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Expert help when you need it- Q1:ments ons Button rations A https://uab.instructure.com/groups/346802/discussion_topics/7329486 pasting it into the reply area of the discussion. De sure to come back and respond to your group members. ib S R Description: UAB Hospital has decided to purchase a unique and very expensive type of MRI scanning equipment (to detect tumors). Look up some information on the internet about what is MRI equipment and how would it be used by a health care provider. The General Electric Company (GE) is a major provider of MRI equipment. It is important for the sales manager of GE Healthcare to understand who is likely to be involved in the buying center for this purchase at UAB Hospital. Each person may influence the purchase and perhaps influence it in different ways. The sales manager needs to see if all of the different buyer center's needs are being met, and if not, why not. Clearly, this kind of thinking can help guide the sales strategy. See the section in the book about "Multiple buying influence in a buying center" for more discussion of the buying center concept. INITIAL POST: In a few sentences, introduce yourself to your new discussion group. . Then address these points related to the case above: . Who might be involved in the buying center? • Explain your answer and describe the type of influence that different people in the buying center at UAB Hospital would have on the purchase.See Answer
- Q2: Review polymers Hybrid-Based Wound Dressings: Combination of Synthetic and Biopolymers Blessing Atim Aderibigbe Citation: Aderibigbe, B.A. Hybrid-Based Wound Dressings: Combination of Synthetic and Biopolymers. Polymers 2022, 14, 3806. https://doi.org/10.3390/ check for updates polym14183806 Academic Editor: Shahin Homaeigohar Received: 3 August 2022 Accepted: 5 September 2022 Published: 12 September 2022 CC Publisher's Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. 4.0/). BY Copyright: © 2022 by the author. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ MDPI Department of Chemistry, University of Fort Hare, Alice 5700, Eastern Cape, South Africa; baderibigbe@ufh.ac.za Abstract: Most commercialized wound dressings are polymer-based. Synthetic and natural polymers have been utilized widely for the development of wound dressings. However, the use of natural polymers is limited by their poor mechanical properties, resulting in their combination with synthetic polymers and other materials to enhance their mechanical properties. Natural polymers are mostly affordable, biocompatible, and biodegradable with promising antimicrobial activity. They have been further tailored into unique hybrid wound dressings when combined with synthetic polymers and selected biomaterials. Some important features required in an ideal wound dressing include the capability to prevent bacteria invasion, reduce odor, absorb exudates, be comfortable, facilitate easy application and removal as well as frequent changing, prevent further skin tear and irritation when applied or removed, and provide a moist environment and soothing effect, be permeable to gases, etc. The efficacy of polymers in the design of wound dressings cannot be overemphasized. This review article reports the efficacy of wound dressings prepared from a combination of synthetic and natural polymers. Keywords: hybrid wound dressings; synthetic polymers; skin regeneration; natural polymers; wound healing; wounds; wound dressings; biomaterials 1. Introduction Wounds are challenging to treat, especially when they have been invaded by mi- crobes [1,2]. Wound healing involves complex mechanisms that require using appropriate wound dressings to induce a timely wound healing process [3]. Polymers have played a huge role in the design of potent wound dressings. Synthetic and natural polymers are used to prepare wound dressings. Synthetic polymers are characterized by features such as easy preparation resulting in controlled physicochemical properties and stability, good mechanical stability with interesting mechanical properties, and degradation in a controlled manner [4,5]. However, they can induce the risk of toxicity and are biologically inert. Natural polymers, on the other hand, do not offer interesting mechanical properties, are biocompatible, biodegradable with interesting biological activities, and can undergo enzymatic degradation to produce by-products that do not trigger toxic reactions [6,7]. However, their high rate of degradation rates is usually challenging to control [5]. The combination of synthetic polymers together with natural polymers has been widely employed to overcome shortcomings common with both types of polymers [8,9]. The combination of both polymers in the design of wound dressings results in hybrid-based wound dressings. Hybrid wound dressings display excellent features such as improved mechanical properties, accelerate wound healing, excellent flexibility, biocompatibility, biodegradability, high adsorption capacity, etc. [10,11]. They are also appropriate for treating high exuding, bleeding, and infected wounds. They are also suitable to promote skin regeneration [12,13]. The further incorporation of bioactive agents and biological molecules into hybrid-based wound dressing has resulted in materials that exhibit excellent wound healing and skin regeneration [14,15]. This review reports the in vitro and in vivo outcomes of hybrid-based wound dressings. Polymers 2022, 14, 3806. https://doi.org/10.3390/polym14183806 https://www.mdpi.com/journal/polymers Polymers 2022, 14, 3806 2. Wound Healing Mechanisms The physiological and architectural restoration of the skin after an injury is based on four important phases: hemostasis, inflammation, proliferation, and remodeling (Figure 1) [16,17]. Wound healing is complex and specialized cells are involved, such as macrophages, fibroblasts, platelets, endothelial cells, etc. There is a significant interaction between the cells and the extracellular matrix. Wound healing is also influenced by the action of growth factors, chemokines, cytokines, chemokines, receptors, etc. [18,19]. Red blood cell White blood cell Blood clot U Hemostasis Microbes Growth Macrophages factor DOO Inflammation Figure 1. The phases of wound healing. Neutrophils ROS Fibroblast Proliferation Epithelial cells Matured scar === DO Remodeling 2 of 31 Keratinocytes -Fibroblast Hemostasis This phase occurs immediately after an injury, whereby the damaged blood vessels constrict rapidly with a blood clot formation to prevent excess loss of blood [20,21]. The platelets play a crucial role in blood clot formation [22]. The constriction of the blood vessels together with the formation of a blood clot results in the lack of oxygen, changes in pH, and glycolysis [23]. After the narrowing of the blood vessels, the widening promotes the invasion of the wound matrix by thrombocytes. The widening of the blood vessels known as vasodilation is characterized by oedema and local redness of the wound [23]. The blood clot is composed of cytokines, fibrin molecules, growth factors, vitronectin, thrombospondins, and fibronectin. The blood clot also acts as a shield against bacteria invasion and as a reservoir for cytokines and growth factors for wound repair [24]. After the formation of a blood clot, the coagulation process terminates to prevent excessive thrombosis and platelet aggregation [24]. In this phase, the repair of smooth muscle cells and endothelial cells also occurs due to the release of platelet-derived growth factors [25]. The platelet-derived growth factors recruit neutrophils and monocytes, which together with transforming growth factor ß (TGF-ß) from the vasculature induce the inflammatory response [26]. Fibroblasts are also recruited by platelet-derived growth factors, and they migrate to the wound site followed by the production of collagen, glycosaminoglycans, and proteins which promote cellular migration and set the stage for subsequent healing events [23,24]. Inflammation The inflammation phase overlaps with the hemostasis phase. In this phase, neutrophils and monocyte infiltration in the wound bed prevent the invasion of microbes, foreign debris, and tissue damage [27,28]. The neutrophils promote the process of phagocytosis of debris and microbes to allow decontamination of the wound. The three known mechanisms of neutrophils in the destruction of debris and bacteria are: directly ingesting followed by destroying the foreign debris by a process known as phagocytosis; via the release of toxic substances, e.g., lactoferrin, cathepsin, proteases, etc. to destroy bacteria and the dead host tissue; and by the production of chromatin and protease 'traps' to capture and destroy bacteria in the extracellular space [29,30]. The by-product of the neutrophil activity, oxygen-free radicals, exhibit bacteriocidal properties to sterilize the wound. However, Polymers 2022, 14, 3806 3 of 31 the oxygen-free radicals can exasperate inflammation of the microenvironment, thereby resulting in delayed wound healing [29,31]. After the completion of the neutrophil activity, the neutrophils undergo apoptosis and are phagocytosed by macrophages or can be sloughed from the wound surface [30]. Macrophages are attracted to the wound and are released from platelets and damaged cells. In this phase, they can withstand the acidic wound environment [32]. Macrophages are composed of significant amounts of growth factors, which include TGF-B and epidermal growth factors, which are crucial in regulating the inflammatory response [30,33,34]. The inflammatory phase of wound healing can be prolonged to remove excess bacteria and debris from the wound. Prolonged inflammation can result in significant tissue damage and delayed proliferation, resulting in the formation of a chronic wound [30]. Multiple factors have been reported to affect the immune response such as lipoxins and the products of arachidonic acid metabolism, thereby hindering the next phase of wound healing [35]. Fur- thermore, the type of immune response plays a crucial role in the formation of hypertrophic scarring or keloid formation. The differentiation of T-helper (Th) cells, major immune mediators in the inflammatory phase to Th2 cell types, results in hypertrophic scarring [36]. In chronic non-healing wounds, the presence of an abundance of neutrophils makes them become proteolytic environments composed of host-derived proteases. Excessive tissue damage is caused by elevated inflammatory cytokines and collagenases [37]. Proliferation In this phase, there is a complex simultaneous combination of fibroblast migration, angiogenesis, and epithelialization together with a wound retraction [38]. A sufficient supply of gas, blood, nutrients, and metabolites is required [38]. The release of vascular endothelial growth factor (VEGF) and cytokines induces the endothelial cells to promote angiogenesis, the formation of new blood vessels and the repair of damaged blood vessels in the wound site. The migration of fibroblast results in the production of fibronectin and collagen with a replacement of the clot with granulation tissue made up of different ranges of collagen [29]. The fibroblasts are converted to myofibroblast phenotype, which is useful in wound contraction. The myofibroblasts also induce angiogenesis, and the collagens produced by the fibroblasts are responsible for providing strength to the tissues. However, the formation of a hypertrophic scar can be induced by an overproduction of collagen [39]. Several macrophage-derived molecules, such as IL-1, ß-bb (PDGF-bb), IL-6, etc. promote pro-re-epithelialization molecules in the fibroblasts. In wounds without a macrophage- derived molecule, IL-6, there is a lack of appropriate inflammatory response, resulting in hampered collagen accumulation, angiogenesis, and re-epithelialization [40]. The migration of the epithelial cells from the edges of the wound forms a sheet of cells that covers the wound, and this process is known as epithelialization [41]. Epithelialization takes place within 24 h in primary wounds, but in secondary closed wounds, the contraction of large areas lacking epithelial cells occurs before complete epithelialization [29,30]. Remodeling This last phase is characterized by a transition from granulation tissue to scar formation with slow angiogenesis and a replacement of type III collagen with type I collagen that is stronger [28]. This remodeling phase is significantly promoted by myofibroblasts developed from fibroblasts, which are responsible for wound contraction [42]. Some reports have shown that fat cells obtained from the differentiation of myofibroblasts replenish the subcutaneous adipose tissue, and this is influenced by the neogenic hair follicles, resulting in the activation of adipocyte transcription factors and bone morphogenic protein (BMP) signaling [43]. Chronic Wounds Chronic wounds are classified as non-healing wounds over a prolonged period, and they can be classified as diabetic, vascular, diabetic, and pressure wounds [44]. Other examples of chronic wounds are gangrenes, ischemia, etc. [45]. The most common type Polymers 2022, 14, 3806 of chronic wound is the diabetic wound, and it affects 15% of the world population who suffer from diabetes [46]. Due to the prolonged healing period of these types of wounds, the patients usually require long medical care, amputation, and in some cases, long hospital stay [47]. Factors that contribute to chronic wounds are reduced blood supply and re- epithelialization, inflammatory responses that are not controllable, and bacterial infections. There are complications associated with diabetes such as foot infections, etc. Un- controlled diabetes reduces the tissue oxygen rate, thereby damaging blood vessels and forming non-healing ulcers [48]. Venous ulcers are common in older patients and affect the lower limbs due to a damaged deep venous system. It is also characterized by increased blood pressure in the vessels, causing leakage and an accumulation of fibrin that blocks the vascular pathway, reducing the flow of oxygen to the surrounding tissues [45,48]. Pres- sure ulcers are also common in older and paralyzed patients. It occurs due to continuous pressure on the skin, decreasing the diffusion of oxygen in the tissues [45]. Pathophysiology of Chronic Wounds Chronic wounds remain in the inflammatory stage of wound healing due to the persis- tent recruitment of neutrophils and macrophages in the wound bed, which also prolongs the wound healing process (Figure 2) [45,49]. The high production of inflammatory molecules and Reactive Oxygen Species (ROS) in chronic wounds affect the synthesis of collagen, decreases proliferation, and causes an abnormal differentiation of keratinocytes [49]. The altered pattern of cytokine in the wound also contributes to delayed wound healing in diabetes patients. The abnormal expression of growth factors is observed in diabetic foot ulcers that disrupt the healing process. Elevated levels of matrix metalloproteinases and reduced levels of tissue inhibitors of metalloproteinases in chronic wounds also affect the wound healing process [45,49]. Matrix metalloproteinases are crucial for the remodeling of the matrix microenvironment by inducing healing responses, such as cellular migra- tion, proliferation, and angiogenic induction. The high levels of protease results in the damage of extracellular matrix (ECM) and growth factors degradation together with their receptors [50]. The damaged ECM prohibits the wound healing process from moving to the proliferative phase. The aforementioned factor results in an inflammation cycle where more proteases are produced. The persistent inflammatory and hypoxic state of chronic wounds induce a high production of ROS that also destroys the ECM proteins. Defective re-epithelialization IFN-y IL-10 TGF-B Mast cell degranulation Keratinocytes Th1/Th17/ Th22 MMPS CD8+ T cells TNF-a Decreased angiogenesis Figure 2. The immune environment of chronic wounds [50]. Bacterial infection ROS M1↑ Macrophages IL-6 10 Neutrophils MMP2/9 NOS IL-13 TNF-a 4 of 31 TNF-a IL-8 IL-13 NETS Polymers 2022, 14, 3806 5 of 31 The presence of senescent macrophages, keratinocytes, fibroblasts, and endothelial cells in chronic wounds contributes to oxidative stress that results in the damage of de- oxyribonucleic acid (DNA) cell cycle arrest and a defect in the intracellular biochemical pathways including GSK-3ß/Fyn/Nrf2 pathway, etc. [49,50]. Chronic wound healing is characterized by reduced angiogenesis and tissue epithelialization [50]. The mesenchymal stem cells also play an important role in wound healing and are recruited into circulation when there is an injury. However, they are defective and deficient in chronic wounds. The absence of effective receptors or promigratory matrix substrates inhibits cell migration and proliferation in chronic wounds. The hallmarks of chronic wounds have been reported to be impaired neovascularization and angiogenesis, resulting in an insufficient supply of nutrients and oxygen for the cells in the wound bed, leading to non-healing wounds [49]. To accelerate the wound healing process of chronic wounds, researchers have employed the use of microbial agents to treat persistent microbial infections [51,52], the delivery of healthy donor-derived functional mesenchymal stem cells to deal with their deficiency [53], and the administration of antioxidants to reduce the ROS to normal levels, thereby reversing the chronic state of the wounds [54,55]. In the treatment of chronic wounds, debridement is performed to remove non-viable tissues [56]. Anti-inflammatory agents are also employed to deal with prolonged inflamma- tion [57], and the use of appropriate wound dressings that addresses the moisture imbalance is crucial for treating chronic wounds [58]. To promote the formation of granulation tissue and epithelialization, the use of growth factors has been used [59,60]. 3. Hybrid Wound Dressings in Wound Healing Hybrid-based wound dressings have been developed from the combination of natural and synthetic polymers. Different types of hybrid-based wound dressings have been developed, such as foams, hydrocolloids, hydrogels, nanofibers, films/membranes, etc. Different preparation techniques have been employed. This section reports the preparation techniques used as well as in vitro and in vivo biological outcomes. 3.1. Foams The commonly used foam wound dressings are polyurethane foams used for moist wound healing (Figure 3a,b) [61]. Silicone foams are not commonly used. Foam wound dressings are characterized by a porous structure and a film-backing, and they are produced with varied thicknesses [62]. They can be adhesive or non-adhesive. The permeability of the film backings varies with a significant influence on water evaporation and gas exchange capacity [62]. The contact of the wound with the foam products is crucial to facilitate the absorption of the exudate. Their adhesion to the surrounding skin of the wound bed is useful in keeping the dressing and preventing the leakage of the exudates that can cause skin irritation [63]. They are suitable as primary or secondary wound dressings. Some of their unique features include their ability to maintain moisture at the wound bed, to be easily removed from the wound, to protect the wound from bacterial invasion, to provide mechanical protection, and to conform to the body shape; they are also non-toxic, easy to use, etc. [62]. Foams are effective for the management of acute and chronic with medium to heavy exudate [64]. They have been prepared from a combination of synthetic and natural polymers. Most reported foam wound dressings are prepared from synthetic polymers [65,66]. Some are prepared from natural polymers only with high porosity but are prone to enzy- matic degradation [67,68]. Hybrid-based foams have been prepared from polyurethane in combination with natural polymers, such as chitosan, sodium alginate, and hydroxypropyl methylcellulose, by Namviriyachote et al. The foams were loaded with silver nanoparticles and asiaticoside. Foams prepared from alginate displayed a high release of silver nanopar- ticles and asiaticoside. Using natural polymers for the preparation of the foams influenced the compressive strength and absorption properties. The foams were non-cytotoxic and compatible, and they improved the rate of wound closure with a significant formation ofSee Answer
- Q3:1. The following questions pertains to the breakdown of glucose for ATP. (15 points) a. What is Glucose? What is Glycogen? b. How many ATP molecules are produced during glycolysis per glucose molecule? Does this ATP production require oxygen? c. Why do muscle cells produce lactic acid during bursts of activity? d. How many ATPs are produced per glucose molecule in the mitochondria during oxidative phosphorylation? Does this ATP production require oxygen? e. Why is there a membrane potential across the inner mitochondrial membrane?See Answer
- Q4:2. The following triglyceride, is composed of 3 Capric Fatty Acid Chains (CH₂O₂) and a glycerol. (20 points) a. How many ATPs would you generate from the complete breakdown of this triglyceride under optimal conditions (assuming cytosolic NADH yields 2.5 ATP/NADH and oxygen is available)? b. If the molecular weight of this triglyceride is 554.84 g/mol, what is the ATP yield per gram of the triglyceride? Compare part b to the ATP yield per gram of glucose under optimal conditions (assuming cytosolic NADH yields 2.5 c. ATP/NADH and oxygen is available)? Glucose molecular weight is 180 g/mol. d. What is the goal of a low carb diet when it comes to your metabolism of ATP?See Answer
