag in 1.4% of patients.

Immune-Mediated Myocarditis

Opdualag can cause immune-mediated myocarditis, which is defined as requiring use of steroids and no clear alternate etiology. The diagnosis of immune-mediated myocarditis requires a high index of suspicion. Patients with cardiac or cardio-pulmonary symptoms should be assessed for potential myocarditis. If myocarditis is suspected, withhold dose, promptly initiate high dose steroids (prednisone or methylprednisolone 1 to 2 mg/kg/day) and promptly arrange cardiology consultation with diagnostic workup. If clinically confirmed, permanently discontinue Opdualag for Grade 2-4 myocarditis.

Myocarditis occurred in 1.7% (6/355) of patients receiving Opdualag, including Grade 3 (0.6%), and Grade 2 (1.1%) adverse reactions. Myocarditis led to permanent discontinuation of Opdualag in 1.7% of patients.

Other Immune-Mediated Adverse Reactions

The following clinically significant IMARs occurred at an incidence of <1% (unless otherwise noted) in patients who received Opdualag or were reported with the use of other PD-1/PD-L1 blocking antibodies. Severe or fatal cases have been reported for some of these adverse reactions: Cardiac/Vascular: pericarditis, vasculitis; Nervous System: meningitis, encephalitis, myelitis and demyelination, myasthenic syndrome/myasthenia gravis (including exacerbation), Guillain-Barr syndrome, nerve paresis, autoimmune neuropathy; Ocular: uveitis, iritis, and other ocular inflammatory toxicities can occur. Some cases can be associated with retinal detachment. Various grades of visual impairment, including blindness, can occur. If uveitis occurs in combination with other IMARs, consider a Vogt-Koyanagi-Haradalike syndrome, as this may require treatment with systemic steroids to reduce the risk of permanent vision loss; Gastrointestinal: pancreatitis including increases in serum amylase and lipase levels, gastritis, duodenitis; Musculoskeletal and Connective Tissue: myositis/polymyositis, rhabdomyolysis (and associated sequelae including renal failure), arthritis, polymyalgia rheumatica; Endocrine: hypoparathyroidism; Other (Hematologic/Immune): hemolytic anemia, aplastic anemia, hemophagocytic lymphohistiocytosis, systemic inflammatory response syndrome, histiocytic necrotizing lymphadenitis (Kikuchi lymphadenitis), sarcoidosis, immune thrombocytopenic purpura, solid organ transplant rejection.

Infusion-Related Reactions

Opdualag can cause severe infusion-related reactions. Discontinue Opdualag in patients with severe or life-threatening infusion-related reactions. Interrupt or slow the rate of infusion in patients with mild to moderate infusion-related reactions. In patients who received Opdualag as a 60-minute intravenous infusion, infusion-related reactions occurred in 7% (23/355) of patients.

Complications of Allogeneic Hematopoietic Stem Cell Transplantation (HSCT)

Fatal and other serious complications can occur in patients who receive allogeneic hematopoietic stem cell transplantation (HSCT) before or after being treated with a PD-1/PD-L1 receptor blocking antibody. Transplant-related complications include hyperacute graft-versus-host disease (GVHD), acute GVHD, chronic GVHD, hepatic veno-occlusive disease after reduced intensity conditioning, and steroid-requiring febrile syndrome (without an identified infectious cause). These complications may occur despite intervening therapy between PD-1/PD-L1 blockade and allogeneic HSCT.

Follow patients closely for evidence of transplant-related complications and intervene promptly. Consider the benefit versus risks of treatment with a PD-1/PD-L1 receptor blocking antibody prior to or after an allogeneic HSCT.

Embryo-Fetal Toxicity

Based on its mechanism of action and data from animal studies, Opdualag can cause fetal harm when administered to a pregnant woman. Advise pregnant women of the potential risk to a fetus. Advise females of reproductive potential to use effective contraception during treatment with Opdualag for at least 5 months after the last dose of Opdualag.

Lactation

There are no data on the presence of Opdualag in human milk, the effects on the breastfed child, or the effect on milk production. Because nivolumab and relatlimab may be excreted in human milk and because of the potential for serious adverse reactions in a breastfed child, advise patients not to breastfeed during treatment with Opdualag and for at least 5 months after the last dose.

Serious Adverse Reactions

In Relativity-047, fatal adverse reaction occurred in 3 (0.8%) patients who were treated with Opdualag; these included hemophagocytic lymphohistiocytosis, acute edema of the lung, and pneumonitis. Serious adverse reactions occurred in 36% of patients treated with Opdualag. The most frequent serious adverse reactions reported in 1% of patients treated with Opdualag were adrenal insufficiency (1.4%), anemia (1.4%), colitis (1.4%), pneumonia (1.4%), acute myocardial infarction (1.1%), back pain (1.1%), diarrhea (1.1%), myocarditis (1.1%), and pneumonitis (1.1%).

Common Adverse Reactions and Laboratory Abnormalities

The most common adverse reactions reported in 20% of the patients treated with Opdualag were musculoskeletal pain (45%), fatigue (39%), rash (28%), pruritus (25%), and diarrhea (24%).

The most common laboratory abnormalities that occurred in 20% of patients treated with Opdualag were decreased hemoglobin (37%), decreased lymphocytes (32%), increased AST (30%), increased ALT (26%), and decreased sodium (24%).

Please see U.S. Full Prescribing Information for OPDUALAG.

OPDIVO U.S. INDICATIONS

OPDIVO (nivolumab), as a single agent, is indicated for the treatment of adult patients with unresectable or metastatic melanoma.

OPDIVO (nivolumab), in combination with YERVOY (ipilimumab), is indicated for the treatment of adult patients with unresectable or metastatic melanoma.

OPDIVO (nivolumab) is indicated for the adjuvant treatment of adult patients with melanoma with involvement of lymph nodes or metastatic disease who have undergone complete resection.

OPDIVO (nivolumab), in combination with platinum-doublet chemotherapy, is indicated as neoadjuvant treatment of adult patients with resectable (tumors 4 cm or node positive) non-small cell lung cancer (NSCLC).

OPDIVO (nivolumab), in combination with YERVOY (ipilimumab), is indicated for the first-line treatment of adult patients with metastatic non-small cell lung cancer (NSCLC) whose tumors express PD-L1 (1%) as determined by an FDA-approved test, with no EGFR or ALK genomic tumor aberrations.

OPDIVO (nivolumab), in combination with YERVOY (ipilimumab) and 2 cycles of platinum-doublet chemotherapy, is indicated for the first-line treatment of adult patients with metastatic or recurrent non-small cell lung cancer (NSCLC), with no EGFR or ALK genomic tumor aberrations.

OPDIVO (nivolumab) is indicated for the treatment of adult patients with metastatic non-small cell lung cancer (NSCLC) with progression on or after platinum-based chemotherapy. Patients with EGFR or ALK genomic tumor aberrations should have disease progression on FDA-approved therapy for these aberrations prior to receiving OPDIVO.

OPDIVO (nivolumab), in combination with YERVOY (ipilimumab), is indicated for the first-line treatment of adult patients with unresectable malignant pleural mesothelioma (MPM).

OPDIVO (nivolumab), in combination with YERVOY (ipilimumab), is indicated for the first-line treatment of adult patients with intermediate or poor risk advanced renal cell carcinoma (RCC).

OPDIVO (nivolumab), in combination with cabozantinib, is indicated for the first-line treatment of adult patients with advanced renal cell carcinoma (RCC).

OPDIVO (nivolumab) is indicated for the treatment of adult patients with advanced renal cell carcinoma (RCC) who have received prior anti-angiogenic therapy.

OPDIVO (nivolumab) is indicated for the treatment of adult patients with classical Hodgkin lymphoma (cHL) that has relapsed or progressed after autologous hematopoietic stem cell transplantation (HSCT) and brentuximab vedotin or after 3 or more lines of systemic therapy that includes autologous HSCT. This indication is approved under accelerated approval based on overall response rate. Continued approval for this indication may be contingent upon verification and description of clinical benefit in confirmatory trials.

OPDIVO (nivolumab) is indicated for the treatment of adult patients with recurrent or metastatic squamous cell carcinoma of the head and neck (SCCHN) with disease progression on or after platinum-based therapy.

OPDIVO (nivolumab) is indicated for the treatment of adult patients with locally advanced or metastatic urothelial carcinoma who have disease progression during or following platinum-containing chemotherapy or have disease progression within 12 months of neoadjuvant or adjuvant treatment with platinum-containing chemotherapy.

OPDIVO (nivolumab), as a single agent, is indicated for the adjuvant treatment of adult patients with urothelial carcinoma (UC) who are at high risk of recurrence after undergoing radical resection of UC.

OPDIVO (nivolumab), as a single agent, is indicated for the treatment of adult and pediatric (12 years and older) patients with microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR) metastatic colorectal cancer (CRC) that has progressed following treatment with a fluoropyrimidine, oxaliplatin, and irinotecan. This indication is approved under accelerated approval based on overall response rate and duration of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in confirmatory trials.

OPDIVO (nivolumab), in combination with YERVOY (ipilimumab), is indicated for the treatment of adults and pediatric patients 12 years and older with microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR) metastatic colorectal cancer (CRC) that has progressed following treatment with a fluoropyrimidine, oxaliplatin, and irinotecan. This indication is approved under accelerated approval based on overall response rate and duration of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in confirmatory trials.

OPDIVO (nivolumab), in combination with YERVOY (ipilimumab), is indicated for the treatment of adult patients with hepatocellular carcinoma (HCC) who have been previously treated with sorafenib. This indication is approved under accelerated approval based on overall response rate and duration of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in the confirmatory trials.

OPDIVO (nivolumab) is indicated for the treatment of adult patients with unresectable advanced, recurrent or metastatic esophageal squamous cell carcinoma (ESCC) after prior fluoropyrimidine- and platinum-based chemotherapy.

OPDIVO (nivolumab) is indicated for the adjuvant treatment of completely resected esophageal or gastroesophageal junction cancer with residual pathologic disease in adult patients who have received neoadjuvant chemoradiotherapy (CRT).

OPDIVO (nivolumab), in combination with fluoropyrimidine- and platinum-containing chemotherapy, is indicated for the first-line treatment of adult patients with unresectable advanced or metastatic esophageal squamous cell carcinoma (ESCC).

OPDIVO (nivolumab), in combination with YERVOY (ipilimumab), is indicated for the first-line treatment of adult patients with unresectable advanced or metastatic esophageal squamous cell carcinoma (ESCC).

OPDIVO (nivolumab), in combination with fluoropyrimidine- and platinum- containing chemotherapy, is indicated for the treatment of adult patients with advanced or metastatic gastric cancer, gastroesophageal junction cancer, and esophageal adenocarcinoma.

IMPORTANT SAFETY INFORMATION

Severe and Fatal Immune-Mediated Adverse Reactions

Immune-mediated adverse reactions listed herein may not include all possible severe and fatal immune-mediated adverse reactions.

Immune-mediated adverse reactions, which may be severe or fatal, can occur in any organ system or tissue. While immune-mediated adverse reactions usually manifest during treatment, they can also occur after discontinuation of OPDIVO or YERVOY. Early identification and management are essential to ensure safe use of OPDIVO and YERVOY. Monitor for signs and symptoms that may be clinical manifestations of underlying immune-mediated adverse reactions. Evaluate clinical chemistries including liver enzymes, creatinine, adrenocorticotropic hormone (ACTH) level, and thyroid function at baseline and periodically during treatment with OPDIVO and before each dose of YERVOY. In cases of suspected immune-mediated adverse reactions, initiate appropriate workup to exclude alternative etiologies, including infection. Institute medical management promptly, including specialty consultation as appropriate.

Withhold or permanently discontinue OPDIVO and YERVOY depending on severity (please see section 2 Dosage and Administration in the accompanying Full Prescribing Information). In general, if OPDIVO or YERVOY interruption or discontinuation is required, administer systemic corticosteroid therapy (1 to 2 mg/kg/day prednisone or equivalent) until improvement to Grade 1 or less. Upon improvement to Grade 1 or less, initiate corticosteroid taper and continue to taper over at least 1 month. Consider administration of other systemic immunosuppressants in patients whose immune-mediated adverse reactions are not controlled with corticosteroid therapy. Toxicity management guidelines for adverse reactions that do not necessarily require systemic steroids (e.g., endocrinopathies and dermatologic reactions) are discussed below.

Immune-Mediated Pneumonitis

OPDIVO and YERVOY can cause immune-mediated pneumonitis. The incidence of pneumonitis is higher in patients who have received prior thoracic radiation. In patients receiving OPDIVO monotherapy, immune-mediated pneumonitis occurred in 3.1% (61/1994) of patients, including Grade 4 (<0.1%), Grade 3 (0.9%), and Grade 2 (2.1%). In patients receiving OPDIVO 1 mg/kg with YERVOY 3 mg/kg every 3 weeks, immune-mediated pneumonitis occurred in 7% (31/456) of patients, including Grade 4 (0.2%), Grade 3 (2.0%), and Grade 2 (4.4%). In patients receiving OPDIVO 3 mg/kg with YERVOY 1 mg/kg every 3 weeks, immune-mediated pneumonitis occurred in 3.9% (26/666) of patients, including Grade 3 (1.4%) and Grade 2 (2.6%). In NSCLC patients receiving OPDIVO 3 mg/kg every 2 weeks with YERVOY 1 mg/kg every 6 weeks, immune-mediated pneumonitis occurred in 9% (50/576) of patients, including Grade 4 (0.5%), Grade 3 (3.5%), and Grade 2 (4.0%). Four patients (0.7%) died due to pneumonitis.

In Checkmate 205 and 039, pneumonitis, including interstitial lung disease, occurred in 6.0% (16/266) of patients receiving OPDIVO. Immune-mediated pneumonitis occurred in 4.9% (13/266) of patients receiving OPDIVO, including Grade 3 (n=1) and Grade 2 (n=12).