- Q5:3. A cell has the following conditions at rest (28 points) [Na], = 155 mM [Na] = 8 mM [Ca] = 1.5 mm [Ca] =0.3 μM [K], 6 mM [K"] = 150 mM a. Calculate En. Eca, and Ex b. If gx = 80 g, and gc. = 0.25 g, calculate Em at rest. c. Calculate the net driving force for Ise. Ik and Ica Hint: net driving force of an ion is En-E. d. Calculate the relative values of Isa. Ik, and Ica (you can leave ga as an unknown). In which direction would the current be flowing for each ion? e. What is the sum of the current produced by all the ions? What does this tell us about the membrane? f. A stimulus causes Ca channels to open, resulting in a sudden increase in Ca** conductance so that now gca = 10 ga. What is the new membrane potential? (Assume ion concentrations stay the same as at rest and Na* & K* conductance don't change) g. If the normal threshold for an action potential is -55 mV, is the stimulus in f enough to cause an action potential?See Answer
- Q6:4. Suppose the internal specific resistance of the axoplasm is 64 0 cm. Calculate the internal resistance of an axon 10 mm long if it has a radius of 0.5 μm. Repeat the calculate for an axon with the same specific resistance and length, but an internal radius of 50 μm. Which axon would have a quicker conduction velocity? (12 points)See Answer
- Q7:6. The action potential on a motor neuron lasts about 1.6 ms. A large motor neuron conducts the action potential at about 200 m/s. (10 points) a. Calculate the distance between the beginning of the action potential and its end. b. The nodes of Ranvier are interruptions in the myelin sheath in myelinated fibers, and typically they are located about 2 mm apart along the length of the axon. The action potential that you calculated in Part A is spread out over how many nodes of Ranvier? c. In Saltatory conduction, what "jumps" from node to node?See Answer
- Q8: Write 12 pages || double spaced MLA || Idea -->do it on how polymers can help brain aneurysm. Write a mock NIH grant discussing a potential research idea surrounding polymers with specific biomedical application. The grant should cover the following: 1. Significance of the idea: what gap does this research fill? 2. Innovation: how is this idea new or exciting? 3. Approach: how and what experiments are required to complete the research? 4. Impact: what will the impact of this research be? When writing the paper, be sure to cover relevant background, including what is currently done in the area and any other pertinent research. The paper should be 12 pages long, inclusive of figures (which are encouraged), but exclusive of references and the abstract. Font is to be Arial 11 with 0.5" margins. Layout of the mock grant should be as follows: 1. Abstract (250 words) - This should be on its own page. a. Describe problem/challenge b. Your approach c. Advantages of your approach over current strategies d. Experimental approach (aims) e. Anticipate results f. Impact 2. Introduction and Background (1 page) 3. Specific Aims (2 page) a. Hypothesis/rationale b. Strategy 4. Significance and Innovation (2 page) 5. Scientific Plan (2 pages) a. General strategy (schematic) b. Experimental strategy (aims) 6. Pitfalls and Alternative Strategies (2 page) 7. Future Directions and Impact (2 page) - 8. References (No page limit) — References should include authors list, title, journal name, year, volume number and page numbers.See Answer
- Q9: ELSEVIER ARTICLE INFO Article history: Received 10 August 2020 Revised 28 September 2020 Accepted 29 September Available online 6 October 2020 Keywords: Bioadhesives Tissue adhesive Musculoskeletal regeneration Bioactive materials controlled delivery Acta Biomaterialia 117 (2020) 77-92 Review article Bioadhesives for musculoskeletal tissue regeneration * Solaiman Tarafder, Ga Young Park, Jeffrey Felix, Chang H. Lee* Regenerative Engineering Laboratory, Center for Dental and Craniofacial Research, Columbia University Irving Medical Center, 630 West 168th Street, VC12-211B, New York, NY 10032, United States 1. Introduction Contents lists available at ScienceDirect Acta Biomaterialia journal homepage: www.elsevier.com/locate/actbio ABSTRACT Bioadhesives are often referred to as natural or synthetic ma- terials that adhere to biological components such as cells, tissues, and organs through physical or chemical conjugation. Bioadhesives have been widely applied as tissue adhesives to bind tissues to- gether in soft tissue wound healing [1-3]. Some types of bioadhe- sives have been used as a hemostatic agent to stop bleeding dur- ing surgical operations or as a tissue sealant for secure gaps or cracks to prevent leakage of liquid or air [3–5]. A number of dif- ferent bioadhesives have been investigated as a tissue glue for skin wound closure to replace suture or wound dressing [1,2,6]. Inter- nal medicine has also utilized various bioadhesives as hemostasis, graft fixation, and sealants in support of surgical treatments [3]. The key properties considered for such bioadhesives, include but not limited to, biocompatibility, biodegradability, toxicity, ad- hesion strength on target surface, and duration of cross-linking [1,3]. Although each aspect may be considered on different weight depending on target application, the adhesive property is likely the most important feature for the abovementioned applications Natural or synthetic materials designed to adhere to biological components, bioadhesives, have received significant attention in clinics and surgeries. As a result, there are several commercially available, FDA- approved bioadhesives used for skin wound closure, hemostasis, and sealing tissue gaps or cracks in soft tissues. Recently, the application of bioadhesives has been expanded to various areas including muscu- loskeletal tissue engineering and regenerative medicine. The instant establishment of a strong adhesion force on tissue surfaces has shown potential to augment repair of connective tissues. Bioadhesives have also been applied to secure tissue grafts to host bodies and to fill or seal gaps in musculoskeletal tis- sues caused by injuries or degenerative diseases. In addition, the injectability equipped with the instant adhesion formation may provide the great potential of bioadhesives as vehicles for localized delivery of cells, growth factors, and small molecules to facilitate tissue healing and regeneration. This review covers recent research progress in bioadhesives as focused on their applications in musculoskeletal tissue repair and regeneration. We also discuss the advantages and outstanding challenges of bioadhesives, as well as the future perspective toward regeneration of connective tissues with high mechanical demand. © 2020 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. C:\Users\Chang\OneDrive - cumc.columbia.edu Research & Study\LAB_Work \Papers\Bioadhesive Review 2020\Revision Bioadhesives Review_Outline Combined rev 2.docx * Corresponding author. E-mail address: chl2109@cumc.columbia.edu (C.H. Lee). Acta BioMaterialia Structure Property Function Relationships in materials https://doi.org/10.1016/j.actbio.2020.09.050 1742-7061/© 2020 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. Check for updates of bioadhesives [1,3]. Functionality of most bioadhesives highly attributed to its adhesion properties on tissues, grafts, or materi- als, providing secure graft fixation, wound closure or dressing. Re- cently, the application of bioadhesives has been expanded for tis- sue repair, tissue engineering, and regenerative medicine [7-14]. To support repair and healing, particularly for musculoskeletal tis- sues, the adhesion strength of bioadhesives is being challenged to move up to the next level [8,9,14-16]. The majority of bioadhe- sives have adhesion strength at a magnitude of kPa but connec- tive tissues such as tendon and knee meniscus show tensile mod- ulus and strength in a MPa range [8,9,14-16]. Such high mechani- cal demands in musculoskeletal tissues also requires a further im- provement not only in the adhesion strength but also in bulk me- chanical properties of bioadhesives [8,9,14-16]. Besides the phys- ical support, bioadhesives have been evolved to deliver bioactive cues and/or cells in addition to provide tissue adhesion [13,14,17– 19]. Bioadhesives applied on injured site can serve as effective lo- calized delivery vehicles as secured in situ [13,14,17-19] or provid- ing physical and/or biochemical environment promoting tissue re- pair [20,21]. Recent improvements in the design and synthesis of bioadhesives have made steps closer to successful applications for musculoskeletal tissues in high mechanical demand. As per PubMed literature search from 2000 to 2019, the num- ber of peer-reviewed publications hit by a keyword "tissue adhe- sives" had increased by 2012 and started to decline from 2013 (Fig. 1A). The number of papers with a keyword "tissue adhesives S. Tarafder, G.Y. Park, J. Felix et al. # of publications A 1000 800 600 400 200 02 2000 2001 2002 2003 Tissue adhesive in vivo 2004 2005 2006 2007 2008 2009 2010 2016 2017 2018 2019 # of publications 250 200 150 100 50 B Bioadhesives in vivo 2000 2001 2002 2003 2004 2005 in vivo" appears to be steady over last decade but only account for -6.7% of total papers searched by "tissue adhesives" (Fig. 1A). The yearly number of publications hit by "bioadhesives" shows a steady increase in last 20 years (Fig. 1B). The number of papers hit by "bioadhesives in vivo" also show a continuous increase (Fig. 1B) that accounts for over 20% of total papers with "bioadhesives". These observations in literature search may suggest a growing re- search interest in adhesive biomaterials containing a designed bi- ological function beyond the traditional role as "tissue adhesive" providing physical bonding, hemostasis, or sealing. Tissue specifi- cally, the publication number of tissue adhesives and bioadhesives was the highest for bone, followed by cartilage, tendon, meniscus and intervertebral discs (IVD) (Fig. 1C). This review summarizes various types of existing bioadhesives and their adhesion mechanisms. It covers the recent advancements in bioadhesives for tissue repair and regeneration, focusing on musculoskeletal tissues. The advantages and outstanding limita- tions of bioadhesives in musculoskeletal repair and regeneration are also discussed with regard to potential and perspective. 