Immune-Mediated Colitis

OPDIVO and YERVOY can cause immune-mediated colitis, which may be fatal. A common symptom included in the definition of colitis was diarrhea. Cytomegalovirus (CMV) infection/reactivation has been reported in patients with corticosteroid-refractory immune-mediated colitis. In cases of corticosteroid-refractory colitis, consider repeating infectious workup to exclude alternative etiologies. In patients receiving OPDIVO monotherapy, immune-mediated colitis occurred in 2.9% (58/1994) of patients, including Grade 3 (1.7%) and Grade 2 (1%). Inpatients receiving OPDIVO 1 mg/kg with YERVOY 3 mg/kg every 3 weeks, immune-mediated colitis occurred in 25% (115/456) of patients, including Grade 4 (0.4%), Grade 3 (14%) and Grade 2 (8%). In patients receiving OPDIVO 3 mg/kg with YERVOY 1 mg/kg every 3 weeks, immune-mediated colitis occurred in 9% (60/666) of patients, including Grade 3 (4.4%) and Grade 2 (3.7%).

Immune-Mediated Hepatitis and Hepatotoxicity

OPDIVO and YERVOY can cause immune-mediated hepatitis. In patients receiving OPDIVO monotherapy, immune-mediated hepatitis occurred in 1.8% (35/1994) of patients, including Grade 4 (0.2%), Grade 3(1.3%), and Grade 2 (0.4%). In patients receiving OPDIVO 1 mg/kg with YERVOY 3 mg/kg every 3 weeks, immune-mediated hepatitis occurred in 15% (70/456) of patients, including Grade 4 (2.4%), Grade 3 (11%), and Grade 2(1.8%). In patients receiving OPDIVO 3 mg/kg with YERVOY 1 mg/kg every 3 weeks, immune-mediated hepatitis occurred in 7% (48/666) of patients, including Grade 4 (1.2%), Grade 3 (4.9%), and Grade 2 (0.4%).

OPDIVO in combination with cabozantinib can cause hepatic toxicity with higher frequencies of Grade 3 and 4ALT and AST elevations compared to OPDIVO alone. Consider more frequent monitoring of liver enzymes as compared to when the drugs are administered as single agents. In patients receiving OPDIVO and cabozantinib, Grades 3 and 4 increased ALT or AST were seen in 11% of patients.

Immune-Mediated Endocrinopathies

Go here to see the original:
Bristol Myers Squibb Receives Positive CHMP Opinion Recommending Approval for LAG-3-Blocking Antibody Combination Opdualag (nivolumab and relatlimab)...

Canine Stem Cell Therapy Market to Witness Growth Acceleration | Aratana Therapeutics, Okyanos, Magellan Stem Cells Travel Adventure Cinema – Travel…

New Jersey (United States) A2Z Market Research published new research on Global Canine Stem Cell Therapy covering the micro-level of analysis by competitors and key business segments (2022-2029). The Global Canine Stem Cell Therapy explores a comprehensive study on various segments like opportunities, size, development, innovation, sales, and overall growth of major players. The research is carried out on primary and secondary statistics sources and it consists of both qualitative and quantitative detailing.

Some of the Major Key players profiled in the study are Aratana Therapeutics, Okyanos, Magellan Stem Cells, Stem Cell Vet, VetStem Biopharma, Medrego, Regeneus Ltd, MediVet Biologic, Cell Therapy Sciences

Get PDF Sample Report + All Related Table and Graphs @:

The global Canine Stem Cell Therapy Market research report delivers a comprehensive analysis of market size, market trends, and market growth prospects. This report also delivers extensive information on the technology expenditure for the forecast period, which gives a unique view of the global Canine Stem Cell Therapy Market across numerous segments. The global Canine Stem Cell Therapy market report also allows consumers recognize market prospects and challenges.

Various factors are responsible for the markets growth trajectory, which are studied at length in the report. In addition, the report lists down the restraints that are posing threat to the global

Global Canine Stem Cell Therapy Market Segmentation:

Market Segmentation: By Type

Allogeneic Stem Cells, Autologous Stem Cells

Market Segmentation: By Application

Veterinary Hospitals, Veterinary Clinics, Veterinary Research Institutes

Key market aspects are illuminated in the report:

Executive Summary: It covers a summary of the most vital studies, the Global Canine Stem Cell Therapy market increasing rate, modest circumstances, market trends, drivers and problems as well as macroscopic pointers.

Study Analysis: Covers major companies, vital market segments, the scope of the products offered in the Global Canine Stem Cell Therapy market, the years measured, and the study points.

Company Profile: Each Firm well-defined in this segment is screened based on a products, value, SWOT analysis, their ability and other significant features.

Manufacture by region: This Global Canine Stem Cell Therapy report offers data on imports and exports, sales, production and key companies in all studied regional markets

Get Special pricing with up to 30% Discount on the first purchase of this report @:

The cost analysis of the Global Canine Stem Cell Therapy Market has been performed while keeping in view manufacturing expenses, labor cost, and raw materials and their market concentration rate, suppliers, and price trend. Other factors such as Supply chain, downstream buyers, and sourcing strategy have been assessed to provide a complete and in-depth view of the market. Buyers of the report will also be exposed to a study on market positioning with factors such as target client, brand strategy, and price strategy taken into consideration.

Highlighting points of Global Canine Stem Cell Therapy Market Report:

Table of Contents

Global Canine Stem Cell Therapy Market Research Report 2022 2029

Chapter 1 Canine Stem Cell Therapy Market Overview

Chapter 2 Global Economic Impact on Industry

Chapter 3 Global Market Competition by Manufacturers

Chapter 4 Global Production, Revenue (Value) by Region

Chapter 5 Global Supply (Production), Consumption, Export, Import by Regions

Chapter 6 Global Production, Revenue (Value), Price Trend by Type

Chapter 7 Global Market Analysis by Application

Chapter 8 Manufacturing Cost Analysis

Chapter 9 Industrial Chain, Sourcing Strategy and Downstream Buyers

Chapter 10 Marketing Strategy Analysis, Distributors/Traders

Chapter 11 Market Effect Factors Analysis

Chapter 12 Global Canine Stem Cell Therapy Market Forecast

Buy the Full Research Report of Global Canine Stem Cell Therapy Market @: :

Contact Us:

Roger Smith

1887 WHITNEY MESA DR HENDERSON, NV 89014

+1 775 237 4147

See the rest here:
Canine Stem Cell Therapy Market to Witness Growth Acceleration | Aratana Therapeutics, Okyanos, Magellan Stem Cells Travel Adventure Cinema - Travel...

Gov. Whitmer announces more budget vetoes, mostly tied to curbs on abortion rights – Detroit Free Press

Show Caption

Hide Caption

Attorney General Dana Nessel discusses Supreme Court ruling on abortion

Nessel has pledged not to enforce Michigan's highly restrictive 1931 abortion law, should it take effect after Friday's U.S. Supreme Court ruling.

Provided by the Michigan Department of the Attorney General

LANSING Gov. Gretchen Whitmer'soffice on Monday detailed more than $20 million in line-item vetoes she plans to make to the state's main 2023 budget billwhen she signs it into law Wednesday.

Most of the vetoed budget itemsin House Bill 5783 relate to the Department of Health and Human Services and involve restrictions on the use of state funds to pay for abortionsor funding for pregnancy and parenting centers that promote alternatives to abortion.Whitmer also vetoed a $2 million tax credit for adoptive parents, a $10 million marketing program to promote adoption as an alternative to abortion, and a $100,000 legal fund in the Michigan Department of Corrections to pay for efforts to prevent the use of state funds for gender-confirmationsurgery or therapies in state prisons.

The announcement drew swift criticism from Republicans. The governor claims to be a voice for choice, but her actions clearly support only one option for women in a crisis pregnancy the deadly choice of abortion," said State Rep. Thomas Albert, R-Lowell, chairman of the House Appropriations Committee.

Monday's announcement follows Whitmer's vetoes Thursday of $6 million from the education budget. The money funded college and university pregnancy and parenting centers that would be prohibited from making abortion referrals and money for "ethical stem cell/tissue research" that would place restrictions on which stem cells and human embryos could be used.

On Wednesday, Whitmer "is expected to veto funding for centers that often purport to offer comprehensive reproductive health care, including abortion, but dont, preying on women at a vulnerable time in their lives," Whitmer spokesman Bobby Leddy said.

The centers, which Leddy said are sometimes known as fake womens health centers or pregnancy resource centers, frequentlyuse deceptive advertising that target young women and women with low incomes who are seeking abortion care, painting themselves as comprehensive, licensed health care clinics that provide all options, and then lie to women about medical facts," Leddy said.

Whitmer "supports legislation that provides every possible resource to women who are pregnant, seeking to start a family, or those who arent ready yet, but she cannot support aspects of a bill that sends millions in taxpayer dollars to fake health centers that intentionally withhold information from women about their health, bodies, and full reproductive freedom," Leddy said.

The budget, approved overwhelmingly by lawmakers from both parties, was the product of negotiations and a consensus the governor reached with Republican legislative leaders. Still, none of the leaders should be surprised by any of the vetoes, Leddy said.

Gideon D'Assandro, a spokesman for House Speaker Jason Wentworth, R-Farwell, confirmed the vetoes are not a surprise.

More: Whitmer vetoes budget items restricting abortion access, stem cell research

More: Whitmer signs $22.2B budget for Michigan K-12 schools, colleges and universities

Albertsaid it is shocking that"helping pregnant women who might consider adoption instead is now a bridge too far," and the vetoes remove help for "expecting mothers and their babies, including those facing a crisis pregnancy, by denying them access to essential care both before and after giving birth."

Whitmer is usingher line-item veto powers for budget bills as tensions are high between herand the Republican-controlled Legislature after the U.S. Supreme Court struck down Roe v. Wade, which recognized abortion rights.

The toppling of Roe would put into effect a 1931 state law that prohibits abortions except to save the life of the mother, but a Court of Claims judge has temporarily preserved the status quo by issuing an injunction against the 1931 law. Whitmer has asked the Michigan Supreme Court to declare that the law violates the state constitution; Republicans are fighting to preserve the law.

Contact Paul Egan: 517-372-8660 or pegan@freepress.com.Follow him on Twitter @paulegan4. Read more on Michigan politics and sign up for our elections newsletter.

Become asubscriber.

Read more:
Gov. Whitmer announces more budget vetoes, mostly tied to curbs on abortion rights - Detroit Free Press

Embryonic Research Could Be the Next Target After Roe – WIRED

Two weeks after the US Supreme Court overturned the federal right to an abortion, Ye Yuan heard from a woman who wanted to reverse her decision to donate her embryos to scientific research. The womanwho contacted Yuan anonymously through a fertility counselorwas fearful that if the law in Colorado changed to make it illegal to discard or experiment on human embryos, then she would be forced to have hers frozen indefinitely. In a year, or five years, might a law change to stop her from having the final say over what happened to them?

This content can also be viewed on the site it originates from.

In states where human embryonic research is legal, people undergoing IVF are often given the choice to donate any excess fertilized embryos to scientific research. These are sometimes used to search for potential treatments for diseases such as diabetes or, as in Yuans case, to research ways to make IVF more successful. Those discarded embryos are really one of the key pieces for us to maintain the high quality of our platform here, says Yuan, who is research director at the Colorado Center for Reproductive Medicine (CCRM). But in the wake of the Dobbs verdict, he is worried that people will be less likely to donate their spare embryos for research and, down the line, that embryonic research could become the next target of antiabortion campaigners.

Its like youre a little girl living in a dark room. You know there are bad guys outside but youre not too worried because the door has been locked, says Yuan. But then somebody tells you that the door has been unlocked. Yuan fears that anything that slows down access to human embryos will ultimately end up slowing progress in IVF, which is responsible for between 1 and 2 percent of all US births annually.

The majority opinion written by Justice Samuel Alito doesnt single out IVF or human embryonic research, but his choice of words to describe abortion could be seen as also being applicable to embryos outside the body, says Glenn Cohen, a bioethicist and professor of law at Harvard Law School. The right to an abortion is distinct from other rights, Alito notes in the opinion, because it destroys potential life and the life of an unborn human being.

The same thing that he uses to distinguish abortion seems to me completely applicable to distinguishing embryos, says Cohen. To me it makes it very, very clear after Dobbs that any state that wants to prohibit the destruction of embryos as part of research is free to do so.

The wording that legislators use to describe the beginning of human life is also important. In at least nine states, trigger lawspieces of legislation designed to restrict abortion quickly after the fall of Roeinclude language that implies an egg cell becomes an unborn child or unborn human being at the precise moment of fertilization. In other words, according to these definitions, every single human embryoincluding donated embryos that might be used in scientific researchis an unborn child. Although most of these trigger laws apply specifically to pregnancy, and so do not regulate embryos outside of the human body, the idea that life begins at the very moment of fertilization could be used to target embryonic research, says Cohen. If you have that view, its not clear to me why you would exempt the destruction of embryos if you prohibit abortion. To me, that wrong is the same.

Read the original:
Embryonic Research Could Be the Next Target After Roe - WIRED

Cell Culture Media Market: Competitive Approach, Breakdown And Forecast by 2027 – Digital Journal

Market Overview

Thecell culture media marketis expected to cross USD 4.33 billion by 2027 at a CAGR of8.33%.

Market Dynamics

The markets growth is being fueled by a diverse range of cell culture media applications, increased research and development in the pharmaceutical industry, an increase in the prevalence of chronic diseases, and increased expansion and product launches by major players. Over the last few decades, advancements in cell culture technology have accelerated. It is widely regarded as one of the most dependable, robust, and mature technologies for biotherapeutic product development.

The high cost of cell culture media and the risk of contamination, on the other hand, are impeding the markets growth. However, the growing emphasis on regenerative and personalized medicine is likely to spur growth in the global cell culture media market.

Get Sample Report: https://www.marketresearchfuture.com/sample_request/4462

Competitive Dynamics

The notable players are the Merck KGaA (Germany), Bio-Rad Laboratories, Inc. (US), Thermo Fisher Scientific Inc. (US), Lonza (Switzerland), GE Healthcare (US), Becton, Dickinson and Company (US), HiMedia Laboratories (India), Corning Incorporated (US), PromoCell (Germany), Sera Scandia A/S (Denmark), The Sartorius Group (Germany), and Fujifilm Holdings Corporation (Japan).

Segmental Analysis

The global market for cell culture media has been segmented according to product type, application, and end user.