2007 2. Types of adhesive 2.1. Cyanoacrylates Cyanoacrylates or acrylic tissue adhesives, synthesized by con- densation of a cyanoacetate with formaldehyde [22,23], have been used as a surgical glue for over 50 years [22,24]. The cyanoacrylate monomers polymerize very rapidly (5-60 s) on contact with tis- sue surfaces to form a film that bonds the apposed wound edges. As summarized in Fig. 2, this polymerization is an exothermic reaction triggered by the hydroxyl groups present on the tissue surface or from the moisture [1,25,26]. Participation from amino groups on the tissue surface can also take place during the poly- merization resulting in a strong bond with the tissue. The gen- eral chemical name and formula of cyanoacrylates are alkyl-2- cyanoacrylates and CH₂=C(CN)-COOR, respectively, where R could be any alkyl group ranging from methyl to decyl [25,27]. The first developed cyanoacrylate adhesive was methyl-2-cyanoacrylate (R= -CH3) (known as Eastman 910) with the shortest chain deriva- tive [22]. It was found that the longer the alkyl chain (the R group) the lower the tissue toxicity from the cyanoacrylate ad- hesives [3,28,29]. While the cyanoacrylate with the shortest alkyl group (-CH3) produces a rigid polymer, flexibility can be improved with the longer alkyl chain and adding plasticizer as well. As a result, many cyanoacrylates with longer chain derivatives have been developed such as ethyl-2-cyanoacrylate (EpigluⓇ; Meyer- Haake, Ober-Morlen, Germany) & Krazy Glue® (Elmer's Products Inc, Columbus, OH), butyl-2-cyanoacrylate (Trufill®; Codman & Years 78 2013 2015 2016 2.2. Fibrin 2017 2018 2019 C # of publications 2000 1500 1000 500 Years Fig. 1. PubMed literature search from 2000 to 2019 with selected key words (A and B) and specific tissue target (C). Bone Acta Biomaterialia 117 (2020) 77-92 IVD Cartilage Tendon Meniscus Shurtleff, Inc., Raynham MA), Indermil® (Connexicon Medical Ltd., Dublin, Ireland), Histoacryl® (B. Braun AG, Melsungen, Germany), and 2-octyl-cyanoacrylate (Dermabond®; Ethicon US, LLC., a John- son & Johnson Company, Cincinnati, OH) & Surgiseal (Adhezion Biomedical, Wyomissing, PA)[22, 29, 30]. Although cyanoacrylates I have been used as tissue adhesives or sealants for decades out- side the U.S., the first cyanoacrylate that was approved by FDA (in 1998) to be used as tissue adhesive was 2-octyl-cyanoacrylate (Dermabond®) [1,22]. Some of the benefits of cyanoacrylate tissue adhesives are the ease of application for first aid, quick adhesion or sealing of wounded tissues, excellent hemostasis, and potential bacterio- static or microbial barrier properties [1,31]. Despite these benefits, cyanoacrylate and its degradation by-products may cause cytotox- icity, foreign body reactions, tissue necrosis, and inflammatory re- sponses [32,33]. Cyanoacrylates degrade via hydrolysis resulting in toxic cyanoacetates and formaldehydes as degradation by-products [26,33]. The inherent brittleness is another setback for cyanoacry- lates. Significant efforts have been made to mitigate such brittle- ness and cytotoxicity by introducing longer alkyl chain derivatives. Because of their cytotoxic and inflammation prone nature, a lim- ited number of cyanoacrylates are approved by FDA, predominately for topical use. Fibrin tissue adhesives or tissue sealants are the most widely used bioadhesives in the U.S. since their first approval by FDA in 1998 [27,30,34,35]. Fibrin sealants, also known as fibrin glue, con- tain two key components derived from plasma coagulation pro- teins, (i) fibrinogen and (ii) thrombin. Upon mixture, these two components mimic the body's natural blood clotting cascades, as thrombin converts soluble fibrinogen into crosslinked, insoluble fibrin [1,34]. Calcium is often added to thrombin to further cat- alyze the clot formation. Although clotting occurs rapidly (within seconds), the clotting time can vary depending on the concentra- tions of fibrinogen and thrombin and the presence of other cat- alyzing and stabilizing components. Fibrin is the only material that is currently FDA approved for use as a hemostat, tissue adhesive, and tissue sealant [36]. Fibrin sealant has wide range of applications. For exam- ple, orthopaedic surgeons frequently use fibrin sealant in autol- ogous chondrocyte implantation (ACI) treatment, where culture- expanded chondrocytes are delivered into a cartilage defect con- fined by a periosteal or collagenous membrane fixated by sutures, followed by sealing the defect boundaries with fibrin sealant. In the suture-free matrix-induced ACI (MACI), type I/type III colla- gen bilayer seeded with chondrocytes is secured directly to the S. Tarafder, G.Y. Park, J. Felix et al. (i) n/H₂C=C1 (ii) Alkyl-2-cyanoacrylates (R could be any alkyl group ranging from methyl to decyl) R H₂C Shorter alkyl (R) group on (i) Cyanoacetate tő-c Poly(methyl-2-cyanoacrylate) (Eastman 910) CH3 OH™ tő-c t-c Toxicity Polycyanoacrylates (i) H Poly(ethyl-2-cyanoacrylate) (EpigluⓇ & Kragy glue®) defect site by fibrin glue [37,38]. Repair of delaminated acetabular articular cartilage using fibrin adhesive was shown to be a useful technique for the early cartilage damage treatment strategy [23]. A cadaveric study showed that improved press-fit fixation of os- teochondral scaffolds can be achieved using fibrin glue [39]. Fibrin glue can improve the meniscus healing when applied to outer zone meniscus defect compared to defect only repair [40,41]. Even bet- ter meniscus healing was observed when fibrin was mixed with bone marrow cells in a rabbit model [40]. A long term follow- up (average of 8 years) of 40 patients showed better repair and healing of arthroscopically repaired meniscal tears using fibrin glue with comparable recurrence rate (10%) compared to repair with su- turing [40]. Fibrin is a unique biopolymer with unique biological and physi- cal characteristics. Fibrin sealants exhibit excellent biocompatibil- Formaldehyde H₂ C-CH3 N 79 R H₂ Longer alkyl (R) group on (i) H-c] C C CH3 H₂ H₂ H₂ Poly(octyl-2-cyanoacrylate) (Dermabond® & Surgiseal®) CH3 Hydrolysis (Degradation) Acta Biomaterialia 117 (2020) 77-92 H₂ H₂ Poly(butyl-2-cyanoacrylate) (Histoacryl®, Indermil® & Trufill®) H₂ Fig. 2. Cyanoacrylate adhesivies: formation of polycyanoacrylates from alkyl-2-cyanoacrylate monomeric units, and the resulting byproducts from the degradation of poly- cyanoacrylates (i); some of the shorter and longer alkyl chain derivatives and their commercial names (ii). CH3 ity, biodegradability, deformability, and elasticity. In addition to that, fibrin adhesives do not trigger any inflammatory responses, foreign body reactions, tissue necrosis, or extensive fibrosis. How- ever, in spite of having all these benefits, fibrin glues have low bond strengths (0.005-0.17 MPa) compared to synthetic tissue ad- hesives [1,35]. This limits their application to the defect site under- going significant tensile loads. Fibrin glue can degrade very rapidly even before the healing process begins because of the proteolytic activity in the musculoskeletal joints [42,43]. This is one of the ma- jor reasons because of their limited applicability for musculoskele- tal tissue repair or regeneration associated with synovial joints. In addition to their uses as sealants and tissue adhesives, fibrin alone, or in combination with other polymers, has also been ex- tensively used for tissue engineering and regenerative medicine applications. S. Tarafder, G.Y. Park, J. Felix et al. 2.3. Aldehyde based bioadhesives Another family of commonly used bioadhesive is based on alde- hyde. For example, gelatin-resorcinol cross-linked with formalde- hyde (GRF) and GRFG (GRF with glutaraldehyde) adhesives are the most commonly applied aldehyde based formulations. Originally developed in Europe in the 1960s, GRF/GRFG have been widely used in Europe and Japan for the past few decades for vascular, thoracoscopic, gastrointestinal, lever, and urinary track surgeries (i) (ii) (iii) (iv) H₂N- H₂N- S H₂N- Gelatin H₂N- Gelatin OH OH -NH₂ + -NH₂ + OH H H Formaldehyde bychom OH Resorcin Formaldehyde Formaldehyde H₂N- H₂N- Ho S... R H Glutaraldehyde H₂N- Glutaraldehyde H₂N- [1,44,45]. The idea of having both formaldehyde and glutaralde- hyde in the same formulation is to obtain the initial strong bond- ing from formaldehyde and the high in vivo stability from glu- taraldehyde. Gelatin contributes to the biodegradability and elas- ticity of the GRF/GRFG glue. Since gelatin crosslinked by formalde- hyde/glutaraldehyde performs poorly in wet condition, resorcin, a phenolic component (1,3-benzenediol), is added to GRFG formula- tion to improve its strength by minimizing the negative effect from an aqueous environment [1] (Fig. 3). Formaldehyde and (or) glu- Schil + Resorcin Formaldehyde Tissue H₂N- H H ΝΗ NH- 3x4 ( t usl +H₂N- H₂N- Tissue H₂N- 80 НО H H NH HN- HO H₂C -N=CH HC=N- R H₂N- H₂N- OH OH CH₂ H₂C NH H₂C fo HO HO H₂C -NH₂ ΝΗ -NH₂ -NH₂ -NH₂ |N=CH HC_N R OH OH OH OH H₂ CH₂ OH OH CH₂ CH, HỌC OH OH HN CH₂ OH Acta Biomaterialia 117 (2020) 77-92 OH CH₂ ΗΝ Fig. 3. Aldehyde based adhesives: formaldehyde and glutaraldehyde act as cross-linkers between gelatin molecules (i) and gelatin and tissue or biological surfaces (ii); crosslinking between resorcin and formaldehyde (iii) and resorcin, formaldehyde and tissue (iv). S. Tarafder, G.Y. Park, J. Felix et al. taraldehyde act as crosslinking agents for both gelatin and resor- cin. Aldehyde groups from formaldehyde and glutaraldehyde re- act with the amine group from gelatin, in addition to the amine group present in the tissue, and thus form a strong bond be- tween GRF/GRFG and tissue. Bonding strength of GRF/GRFG can be achieved to the level of cyanoacrylates. In spite of its ex- cellent hemostatic and adhesive properties and widespread us- age in Europe and Japan for decades, GRF/GRFG glues have not been approved by FDA to be used for clinical applications in the U.S.[3] This is likely due to the potential cytotoxicity, mutagenicity, and carcinogenicity caused by formaldehydes, which either can be caused by the residue of unreacted formaldehyde molecules or by the degradation byproducts [46,47]. As a result, some formulations with less toxic glutaraldehyde glyoxal or glutaric acid may improve the biosafety of GRF/GRFG [1,48,49]. BioGlue® (Cryolife, Kennesaw, GA), a protein-aldehyde system (PAS), is a commercially available glutaraldehyde-based formula- tion. It has two components, bovine serum albumin (BSA) and glu- taraldehyde, and the gluing mechanism is similar to GRF/GRFG. BioGlue® has been approved by FDA in 1999 to be used in the U.S. as adjunct to suturing or stapling for acute thoracic aortic dissection and cardiac surgery [27,35]. In vivo degradation rate of BioGlue® is slower than GRF/GRFG. However, the potential cytotoxicity of glutaraldehyde has led to the use of alternative crosslinking agents in other albumin based formulations. For in- stance, PreveLeak™ (Baxter Healthcare, Deerfield, IL) is composed of BSA and polyaldehyde [50] and Progel® (Neomend, Inc., Irvine, CA) is composed of human serum albumin and a polyethyleneg- lycol (PEG) crosslinker functionalized with succinate groups (PEG- (SS)₂), where N-hydroxysuccinimide (NHS) ester groups are at- tached to each end of the PEG [51]. Both PreveLeak and Progel® (i) Tetra-sussinimidyl polyethyleneglycol(4S-PEG): LICHA BOICH SCHO) H₂O) n -OC(CH 2) 3CO(CH₂CH₂O) n Tissue HSCH₂CH₂O(CH2CH₂O) n CH₂ HSCH₂CH₂O(CH2CH₂O) n (ii) Tetra-thiol polyethyleneglycol (4T-PEG): CH₂ H₂C CH₂ H₂C #CHOICH=CH-01/1/20 CH₂ CH₂ (OCH₂CH₂) OC( (CH₂CH₂), (OCH2CH₂) OC(CH2) 3CO- CH₂ CH₂ (OCH2CH₂) OCH₂CH₂SH CH₂ , (OCH2CH₂) OCH₂CH₂SH (1904, 04, DEICH) CH₂ N CH₂ C-SCH2CH₂O(CH2CH₂O) n are FDA approved for vascular reconstructions and intraoperative use during pulmonary resection, respectively. 2.4. Polyethylene glycol (PEG) based adhesives Polyethylene glycol (PEG) based adhesives are a highly water- absorptive hydrogel which have been widely used as fluid barri- ers and hemostatic adhesives. The first commercially available PEG based adhesive was FocalSealⓇ® (Genzyme Biosurgery Inc., Cam- bridge, MA) [52]. It was activated by light and intended to be used as a lung sealant. However, FocalSeal® is no longer available in the market due to its difficulty to use. Currently there are two FDA ap- proved PEG based adhesives available in the market, Coseal (Bax- ter International Inc., Deerfield, IL) and DuraSeal® (Integra Life- Sciences, Princeton, NJ) [53–55]. Coseal is a fully synthetic adhesive that contains two biocompatible functionalized polyethylene gly- cols (PEG), tetra-succinimidyl (4S) and tetra-thiol (4T)-derivatized polyethyleneglycol (4S-PEG and 4T-PEG) [52]. A covalently bonded hydrogel forms when 4S-PEG and 4T-PEG are mixed together. Gel formation occurs through the reaction between the thiol groups and the carbonyl groups of the succinimidyl esters resulting in the formation of a thio-ester covalent network between PEG molecules (Fig. 4). Free N-hydroxy-succinimide molecules are liberated from the reactions. It is indicated for use in vascular reconstructions to achieve adjunctive hemostasis by mechanically sealing areas of leakage. Duraseal contains polyethylene glycol (PEG) ester solution and a trilysine amine solution. This has been used as an adjunct for dural closure to prevent cerebrospinal fluid (CSF) leakage dur- ing brain and spine surgeries. PEG based adhesives are hydrophilic, biocompatible, and biodegradable. However, these adhesives ex- hibit high swelling ratio of up to 400% and thus need to be very (iii) Schematic representation of how a covalently bonded hydrogel forms when 4S-PEG and 4T-PEG are mixed together: CH₂ H₂C CH₂ CH₂ (iv) Schematic representation of how the hydrogel formed from 4S-PEG and 4T-PEG interacts with the biological tissue: (OCH_CH),OCH/CH,S-CICH+;) C&CH/C+01 CH₂CH₂S- 81 H₂C (OCH2CH₂) OCH₂CH₂SH CH₂ CH₂ 4T-PEG (OCH=CH), OCH(CHS C&H) C&CH CHOIR CH₂ CH₂ H₂C Acta Biomaterialia 117 (2020) 77-92 CH₂ LỊCH LỊCH CHỌN CH₂ CH₂ CH₂ H₂C CH₂ (OCH₂CH₂),OC(CH 2) 3C- CH₂ H₂C CH₂ CH₂ 4S-PEG -OH N-hydroxy-succinimide Tissue Fig. 4. Polyethylene glycol (PEG) based adhesives: Chemical structure of tetra-succinimidyl (4S) and tetra-thiol (4T)-derivatized polyethyleneglycol 4S-PEG (i) and 4T-PEG (ii); crosslinking between 4S-PEG and 4T-PEG (iii); and interactions between tissue/biological surfaces with the hydrogel formed from 4S-PEG and 4T-PEG (iv).See Answer
- Q10: Individual Assignment – Mini Literature Review (700-900 words) (20% of final module mark) This report will be a literature review on a research topic of interest covered in the 'Biomaterials' classes. General guidelines: The purpose of this review is to give a brief overview of the studies performed on the selected topic and to provide a critical evaluation of the literature in a clear and concise manner. It should be an original piece of work. Only peer review journal experimental articles should be included in the review, which should be clearly cited. Students should not use other review papers to form the basis of their review. The overall objective of the assignment is to familiarise students with the scientific literature, understand these articles and critique their content. A good review identifies what information is important and condenses that information for the reader. Better understanding of the subject will make it easier to explain it thoroughly and briefly. A minimum of 8 experimental scientific papers should be used and referenced in the review (no need to submit these papers with your report). This mini-review should be between 700-900 words (excluding references). The review should: be written in your own words; contain the main arguments and critical evaluation of the papers; • be clearly structured, have an introduction and a conclusion; • • indicate where you agree or disagree with the authors. The review should not: use other review papers published in the literature as the basis of this report; • • be produced using cut-and-paste at any stage; . contain any sentences (or phrases more than a few words long) occurring in the original paper (quoted or not). Provide all references used at the end of your report. Use an appropriate referencing style, for example the ACS style (link given below) or a different one: https://pubs.acs.org/doi/full/10.1021/acsguide.40303See Answer
- Q11: Research Methodology Spring 2023- 2024 Assignment on "Materials and Methods" Let's examine the "Materials and Methods" sections of the published papers that you chose. How are they similar and different?? You need to select two different papers addressing similar problems. Select one common experiment that was done in both papers, and compare between the two papers on the similarities and differences of this experiment using the below form. 13 > - + ** Paper 1 Title: Link: Materials and Methods of the experiment you selected: Paper 2 Title: Link: Materials and Methods: Materials and Methods of the experiment you selected: Table 1: Differences in materials and methods of your selected experiment between Paper 1 and Paper 2. Paper 1 Paper 2 Table 2: Similarities in materials and methodology of your selected experiment between Paper 1 and Paper 2. + + Your feedback/ opinionSee Answer
- Q12:11:53 < CHE3008 Individual Assig... Q L 5G 62 Individual Assignment - Mini Literature Review (700-900 words) (20% of final module mark) This report will be a literature review on a research topic of interest covered in the 'Biomaterials' classes. Suomasion dreadme General guidelines: The purpose of this review is to give a brief overview of the studies performed on the selected topic and to provide a critical evaluation of the literature in a clear and concise manner. It should be an original piece of work. Only peer review journal experimental articles should be included in the review, which should be clearly cited. Students should not use other review papers to form the basis of their review. The overall objective of the assignment is to familiarise students with the scientific literature, understand these articles and critique their content. A good review identifies what information is important and condenses that information for the reader. Better understanding of the subject will make it easier to explain it thoroughly and briefly. A minimum of 8 experimental scientific papers should be used and referenced in the review (no need to submit these papers with your report). This mini-review should be between 700-900 words (excluding references). The review should: ⚫be written in your own words; ⚫be clearly structured, have an introduction and a conclusion; contain the main arguments and critical evaluation of the papers; ⚫ indicate where you agree or disagree with the authors. The review should not. ⚫use other review papers published in the literature as the basis of this report; be produced using cut-and-paste at any stage; ⚫contain any sentences (or phrases more than a few words long) occurring in the original paper (quoted or not). Provide all references used at the end of your report. Use an appropriate referencing style, for example the ACS style (link given below) or a different one: https://pubs.acs.org/doi/full/10.1021/acsguide.40303 2 35 Dashboard Calendar To-do Notifications InboxSee Answer
- Q13: Dovepress INTERNATIONAL JOURNAL OF NANOMEDICINE OPEN ACCESS International Journal of Nanomedicine Growth Factor and Its Polymer Scaffold-Based Delivery System for Cartilage Tissue Engineering a ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/dijn20 Li Chen, Jiaxin Liu, Ming Guan, Tongqing Zhou, Xin Duan & Zhou Xiang To cite this article: Li Chen, Jiaxin Liu, Ming Guan, Tongqing Zhou, Xin Duan & Zhou Xiang (2020) Growth Factor and Its Polymer Scaffold-Based Delivery System for Cartilage Tissue Engineering, International Journal of Nanomedicine,, 6097-6111, DOI: 10.2147/IJN.S249829 To link to this article: https://doi.org/10.2147/IJN.S249829 CrossMark © 2020 Chen et al. Published online: 14 Aug 2020. bmit your article to this journal Article views: 433 QView related articles View Crossmark data Citing articles: 61 View citing articles Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=dijn20 Taylor & Francis Taylor & Francis Group International Journal of Nanomedicine Open Access Full Text Article Growth Factor and Its Polymer Scaffold-Based Delivery System for Cartilage Tissue Engineering 1,2,* Li Chen Jiaxin Liu¹›* Ming Guan 2,3 Tongqing Zhou² Xin Duan' I Zhou Xiang 'Department of Orthopedics, National Clinical Research Center for Geriatrics, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, People's Republic of China; 2School of Dentistry, University of Michigan, Ann Arbor, MI, 48109, USA; ³Department of Orthopedics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, People's Republic of China *These authors contributed equally to this work Correspondence: Xin Duan Tel +86-28-85422426 Email dxbaal@hotmail.com Zhou Xiang Tel +86-28-85422605 Email xiangzhou15@hotmail.com submit your manuscript | www.dovepress.com DovePress in http://doi.org/10.2147/1JN.S249829 This article was published in the following Dove Press journal: International Journal of Nanomedicine Dovepress open access to scientific and medical research REVIEW Abstract: The development of biomaterials, stem cells and bioactive factors has led to cartilage tissue engineering becoming a promising tactic to repair cartilage defects. Various polymer three-dimensional scaffolds that provide an extracellular matrix (ECM) mimicking environment play an important role in promoting cartilage regeneration. In addition, numer- ous growth factors have been found in the regenerative process. However, it has been elucidated that the uncontrolled delivery of these factors cannot fully exert regenerative potential and can also elicit undesired side effects. Considering the complexity of the ECM, neither scaffolds nor growth factors can independently obtain successful outcomes in carti- lage tissue engineering. Therefore, collectively, an appropriate combination of growth factors and scaffolds have great potential to promote cartilage repair effectively; this approach has become an area of considerable interest in recent investigations. Of late, an increasing trend was observed in cartilage tissue engineering towards this combination to develop a controlled delivery system that provides adequate physical support for neo-cartilage formation and also enables spatiotemporally delivery of growth factors to precisely and fully exert their chon- drogenic potential. This review will discuss the role of polymer scaffolds and various growth factors involved in cartilage tissue engineering. Several growth factor delivery strategies based on the polymer scaffolds will also be discussed, with examples from recent studies highlighting the importance of spatiotemporal strategies for the controlled delivery of single or multiple growth factors in cartilage tissue engineering applications. Keywords: polymer scaffold, growth factor, delivery, cartilage repair Introduction Articular cartilage is a specific type of connective tissue that covers the articular surfaces of the bone; it is mainly composed of a dense extracellular matrix (ECM) and a sparse cell population. It plays an essential role in the biomechanical functions of the joints, including shock absorption, sheer resistance and load bearing.