The market has been segmented by product type into classical media, stem cell media, serum-free media, and others.

Further subcategories of stem cell culture media include bone marrow, embryonic stem cells, mesenchymal stem cells, and neural stem cells.

The market is segmented into four application segments: drug discovery and development, cancer research, genetic engineering, and tissue engineering and biochemistry.

The market is segmented by end user into biochemistry and pharmaceutical companies, research laboratories, academic institutions, and pathology laboratories.

Regional Overview

According to region, the global cell culture media market is segmented into the Americas, Europe, Asia-Pacific, and the Middle East & Africa.

The Americas dominated the global cell culture media market. The large share is attributed to the presence of major manufacturers, rising disease prevalence resulting in increased demand for drugs and other medications, technological advancements in the preclinical and clinical segments, growing public awareness, and high disposable income.

Europe ranks second in terms of market size for cell culture media. Factors such as an increase in the biopharmaceutical sector in the European region, increased government initiatives to promote research to find a cure for the growing number of chronic diseases, an increase in the number of pharmaceutical manufacturers, improving economies, a high disposable income per individual, and increased healthcare spending are all contributing to the markets growth in this region. The European market is expected to be driven by expanding R&D activities and a developing biopharmaceutical sector.

Asia-Pacific held the third-largest market share, owing to the presence of numerous research organizations, low manufacturing costs, low labor costs, developing healthcare infrastructure, and increased investment by American and European market giants in Asian countries such as China and India.

The Middle East and Africa, with limited economic development and extremely low income, held the smallest market share in 2019 but is expected to grow due to growing public awareness and demand for improved healthcare facilities in countries, as well as rising disposable income.

Browse Full Reports: https://www.marketresearchfuture.com/reports/cell-culture-media-market-4462

Related Report: Dysmenorrhea Treatment Market Research Report- Global Forecast to 2027

Syringe and Needle Market Research Report Global Forecast to 2027

Global Medical Robotics Market Research Report- Forecast To 2027

About US:

Market Research Future (MRFR) enable customers to unravel the complexity of various industries through Cooked Research Report (CRR), Half-Cooked Research Reports (HCRR), Raw Research Reports (3R), Continuous-Feed Research (CFR), and Market Research & Consulting Services.

Contact us:

Market Research Future (part of Wantstats Research and Media Private Limited),

99 Hudson Street,5Th Floor, New York,

New York 10013

See the article here:
Cell Culture Media Market: Competitive Approach, Breakdown And Forecast by 2027 - Digital Journal

Stem Cell Therapy Market Size, Scope, Growth Opportunities, Trends by Manufacturers And Forecast to 2029 This Is Ardee – This Is Ardee

New Jersey, United States TheStem Cell TherapyMarket research guides new entrants to obtain precise market data and communicates with customers to know their requirements and preferences. It spots outright business opportunities and helps to bring new products into the market. It identifies opportunities in the marketplace. It aims at doing modifications in the business to make business procedures smooth and make business forward. It helps business players to make sound decision making. Stem Cell Therapy market report helps to reduce business risks and provides ways to deal with upcoming challenges. Market information provided here helps new entrants to take informed decisions making. It emphasizes on major regions of the globe such as Europe, North America, Asia Pacific, Middle East, Africa, and Latin America along with their market size.

Such unique Stem Cell Therapy Market research report offers some extensive strategic plans that help the players to deal with the current market situation and make your position. It helps in strengthening your business position. It offers better understanding of the market and keep perspective to aid one remain ahead in this competitive market. Organizations can gauze and compare their presentation with others in the market on the basis of this prompt market report. This market report offers a clarified picture of the varying market tactics and thereby helps the business organizations gain bigger profits. You get a clear idea about the product launches, trade regulations and expansion of the market place through this market report.

Get Full PDF Sample Copy of Report: (Including Full TOC, List of Tables & Figures, Chart) @https://www.verifiedmarketresearch.com/download-sample/?rid=24113

Key Players Mentioned in the Stem Cell Therapy Market Research Report:

Osiris Therapeutics Medipost Co. Ltd., Anterogen Co. Ltd., Pharmicell Co. Ltd., HolostemTerapieAvanzateSrl, JCR Pharmaceuticals Co. Ltd., Nuvasive RTI Surgical Allosource

Stem Cell TherapyMarket report consists of important data about the entire market environment of products or services offered by different industry players. It enables industries to know the market scenario of a particular product or service including demand, supply, market structure, pricing structure, and trend analysis. It is of great assistance in the product market development. It further depicts essential data regarding customers, products, competition, and market growth factors. Stem Cell Therapy market research benefits greatly to make the proper decision. Future trends are also revealed for particular products or services to help business players in making the right investment and launching products into the market.

Stem Cell TherapyMarket Segmentation:

Stem Cell Therapy Market, By Cell Source

Adipose Tissue-Derived Mesenchymal Stem Cells Bone Marrow-Derived Mesenchymal Stem Cells Cord Blood/Embryonic Stem Cells Other Cell Sources

Stem Cell Therapy Market, By Therapeutic Application

Musculoskeletal Disorders Wounds and Injuries Cardiovascular Diseases Surgeries Gastrointestinal Diseases Other Applications

Stem Cell Therapy Market, By Type

Allogeneic Stem Cell Therapy Autologous Stem Cell Therapy

Inquire for a Discount on this Premium Report@ https://www.verifiedmarketresearch.com/ask-for-discount/?rid=24113

For Prepare TOC Our Analyst deep Researched the Following Things:

Report Overview:It includes major players of the Stem Cell Therapy market covered in the research study, research scope, market segments by type, market segments by application, years considered for the research study, and objectives of the report.

Global Growth Trends:This section focuses on industry trends where market drivers and top market trends are shed light upon. It also provides growth rates of key producers operating in the Stem Cell Therapy market. Furthermore, it offers production and capacity analysis where marketing pricing trends, capacity, production, and production value of the Stem Cell Therapy market are discussed.

Market Share by Manufacturers:Here, the report provides details about revenue by manufacturers, production and capacity by manufacturers, price by manufacturers, expansion plans, mergers and acquisitions, and products, market entry dates, distribution, and market areas of key manufacturers.

Market Size by Type:This section concentrates on product type segments where production value market share, price, and production market share by product type are discussed.

Market Size by Application:Besides an overview of the Stem Cell Therapy market by application, it gives a study on the consumption in the Stem Cell Therapy market by application.

Production by Region:Here, the production value growth rate, production growth rate, import and export, and key players of each regional market are provided.

Consumption by Region:This section provides information on the consumption in each regional market studied in the report. The consumption is discussed on the basis of country, application, and product type.

Company Profiles:Almost all leading players of the Stem Cell Therapy market are profiled in this section. The analysts have provided information about their recent developments in the Stem Cell Therapy market, products, revenue, production, business, and company.

Market Forecast by Production:The production and production value forecasts included in this section are for the Stem Cell Therapy market as well as for key regional markets.

Market Forecast by Consumption:The consumption and consumption value forecasts included in this section are for the Stem Cell Therapy market as well as for key regional markets.

Value Chain and Sales Analysis:It deeply analyzes customers, distributors, sales channels, and value chain of the Stem Cell Therapy market.