² Once articular cartilage is damaged, it has a limited potential for sponta- neous repair due to the lack of vascularity, nerves and lymphatics. This can result in joint pain, swelling, dysfunction, and eventually lead to osteoarthritis (OA).³,4 In the past two decades, OA has been the most common form of arthritis accounting for approximately 300 million patients worldwide and undoubtedly has been considered as one of the most significant health problems that also pose a substantial financial burden on the public health system, and the patients themselves. Currently, conservative treatments, including pharmacological and non-pharmacological therapies, are commonly applied to improve joint pain, reduce stiffness and improve physical function in patients with OA. However, the therapies сс International Journal of Nanomedicine 2020:15 6097-6111 02 Ⓒ2020 Chen et al. This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms.php NC and incorporate the Creative Commons Attribution - Non Commercial (unported, v3.0) License (http://creativecommons.org/licenses/by-nc/3.0/). By accessing the work you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms (https://www.dovepress.com/terms.php). 6097 Chen et al 7-10 cannot prevent further joint degeneration. Surgical strate- gies are also designed to treat cartilage defects, including autologous chondrocyte implantation (ACI), microfrac- ture, osteochondral grafts and total joint arthroplasty. However, they have some inherent shortcomings such as the requirement of secondary surgery, immunogenic responses, shortage of donor tissues and pathogen trans- mission risks. Alternatively, cartilage tissue engineer- ing, which involves combining cells, scaffolds and growth factors, has emerged as a promising strategy for cartilage repair.¹¹ Meanwhile, the methods of minimally invasive surgery, including the implantation and injection, are an important component of clinical translation of tissue engi- neering techniques which have been verified in vitro. ¹2 In general, scaffolds as biologically active ECM provide mechanical support for cell growth and chondrogenic dif- ferentiation, which could be beneficial for stimulating and accelerating the cartilage regeneration process. With the development of chemistry and processing, numerous synthesized and natural materials have been applied to fabricate scaffolds that successfully promote the cartilage regeneration without noticeable signs of immune response and rejection. ¹3-15 12 16,17 While biomimetic three-dimensional scaffolds have been made, they cannot create high-quality cartilage tissue independently. Stem cells, pluripotent cells and native progenitor cells are commonly used in combination with scaffolds to accelerate and improve the regeneration process. Moreover, cell-based therapies are influenced by the cellular microenvironment to some extent. Growth factors are of high importance as they have the potency to induce and enhance cellular responses, which is beneficial for the cells as they need to differentiate into desired lineages.¹8 Although scaffolds can obtain sufficient growth factors from the culture medium under in vitro conditions, the incorporated growth factors can spread out of the scaffolds and degrade in a short time in vivo. Besides, different dosages and delivery rates are required for dif- ferent growth factors to induce the cells in in vitro or in vivo conditions. Today, a plethora of studies have been conducted to investigate the delivery of single or multiple growth factors from the scaffolds in a defined 18 19,20 manner. This review examined the delivery of growth factors for cartilage tissue engineering, with an emphasis on the polymer scaffold-based approaches. First, the aim is to enable an understanding of current applications of polymer scaffolds, following with the descriptions of different 6098 submit your manuscript | www.dovepress.com DovePress Dovepress growth factors involved in cartilage tissue engineering. A latter section will place a particular emphasis on the growth factor delivery strategies associated with polymer scaffolds. Finally, the current challenges and suggestions of polymer scaffold-based growth factor delivery for car- tilage tissue engineering are explained. Polymer Scaffolds Articular cartilage, with its unique mechanical properties pro- vides the contact surfaces for load transfer between bones, which enables the joint to withstand weight-bearing. The ability to do so is attributed to its complex structure comprised of a fluid phase and a solid matrix that is composed mainly of a depth-dependent collagen fibrous network and proteogly- cans, as well as other types of proteins, lipids, and cells. Therefore, the scaffold suitable for cartilage tissue engineering should have good biocompatibility for cell adhesion, migra- tion and proliferation, and also provide appropriate mechanical and structural support. In addition, biodegradability and being free of adverse reactions are basic properties required for a three-dimensional scaffold mimicking physiological characteristics.2¹ Currently, a wide range of natural and syn- thetic polymers play an important role in the development of scaffolds for cartilage tissue engineering. Due to superior biocompatibility and biodegradation, natural polymers like collagen, chitosan, silk fibroin, alginate, hyaluronic acid and chondroitin sulfate are suitable for initiating a fast regeneration process. However, potential pathogen transmission, immuno- genicity and poor mechanical properties limit their clinical application. On the other hand; synthesized polymers can artificially regulate the degree of polymerization, thereby con- trolling its mechanical properties, internal structure and degra- dation, which can effectively promote the regeneration process. Poly (lactic acid) (PLA), poly glycolic acid (PGA), poly lactide-co-glycolic acid (PLGA) and poly caprolactone (PCL) are the most commonly synthesized polymers in the application of three-dimensional scaffolds for cartilage tissue engineering.23 When comparing these to natural polymers, the properties of synthetic polymer-based scaffolds are consider- ably different in terms of their tunable properties, such as molecular weight, transition temperatures and crystallinity.24 Polymer nanofibers have been extensively studied due to their ability to encapsulate and deliver growth factors for different tissue regeneration purposes. Nanofiber scaf- folds with high surface to volume ratio and interconnected porous structure, seem to hold the lead position as the ideal candidate for cartilage tissue engineering. They play a role in stimulating the ECM environment, allowing 21 International Journal of Nanomedicine 2020:15 Dovepress cells to populate empty spaces and organize themselves, and mechanical stimulation can be applied to this porous structure to orient the cells and maintain a chondrocyte phenotype. As a result, scaffolds will be degraded and replaced by newly formed ECM, without producing adverse effects due to the degradation products. To date, various technologies such as electrospinning, phase separation, self-assembly, drawing and template synthesis have been applied in attempts to optimize nanofiber scaf- folds to make them more consistently bioactive and mechanically stable for effective tissue regeneration application. For example; a nanofibrous scaffold was developed that was highly porous, interconnected and degradable. It was developed using phase separation of poly l-lactic acid (PLLA) solutions combined with poro- gen leaching techniques. Through a series of characteristic tests, chondrogenic evaluations in vitro and in vivo demonstrated that this nanofibrous PLLA scaffold is an excellent candidate providing an advantageous three- dimensional microenvironment for a wide variety of car- tilage repair strategies (Figure 1).² 27-29 Growth Factors 30 Growth factors are a group of peptides that mediate cel- lular proliferation, migration and differentiation by bind- ing to transmembrane receptors located on target cells. When a sufficient number of receptors are activated, the signaling transduction process may trigger a series of specific cellular activities.³0 Concerning cartilage develop- ment, growth factors play an essential role in regulating the processes of chondrogenesis and hypertrophy, such as the members of transforming growth factor-ß (TGF-B) superfamily, insulin-like growth factor-1 (IGF-1), fibro- blast growth factor (FGF) family and platelet-derived growth factor (PDGF). In order to provide a better under- standing of their potential, descriptions of their roles involved in the regeneration and maintenance of articular cartilage will now be described (Table 1). Transforming Growth Factor-B Superfamily The TGF-ß superfamily is comprised of more than 30 closely related polypeptides, mainly including typical TGF-ßs, bone morphogenetic proteins (BMPs), growth and differentiation factors (GDFs) and activin/inhibin, which regulate multiple cell functions from early develop- ment to regulating homeostasis throughout adult life.