Key Findings:This section gives a quick look at the important findings of the research study.

For More Information or Query or Customization Before Buying, Visit @ https://www.verifiedmarketresearch.com/product/stem-cell-therapy-market/

About Us: Verified Market Research

Verified Market Research is a leading Global Research and Consulting firm that has been providing advanced analytical research solutions, custom consulting and in-depth data analysis for 10+ years to individuals and companies alike that are looking for accurate, reliable and up to date research data and technical consulting. We offer insights into strategic and growth analyses, Data necessary to achieve corporate goals and help make critical revenue decisions.

Our research studies help our clients make superior data-driven decisions, understand market forecast, capitalize on future opportunities and optimize efficiency by working as their partner to deliver accurate and valuable information. The industries we cover span over a large spectrum including Technology, Chemicals, Manufacturing, Energy, Food and Beverages, Automotive, Robotics, Packaging, Construction, Mining & Gas. Etc.

We, at Verified Market Research, assist in understanding holistic market indicating factors and most current and future market trends. Our analysts, with their high expertise in data gathering and governance, utilize industry techniques to collate and examine data at all stages. They are trained to combine modern data collection techniques, superior research methodology, subject expertise and years of collective experience to produce informative and accurate research.

Having serviced over 5000+ clients, we have provided reliable market research services to more than 100 Global Fortune 500 companies such as Amazon, Dell, IBM, Shell, Exxon Mobil, General Electric, Siemens, Microsoft, Sony and Hitachi. We have co-consulted with some of the worlds leading consulting firms like McKinsey & Company, Boston Consulting Group, Bain and Company for custom research and consulting projects for businesses worldwide.

Contact us:

Mr. Edwyne Fernandes

Verified Market Research

US: +1 (650)-781-4080 UK: +44 (753)-715-0008 APAC: +61 (488)-85-9400 US Toll-Free: +1 (800)-782-1768

Email: sales@verifiedmarketresearch.com

Website:- https://www.verifiedmarketresearch.com/

Read the original:
Stem Cell Therapy Market Size, Scope, Growth Opportunities, Trends by Manufacturers And Forecast to 2029 This Is Ardee - This Is Ardee

Human iPSC co-culture model to investigate the interaction between microglia and motor neurons | Scientific Reports – Nature.com

Ethics statement

All human material (blood RNA, primary microglia RNA, iPSCs) used in this study was derived after signed informed consent: for blood, according to University of Oxford OHS policy document 1/03; all procedures related to the use of the primary microglia followed established institutional (McGill University, Montreal, QC, Canada) and Canadian Institutes of Health Research guidelines for the use of human cells; for iPSC, with approval from the South Central Berkshire Research Ethics Committee, U.K. (REC 10/H0505/71). The blood RNA and primary microglia RNA samples have been published previously26, as have the iPSC lines (see below).

Four healthy control iPSC lines, SFC840-03-03 (female, 67years old,35), SFC841-03-01 (male, 36,18), SFC856-03-04 (female, 78,36), OX3-06 (male, 49,37), generated from skin biopsy fibroblasts and characterized as described before, were used in this study. Additionally, the previously reported26 line AH016-3 Lenti_IP_RFP (male, 80years old), which constitutively expresses Red Fluorescent Protein (RFP) under continuous puromycin selection, was used for some live-imaging experiments.

iPSCs were cultured in mTeSR1 (StemCell Technologies) or OXE8 medium38 on Geltrex (Thermo Fisher)-coated tissue culture plates with daily medium changes. Passaging was done as clumps using EDTA in PBS (0.5mM). Cells were initially expanded at low passage to create a master stock, which was used for all experiments to ensure consistency. Cells were regularly tested negative for mycoplasma using MycoAlert Mycoplasma Detection Kit (Lonza).

iPSCs were differentiated to MNs according to our previously published protocol18,19,27. Briefly, neural induction of iPSC monolayers was performed using DMEM-F12/Neurobasal 50:50 medium supplemented with N2 (1X), B27 (1X), 2-Mercaptoethanol (1X), AntibioticAntimycotic (1X, all ThermoFisher), Ascorbic Acid (0.5M), Compound C (1M, both Merck), and Chir99021 (3M, R&D Systems). After two days in culture, Retinoic Acid (RA, 1M, Merck) and Smoothened Agonist (SAG, 500nM, R&D Systems) were additionally added to the medium. Two days later, Compound C and Chir99021 were removed from the medium. After another 5days in culture, neural precursors were dissociated using accutase (ThermoFisher), and split 1:3 onto Geltrex-coated tissue culture plates in medium supplemented with Y-27632 dihydrochloride (10M, R&D Systems). After one day, Y-27632 dihydrochloride was removed from the medium, and then the cells were cultured for another 8days with medium changes every other day. For terminal maturation, the cells were dissociated on day in vitro (DIV) 18 using accutase and plated onto coverslips or tissue culture plates coated with polyethylenimine (PEI, 0.07%, Merck) and Geltrex or tissue culture dishes coated with PDL (Sigma-Aldrich)/ Laminin (R&D Systems)/ Fibronectin (Corning). For this step, the medium was additionally supplemented with BDNF (10ng/mL), GDNF (10ng/mL), Laminin (0.5g/mL, all ThermoFisher), Y-27632 dihydrochloride (10M), and DAPT (10M, R&D Systems). Three days later, Y-27632 dihydrochloride was removed from the medium. After another three days, DAPT was removed from the medium. Full medium changes were then performed every three days.

For MNs differentiated in co-culture medium alone, all steps were performed similarly until three days after the terminal re-plating (D21). MNs were then cultured in co-culture medium as described below.

iPSCs were differentiated to macrophage/microglia precursors as described previously20,21. Briefly, embryoid body (EB) formation was induced by seeding iPSCs into Aggrewell 800 wells (STEMCELL Technologies) in OXE838 or mTeSR1 medium supplemented with Bone Morphogenetic Protein 4 (BMP4, 50ng/mL), Vascular Endothelial Growth Factor (VEGF, 50ng/mL, both Peprotech), and Stem Cell Factor (SCF, 20ng/mL, Miltenyi Biotec). After four days with daily medium changes, EBs were transferred to T175 flasks (~150 EBs each) and differentiated in X-VIVO15 (Lonza), supplemented with Interleukin-3 (IL-3, 25ng/mL, R&D Systems), Macrophage Colony-Stimulating Factor (M-CSF, 100ng/mL), GlutaMAX (1X, both ThermoFisher), and 2-Mercaptoethanol (1X). Fresh medium was added weekly. After approximately one month, precursors emerged into the supernatant and could be harvested weekly. Harvested cells were passed through a cell strainer (40M, Falcon) and either lysed directly for RNA extraction or differentiated to microglia in monoculture or co-culture as described below.

Three days after the final re-plating of differentiating MNs (DIV21), macrophage/microglia precursors were harvested as described above and resuspended in co-culture medium comprised of Advanced DMEM-F12 (ThermoFisher) supplemented with GlutaMAX (1X), N2 (1X), AntibioticAntimycotic (1X), 2-Mercaptoethanol (1X), Interleukin-34 (IL-34, 100ng/mL, Peprotech), BDNF (10ng/mL), GDNF (10ng/mL), and Laminin (0.5g/mL). MNs were quickly rinsed with PBS, and macrophage/microglia precursors re-suspended in co-culture medium were added to each well. Co-cultures were then maintained for at least 14days before assays were conducted as described below. Half medium changes were performed every 23days.

For comparisons between co-cultures and monocultures, MNs and monocultured microglia were also differentiated alone in co-culture medium.

Cells cultured on coverslips were pre-fixed with 2% paraformaldehyde in PBS for 2min and then fixed with 4% paraformaldehyde in PBS for 15min at room temperature (RT). After permeabilization and blocking with 5% donkey/goat serum and 0.2% Triton X-100 in PBS for 1h at RT, the coverslips were incubated with primary antibodies diluted in 1% donkey/goat serum and 0.1% Triton X-100 in PBS at 4C ON. The following primary antibodies were used: rabbit anti-cleaved caspase 3 (1:400, 9661S, Cell Signaling), mouse anti-ISLET1 (1:50, 40.2D6, Developmental Studies Hybridoma Bank), mouse anti-TUJ1 (1:500, 801201, BioLegend), rabbit anti-TUJ1 (1:500, 802001, BioLegend), chicken anti-TUJ1 (1:500, GTX85469, GeneTex), rabbit anti-IBA1 (1:500, 019-19741, FUJIFILM Wako Pure Chemical Corporation), goat anti-IBA1 (1:500, ab5076, abcam), rabbit anti-synaptophysin (1:200, ab14692, abcam), goat anti-ChAT (1:100, ab114P, abcam), rat anti-TREM2 (1:100, MAB17291-100, R&D Systems), rabbit anti-TMEM119 (1:100, ab185337, abcam), rat anti-CD11b (1:100, 101202, BioLegend).

After three washes with PBS-0.1% Triton X-100 for 5min each, coverslips were incubated with corresponding fluorescent secondary antibodies Alexa Fluor 488/568/647 donkey anti-mouse/rabbit/rat/goat, goat anti-chicken (all 1:1000, all ThermoFisher). Coverslips were then washed twice with PBS-0.1% Triton X-100 for 5min each and incubated with 4,6-diamidino-2-phenylindole (DAPI, 1g/mL, Sigma-Aldrich) in PBS for 10min. After an additional 5min-washing step with PBS-0.1% Triton X-100, the coverslips were mounted onto microscopy slides using ProLong Diamond Antifade Mountant (ThermoFisher). Confocal microscopy was then performed using an LSM 710 microscope (Zeiss).

For the analysis of neuronal and MN markers after differentiation, three z-stacks (2m intervals) of randomly selected visual fields (425.1425.1m) were taken for each coverslip at 20magnification. The ratios of TUJ1-positive, ChAT-positive, ISLET1-positive, ChAT-positive/ TUJ1-positive, and ISLET1-positive/ TUJ1-positive cells were then quantified using Fiji in a blinded fashion.

For the analysis of microglial markers in monoculture and co-culture, three z-stacks (1m intervals) of randomly selected visual fields (212.55212.55m) were taken for each coverslip at 40magnification. The expression of CD11b, TMEM119, and TREM2 in IBA1-positive cells in monoculture and co-culture was then quantified using Fiji.

For the analysis of apoptosis in neurons, five z-stacks images of randomly selected visual fields (212.55212.55m) were taken at 40magnification for each coverslip. The ratios of cleaved caspase 3/ TUJ1-positive cells were then quantified for neurons in monoculture and co-culture in a blinded fashion. For the analysis of apoptosis in microglia, three z-stacks images of randomly selected visual fields (212.55212.55m) were taken at 40magnification for each coverslip. The ratios of cleaved caspase 3/ IBA1-positive cells were then quantified for microglia in monoculture and co-culture.

For the analysis of microglial ramifications, five z-stacks images of randomly selected visual fields (212.55212.55m) were taken at 40magnification for each coverslip. To analyze the branching of IBA1-positive microglia in monoculture and co-culture, the average branch length, number of branch points and number of branch endpoints was determined using 3DMorph39, a Matlab-based script for the automated analysis of microglial morphology.

From the same harvest, macrophage precursors (pMacpre) were either lysed directly or differentiated to microglia in monoculture (pMGL) or microglia in co-culture with MNs (co-pMG) for 14days. pMGL were rinsed with PBS and directly lysed in the dish. For both pMacpre and pMGL, RNA was extracted using an RNAeasy Mini Plus kit (Qiagen) according to the manufacturers instructions. Co-cultures were first dissociated by 15min incubation with papain (P4762, Sigma-Aldrich) diluted in accutase (20 U/mL) and gentle trituration based on a previously published protocol40. The cell suspension was then passed through a cell strainer (70m, Falcon) to remove cell clumps. To extract co-pMG, magnetic-activated cell sorting (MACS) was then performed using CD11b-MACS beads (130093-634, Miltenyi Biotec) according to the manufacturers instructions. The panned cell population was lysed for RNA extraction using an RNAeasy Micro kit (Qiagen) according to the manufacturers instructions. In addition, RNA from human fetal microglia and blood monocytes from three different healthy genetic backgrounds wasre-used from our previous study26.

RNA from the four different healthy control lines (listed earlier) per condition (pMacpre, pMGL, co-pMG) was used for RNA sequencing analysis. Material was quantified using RiboGreen (Invitrogen) on the FLUOstar OPTIMA plate reader (BMG Labtech) and the size profile and integrity analysed on the 2200 or 4200 TapeStation (Agilent, RNA ScreenTape). RIN estimates for all samples were between 9.2 and 9.9. Input material was normalised to 100ng prior to library preparation. Polyadenylated transcript enrichment and strand specific library preparation was completed using NEBNext Ultra II mRNA kit (NEB) following manufacturers instructions. Libraries were amplified (14 cycles) on a Tetrad (Bio-Rad) using in-house unique dual indexing primers (based on41). Individual libraries were normalised using Qubit, and the size profile was analysed on the 2200 or 4200 TapeStation. Individual libraries were normalised and pooled together accordingly. The pooled library was diluted to~10nM for storage. The 10nM library was denatured and further diluted prior to loading on the sequencer. Paired end sequencing was performed using a NovaSeq6000 platform (Illumina, NovaSeq 6000 S2/S4 reagent kit, v1.5, 300 cycles), generating a raw read count of a minimum of 34M reads per sample.

Further processing of the raw data was then performed using an in-house pipeline. For comparison, the RNA sequencing data (GSE89189) fromAbud et al.28 and the dataset (GSE85839) fromMuffat et al.29 were downloaded and processed in parallel. Quality control of fastq files was performed using FastQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and MultiQC42. Paired-end reads were mapped to the human GRCh38.p13 reference genome (https://www.gencodegenes.org) using HISAT2 v2.2.143. Mapping quality control was done using SAMtools44 and Picard (http://broadinstitute.github.io/picard/) metrics. The counts table was obtained using FeatureCounts v2.0.145. Normalization of counts and differential expression analysis for the comparison of pMGL and co-pMG was performed using DESeq2 v1.28.146 in RStudio 1.4.1103, including the biological gender in the model and with the BenjaminiHochberg method for multiple testing correction. Exploratory data analysis was performed following variance-stabilizing transformation of the counts table, using heat maps and hierarchical clustering with the pheatmap 1.0.12 package (https://github.com/raivokolde/pheatmap) and principal component analysis. Log2 fold change (log2 fc) shrinkage for the comparison of pMGL and co-pMG was performed using the ashr package v2.2-4747. Genes with |log2 fc|>2 and adjusted p value<0.01 were defined as differentially expressed and interpreted with annotations from the Gene Ontology database using clusterProfiler v3.16.148 to perform over-representation analyses.