³1 31 International Journal of Nanomedicine 2020:15 Chen et al A large number of studies have shown that they have significant regulatory effects on the homeostasis and repair of articular cartilage. Transforming Growth Factor-B TGF-B is a dimer with a molecular weight of 25 kilo Daltons (kDa) that is composed of two identical or similar chains. There are three isoforms (1-3) that are generally considered to be potent stimulators in all stages of chon- drogenesis with a function of inducing proteoglycans and type II collagen synthesis. ³2 TGF-ß signaling transduction is based on the membrane-bound heteromeric receptors (type I and type II). Binding to type II receptors leads to the phosphorylation of type I receptors, causing the phos- phorylation of TGF-B specific Smad proteins, particularly Smad 2 and 3. In addition, some Smad-independent pathways, including p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (Erk) and stress-activated protein kinase/c-Jun NH(2)-terminal kinase (SAPK/JNK) can also be activated by TGF-B.³4 33 34 35 TGF-B is one of the main initiators of chondrogenesis of mesenchymal precursor cells, and the differentiation of mesenchymal stem cells (MSC) into chondrocytes also requires its stimulation. The expression of N-cadherin was induced by strong stimulation of TGF-ß to enhance cell adhesion and aggregation, and subsequently promote cell proliferation, differentiation and deposition of the cartilage- specific extracellular matrix.³5 Among these three isoforms, TGF-B1 was the first to be discovered, and TGF-ß1 and TGF- 33 have been used in a large number of studies to explore the effect of TGF-B on the repair of cartilage after it defects. Although some studies suggest that the ability of TGF-32 and TGF-33 to promote cartilage differentiation may be more superior to that of TGF-31, there is a consensus that there is no significant difference among the three TGF-B isoforms regarding their ability to promote cartilage differentiation. 36,37 In a Sprague-Dawley rat full-thickness cartilage defect model, Lentivirus-TGF-ß1-EGFP transduced BMSCs/calcium alginate gel significantly improved the amount of glycosaminoglycan (GAG) and type II collagen in the defect area in the early stage via activating the Smad pathway, when compared to a BMSCs/calcium alginate gel without TGF-31 transfection. Hypertrophy markers gene expression of chondrocytes were also inhibited by increasing Yes-associated protein-1 (YAP-1).38 Additionally, TGF- 31-incorporated collagen vitrigel had a better effect on mana- ging the early pain mitigation and osteochondral defect repair compared to collagen vitrigel alone.39 Moreover, BMSC submit your manuscript | www.dovepress.com DovePress 6099 Chen et al A Acc. V Magn 10.0kV 100x C 6100 E 200m were induced for the first 4 days with transient soluble TGF- B1, in which the accumulation of proteoglycans was 10-fold higher than TGF-B1-free culture after 3 weeks. These results suggest that TGF-ß promotes chondrogenic differentiation mainly depends on the extent of stimulation of the first week. 40 Nevertheless, there are still some studies that do not support the role of TGF-ß in cartilage repair in vivo. In a rabbit osteochondral defect model, oligo polyethene glycol B D submit your manuscript | www.dovepress.com DovePress F Figure I Nanofibrous PLLA scaffolds induce cartilage regeneration in vitro and in vivo. (A) SEM micrographs of nanofibrous PLLA scaffolds with macro-porous structures (Scale bar: 200 µm). (B) SEM micrographs of the nanofibrous microstructure of the pore walls at a higher magnification (Scale bar: 10 µm). (C) H&E staining showed that BMSCs grew throughout the whole scaffolds after 4 weeks in vitro chondrogenic culture on nanofibrous PLLA scaffold (Scale bar: 200um). (D) Alcian blue staining showed a dense GAG matrix deposition after 4 weeks in vitro chondrogenic culture on nanofibrous PLLA scaffold (Scale bar: 100 µm). (E) H&E staining revealed that BMSCs/ nanofibrous PLLA scaffold constructs had typical cartilage morphology after 8 weeks implanted in nude mice (Scale bar: 200 µm). (F) Safranin-O staining showed that BMSCs/nanofibrous PLLA scaffold constructs were positive for GAG-containing matrix in vivo (Scale bar: 200 µm). 10 mm. Notes: Reprinted from Gupte MJ, Swanson WB, Hu J, et al. Pore size directs bone marrow stromal cell fate and tissue regeneration in nanofibrous macroporous scaffolds by mediating vascularization. Acta Biomater. 2018;82:1-11. Copyright (2018), with permission from Elsevier.27 Dovepress (PEG) fumarate (OPF) hydrogel composites containing gela- tin microparticles (GMPs) loaded with MSCs with or without TGF-B1 did not improve cartilage morphology. Besides, undesirable side effects such as synovial fibrosis, endochon- dral ossification and hypertrophic scars were observed in vivo after a continuous stimulation by TGF-B1.4 Therefore, it is crucial to properly deliver and present TGF- ẞs in vivo for cartilage regeneration. 42-44 International Journal of Nanomedicine 2020:15See Answer
- Q14: Downloaded via UNIV OF SHARJAH on February 25, 2024 at 21:05:23 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. ACS! Biomaterials SCIENCE & ENGINEERING pubs.acs.org/journal/abseba Engineering a Cortisol Sensing Enteric Probiotic Vaughn Litteral,* Rebecca Migliozzi, David Metzger, Craig McPherson, and Roland Saldanha Cite This: ACS Biomater. Sci. Eng. 2023, 9, 5163-5175 Metrics & More ACCESSI ABSTRACT: Chronic stress can lead to prolonged adrenal gland secretion of cortisol, resulting in human ailments such as anxiety, post-traumatic stress disorder, metabolic syndrome, diabetes, immunosuppression, and cardiomyopathy. Real time monitoring of chronic increases in cortisol and intervening therapies to minimize the physiological effects of stress would be beneficial to prevent these endocrine related illnesses. Gut microbiota have shown the ability to secrete, respond, and even regulate endocrine hormones. One such microbe, Clostridium scindens, responds transcriptionally to cortisol. We engineered these cortisol responsive genetic elements from C. scindens into an enteric probiotic, E. coli Nissle 1917, to drive the expression of a fluorescent reporter allowing for the designing, testing, and building of a robust and physiologically relevant novel cortisol probiotic sensor. This smart probiotic was further engineered to be more sensitive and to respond to elevated cortisol by expressing tryptophan decarboxylase, thereby bestowing the ability to generate tryptamine and serotonin. Here we show that upon cortisol treatment the smart probiotic produces measurable amounts of tryptamine. Accumulated levels of these neuromodulators should improve mood, anxiety, and depression and drive down cortisol levels. Importantly, this work can serve as a model for the engineering of a sense-and-respond probiotic to modulate the gut-brain axis. KEYWORDS: engineered probiotic, probiotics, smart probiotic, gut brain axis, Escherichia coli Nissle 1917, EcN, cortisol, glucocorticoid, 5-alpha-tetrahydrocortisol, adrenal, chronic stress, tryptophan, tryptophan decarboxylase, 5-HT, serotonin, trypta lysR transcription factor, bacterial sensor, Clostridium scindens, bacterial targeted gene integration, biosensor, sense and respond circuit INTRODUCTION The prevalence of anxiety, stress, and depression is ever increasing in modern societies. Mitigating the endocrine response in times of chronic anxiety, stress, and depression has historically been a challenge due to the endocrine system's intimate role in physiological homeostasis. Neuman et al.² demonstrated that endogenous microbial sentinels have been shown to both monitor and influence the endocrine system and ultimately human physiology. Probiotics show promise in restoring homeostasis in endocrine and immunological dysregulation. More recently, engineering probiotics as living diagnostics and therapeutics has allowed for a whole cell therapeutic approach with a beneficial metabolic potential that seems unlimited. Decades of research into the interplay between the gastrointestinal tract and the nervous system referred to as the gut brain axis (GBA) has revealed an intricate communication network shown to have a great impact in human health, development, and physiology. The GBA is a bidirectional communication system between the nervous system (central nervous system, autonomic nervous system, and enteric nervous system), and hypothalamus pituitary adrenal (HPA) axis with gut microbiota symbionts. Sudo et al. demonstrated that microbiota were essential for HPA develop- ACS Publications © 2023 The Authors. Published by American Chemical Society Read Online Article Recommendations 5163 T SI Supporting Information E. coli Nissle 1917 Ato/C:lysRtdc Chromosomal DNA lysR tryptophan tdc Received: November 1, 2022 Accepted: April 17, 2023 Published: August 30, 2023 LysR tryptophan decarboxylase (TDC) Article an tryptamine. Special Issue: Design and Evaluation of Engineered Probiotics Cortisol ment in germ free mice. Stressors, such as from the host inflammatory response and environmental factors, activate the HPA through the hippocampus release of CRF (corticotropin releasing factor), which in turn stimulates the pituitary to release ACTH (adrenal corticotropic hormone). ACTH stimulates specific cellular receptors, resulting in adrenal secretion of the stress hormone cortisol. While acute stress can be beneficial, chronic stress stimuli can lead to chronic cortisol secretion with detriments to human health and performance (i.e., anxiety, stress, post-traumatic stress disorder, diabetes, immunosuppression, cardiomyopathy, weight gain, and alcoholism). About 10% of cortisol is in active form and present in the blood circulation, whereas the remainder is bound to albumin or cortisol binding globulin protein. Cortisol and associated glucocorticoid metabolites regulate nearly every cell type in the body through the glucocorticoid 15 TOIC SACS materials https://doi.org/10.1021/acsbiomaterials.2c01300 ACS Biomater. Sci. Eng. 2023, 9, 5163-5175 ACS Biomaterials Science & Engineering 16 and mineralocorticoid receptors acting as seemingly global transcription factors in gene expression. Traditional ther- apeutic interventions into ameliorating the endocrine response is met with many adverse events in off target tissues as cortisol exerts this global but tissue specific gene expression response. ¹7 18-20 Noninvasive monitoring of free cortisol in sweat and saliva has evolved in recent years as an interest in the human performance and health monitoring industry as well as the United States Department of Defense. Cortisol in saliva, sweat and tears is indicative of blood levels and is a predictive biomarker in determining real time psychological stress.