Equal amounts of RNA (30ng) were reverse-transcribed to cDNA using the High-Capacity cDNA Reverse Transcription Kit (ThermoFisher) according to the manufacturers instructions. Quantitative real-time PCR was performed with Fast SYBR Green Master Mix (ThermoFisher) according to the manufacturers instructions using a LightCycler 480 PCR System (Roche). The following primers (ChAT from Eurofins Genomics, all others from ThermoFisher) were used:

Quantification of the relative fold gene expression of samples was performed using the 2Ct method with normalization to the GAPDH reference gene.

AH016-3 Lenti-IP-RFP-microglia were co-cultured with healthy control motor neurons in PEI- and Geltrex-coated glass bottom dishes for confocal microscopy (VWR). The RFP signal was used to identify microglia in co-culture. To visualize microglial movement, images of the RFP signal and brightfield were taken every~30s for 1h (22 stitched images, 20magnification) using a Cell Observer spinning disc confocal microscope (Zeiss) equipped with an incubation system (37C, 5% CO2). To image phagocytic activity, co-cultures were rinsed with Live Cell Imaging Solution (1X, ThermoFisher), and pHrodo Green Zymosan Bioparticles Conjugates (P35365, ThermoFisher) diluted in Live Cell Imaging Solution (50g/mL), which become fluorescent upon phagocytic uptake, were added. The dish was immediately transferred to the spinning disc confocal microscope, and stitched images (33, 20magnification) were acquired every 5min for 2h.

To induce pro-inflammatory (M1) or anti-inflammatory (M2) microglial phenotypes, cells were treated with Lipopolysaccharides (LPS, 100ng/mL, Sigma) and Interferon- (IFN-, 100ng/mL, ThermoFisher), or Interleukin-4 (IL-4, 40ng/mL, R&D Systems) and Interleukin-13 (IL-13, 20ng/mL, Peprotech), respectively, for 18h. Vehicle-treated (co-culture medium) cells were used as an unstimulated (M0) control.

To analyze the clustering of microglia upon pro-inflammatory and anti-inflammatory stimulation, RFP-positive microglia were imaged directly before the addition of M1/M2 inducing agents, and at 9h and 18h post-stimulation using the Cell Observer spinning disc confocal microscope (55 stitched images, 10magnification). The number of individual microglial cells and size of microglial clusters was quantified using the analyze particle function in Fiji.

After stimulation with M1/M2-inducing agents, culture supernatants were collected and spun down at 1200g for 10min at 4C. Pooled samples from three different healthy control lines for each cell type were analyzed using the Proteome Profiler Human XL Cytokine Array Kit (R&D Systems) according to the manufacturers instructions. The signal was visualized on a ChemiDoc MP imaging system (Bio-Rad) and analyzed using ImageStudioLite v5.2.5 (LI-COR). Data was then plotted as arbitrary units using the pheatmap 1.0.12 package in RStudio 1.4.1103.

In addition, to confirm the relative expression of Serpin E1 and CHI3L1 in cell culture supernatants, the Human Human Chitinase 3-like 1 Quantikine ELISA Kit (DC3L10) and Human Serpin E1/PAI-1 Quantikine ELISA Kit (DSE100, both R&D Systems) were used according to the manufacturers instructions.

pNeuron, pMGL and co-cultures were plated and maintained in WillCo-dish Glass Bottom Dishes (WillCo Wells) for 14days. Calcium transients were measured using the fluorescent probe Fluo 4-AM according to the manufacturers instructions (ThermoFisher). Cells were incubated with 20M Fluo 4-AM resuspended in 0.2% dimethyl sulfoxide for 30min at RT in Live Imaging Solution (ThermoFisher). After a washing step with Live Imaging Solution, cells were allowed to calibrate at RT for 1520min before imaging. Ca2+ images were taken by fluorescence microscopy at RT. The dye was excited at 488nm and images were taken continuously with a baseline recorded for 30s before stimulation. The stimuli used for calcium release were 50mM KCl (Sigma-Aldrich) for 30s, followed by a washing step for one minute. Microglial calcium release was stimulated by 50M ADP (Merck) under continuous perfusion for 1min, followed by a 1-min wash. Analysis of fluorescence intensity was performed using Fiji. Fluorescence measurements are expressed as a ratio (F/Fo) of the mean change in fluorescence (F) at a pixel relative to the resting fluorescence at that pixel before stimulation (Fo). The responses were analysed in 2040 cells per culture.

MNs on DIV 3345 were maintained in a bath temperature of 25C in a solution containing 167mM NaCl, 2.4mM KCl, 1mM MgCl2, 10mM glucose, 10mM HEPES, and 2mM CaCl2 adjusted to a pH of 7.4 and 300mOsm. Electrodes with tip resistances between 3 and 7M were produced from borosilicate glass (0.86mm inner diameter; 1.5mm outer diameter). The electrode was filled with intracellular solution containing 140mMK-Gluconate, 6mM NaCl, 1mM EGTA, 10mM HEPES, 4mM MgATP, 0.5mM Na3GTP, adjusted to pH 7.3 and 290mOsm. Data acquisition was performed using a Multiclamp 700B amplifier, digidata 1550A and clampEx 6 software (pCLAMP Software suite, Molecular Devices). Data was filtered at 2kHz and digitized at 10kHz. Series resistance (Rs) was continuously monitored and only recordings with stable<50 M and Rs<20% were included in the analysis. Voltage gated channel currents were measured on voltage clamp, neurons were pre-pulsed for 250ms with 140mV and subsequently a 10mV-step voltage was applied from 70 to+70mV. Induced action potentials were recorded on current clamp, neurons were held at 70mV and 8 voltage steps of 10mV, from 10 to 60mV, were applied. Data was analyzed using Clampfit 10.7 (pCLAMP Software suite).

Statistical analyses were conducted using GraphPad Prism 9 (GraphPad Software, San Diego, California USA, http://www.graphpad.com). Comparisons of two groups were performed by two-tailed unpaired t-tests and multiple group comparisons by one-way or two-way analysis of variance (ANOVA) with appropriate post-hoc tests as indicated in the figure legends. The statistical test and number of independent experiments used for each analysis are indicated in each figure legend. Data are presented as single data points and meansSEM. Differences were considered significant when P<0.05 (*P<0.05; **P<0.01; ***P<0.001; ns: not significant). GraphPad Prism 9 or RStudio 1.4.1103 were used to plot data. Final assembly and preparation of all figures was done using Adobe Illustrator 25.4.1.

Read this article:
Human iPSC co-culture model to investigate the interaction between microglia and motor neurons | Scientific Reports - Nature.com