ª Gastro-intestinal glucocorticoid levels have been difficult to ascertain, wherein bacterial secondary metabolism of cortisol and resultant desmolysis occurs beginning in the large intestine. Ridlon et al. employed RNA-seq to investigate cortisol metabolism and its regulated gene expression in one such gut bacterium, Clostridium scindens. C. scindens demon- strated robust response in gene expression to the stress hormone, cortisol, and observed side chain cleavage of cortisol resulting in androgen gut metabolites. These studies showed further evidence that the gut microbiota have the innate ability to monitor the host endocrine stress molecule, cortisol, and influence the metabolic fate of these endocrine hormones. 21 23-25 26,27 In addition, it has been increasingly demonstrated that gut microbiota have additional and significant neuroendocrine roles in the GBA through secondary metabolism.²2 For example, the metabolic fate in the gut of ingested tryptophan, Trp, an essential amino acid, results in indoles, serotonin (5- hydroxytryptamine, 5-HT), tryptamines, melatonin, and kynurenine. All of these metabolites have a profound effect on host mood, anxiety, sleep, and/or stress. Trp metabolism is accomplished primarily through the kynurenine pathway (95%) resulting in nicotinamide dinucleotide (NAD +) production, while only minor amounts (1%) are converted to 5-HT. Dysregulation of the kynurenine pathway can lead to aging, various mental and neurodegenerative disorders, and chronic fatigue syndrome." Whereas 5-HT has been researched for more the 70 years, its intimate role in gut physiology is just recently being understood. 28 Cortisol stimulates 5-HT reuptake in peripheral circulation through Serotonin-selective Re-uptake Transporters (SERT) further affecting 5-HT levels.30 Ninety percent of 5-HT production occurs in intestinal enterochromaffin cells (EC), and 5-HT synthesis can be stimulated through 5-HTR4 activation by the trace amine tryptamine, wherein the gut-produced 5-HT is absorbed, stored, and distributed by platelets.³ Five of the seven known classes of serotonergic receptors (5-HT1 thru 5- HT7) are expressed throughout the gut. Activation of 5-HT2a receptor by intestinal 5-HT results in a block of TNFa and subsequent increase in SERT.³ The 5-HT is distributed by platelets throughout the circulation and plays an important role in hemostasis, peristaltic reflex, and gut physiology.³5 While 5- HT does not have the ability to cross the blood brain barrier, Trp does have the ability to cross and therefore directly contributes to central and peripheral 5-HT and melatonin levels. 35,36 Sequestering of Trp through microbial secondary metabolism in the gut leads to decreased Trp in the brain and can greatly affect its bioavailability for neuroendocrine roles. Corynebacterium spp., Streptococcus spp., and Escherichia coli have been shown to synthesize 5-HT in culture. Williams et al. evaluated bacterial tryptophan decarboxylase activity (a rare functionality for gut bacteria). In fact, there is an approximately 10% prevalence in the population as discovered by Fishbach in 31,32 33,34 35 37 29 5164 Article pubs.acs.org/journal/abseba 38 39 analyzing Human Microbiome Project data. Allowing for the tryptophan decarboxylase activity in the gut would result in an increase in tryptamine and subsequent stimulation of 5-HT production from EC cells, as well as biotransformations of 5- hydroxytryptophan directly to 5-HT. In the case of chronic stress, both in animal models and human studies, subjects greatly benefited through decreasing circulating cortisol levels by increasing 5-HT levels with SRIs (Serotonin Reuptake Inhibitors).36,40 41 42 In order to mitigate the debilitating effects of chronic stress, a promising transient strategy herein was conceived to restore normal physiology and function in the gut with a whole cell engineering approach. This study utilizes a well-characterized probiotic chassis that is able to withstand and colonize the harsh gastric intestinal environment. The only commercially available Gram-negative probiotic to date, E. coli Nissle 1917, EcN, was originally isolated from a World War I soldier that was able to surprisingly survive with dysentery.4¹ The EcN strain was also recently shown to increase 5-HT extracellular concentrations in an in vitro gut tissue model." An EcN smart probiotic with elements of Clostridium scindens to sense active glucocorticoids and respond by contributing the rare Trp decarboxylase activity (similar to Clostridium sporogenes) resulted in decarboxylase activity with some substrate promiscuity toward 5-hydroxytryptophan that would result in 5-HT metabolites as well as dietary tryptophan biotransforma- tion to tryptamine. In periods of chronic stress and elevated cortisol production, we predict the engineered probiotic in the gut would increase tryptamine and ultimately intestinal 5-HT production and distribution via induced promiscuous tryptophan decarboxylase activity. The intestinal and periph- erally distributed 5-HT would drive down circulating cortisol levels and likely would improve mood, anxiety, and depression. 43 Importantly, this effort models a stress-sensing and potentially neuro-modulating probiotic in in vitro systems that prove to mimic human physiology. 44 MATERIALS AND METHODS Materials. Restriction, DNA modifying enzymes, and polymerases were from New England Biolabs (Ipswich, MA, United States). Plasmid miniprep and PCR purification kits were from QIAGEN (Germantown, MD, United States). Synthetic DNA and oligonucleo- tides were obtained from Integrated DNA Technologies (IDT; Coralville, IA, United States). Unless otherwise indicated, all other chemicals and reagents were from Sigma (St. Louis, MO, United States) or Thermo Fisher Scientific (Waltham, MA, United States). 52 Bacterial Strains and Culture Conditions. Table S1 lists the E. coli strains used in this study. Figures S9-S11 display the workflow utilized for recombinant strain construction as further detailed by Yang et al. The wild type E. coli strain Nissle 1917, referenced herein as EcN (Mutaflor, DSM 6601, and serotype 06:K5:H1), was kindly supplied by Dr. A. Breedon. BW2511, JW0451-2, and JW5503-1 were obtained from the CGSC (Yale University, Dept. of MCB), E. coli NEBSalpha (cat# C2987H) strains were obtained from NEB. The E. coli strains were routinely maintained in Luria-Bertani (LB) or M9 minimal salts medium with or without 1.5% Bacto Agar (Difco Laboratories, Detroit, Mich., United States). Electrocompetent cells were prepared using Biorad's MicroPulser (BIORAD Hercules, CA) protocol for the preparation of E. coli electrocompetent cells; see Aususbel et al. and Miller and Nickoloff. 56,57 Ampicillin (50 ug/mL), apramycin (50-100 ug/uL), chloramphenicol (30 ug/mL), and kanamycin 25-50 ug/mL) were used for antibiotic selection in this study. Biotek Plate Assay. Mid log E. coli strains were used to inoculate a fresh culture in early log (OD 600 = 0.02). Glucocorticoids https://doi.org/10.1021/acsbiomaterials.2c01300 ACS Biomater. Sci. Eng. 2023, 9, 5163-5175 ACS Biomaterials Science & Engineering dissolved in culture media in 14 mm Falcon tubes (cat# 14-959-11B) with normalized DMSO 0.02-0.2% as a cosolvent and samples exposed to serially diluted glucocorticoid doses or DMSO and respective antibiotic selection. A kinetic plate assay was carried out with black/clear bottom Costar plates (cat# 3601) on a Biotek Neo2 multimodal plate reader. Both the OD 600 and the fluorescent wavelength(s) were monitored. The glucocorticoid dose response was normalized by OD600 and the EC50 was calculated with a nonlinear regression model. Data analysis was performed in Graphpad Prism 9.0. Tryptophan and Tryptamine Extraction. Following induction of tryptamine production, the culture samples were centrifuged at 16 000g for 1 min and then filtered through a 0.2 um filter, then the filtrate was treated with a 2x volume of 4N sodium hydroxide at pH 11. Next, phase extraction was performed with 1:1 with ethyl acetate, followed by mixing and centrifugation at 16 000g for 1 min to allow for phase separation. The resulting aqueous phase contained tryptophan and the organic phase captured tryptamine. Samples were subsequently prepared for HPLC analysis. Cloning and Construct Assembly. The overall cloning strategy used 2-5 DNA fragments produced from either DNA synthesis (gblocks), oligos or high-fidelity polymerase generated amplicons purified though Qiagen PCR or Qiaex gel extraction protocols. Following purification, fragments were assembled with NEB Builder HiFi Gibson Assembly using a total reaction volume of 5-10 µL. 10% of the HiFi reaction was transformed into NEB5a chemical competent cells and plated on selective media. After the plates were incubated for 18-24 h at 37 °C, individual colonies were selected for evaluation Bacterial Plasmid Construction. pLysE2Cr. The E2 crimson fluorescent reporter was designed to be under the control of LysR using the synthesized lysR with its bidirectional promoter (Genbank WP_004606447.1) included in the 250 bp upstream sequence (Figure S1) all cloned into the supplied pIDT bacterial expression vector, pIDT (kan¹), resulting in plasmid pLysRE2Cr. The lysR open reading frame (ORF) was codon optimized for E. coli expression using online in silico analysis tools provided by Integrated DNA Technologies (IDT, Coralville, IA). pLysRsfGFP. This plasmid was a gift from the U.S. Army Lab (Dr. Steve Blum) and was created by swapping the ORF of the sfGFP reporter for that of the E2 crimson reporter in plasmid pLysRE2Cr. pLysRTDC. The pLysRE2Cr plasmid was used as a PCR template for a 900bp fragment of the lysR gene (C. scindens), primed with lysR- F and lysR-R and amplified with Q5 high fidelity polymerase (NEB #M0491S). The tdc-1 (Tryptophan decarboxylase-1 from Oryza sativa) gene was synthesized by IDT as a 1.5kb fragment (see the supplemental sequence list for details) and was likewise used as a PCR template and amplified with Q5 high fidelity polymerase. The lysR and tdc-1 containing fragments were assembled via an NEB Builder HiFi Assembly kit into the XhoI/NotI sites of pAME200 (this plasmid was a gift from Dr. A. Breedon, USAF RHB) resulting in plasmid pLysTDC. PUC19lysRsfGFP-Plasmid. pLysRsfGFP was used as template with oligos to generate a linear fragment both assembled with either 200 bp or 1000bp of flanking tolC homology gblocks (See the supplemental sequence information for gblocks from IDT) assembled into the pUC19 BamH1/EcoR1 vector. PUC19lysRE2Crimson Plasmid. pLysRE2Crimson was used as templates with oligos to generate a linear fragment both assembled with either 200bp or 1000bp of flanking tolC homology gblocks (see supplemental sequence information for gblocks from IDT) assembled into the pUC19 BamH1/EcoR1 vector. PUC19lysRTDC Plasmids. pLysRE2Crimson was used as templates with oligos to generate a linear fragment both assembled with either 200 bp or 1000bp of flanking tolC homology gblocks (See Supplemental Sequence information for gblocks from IDT) assembled into the pUC19 BamH1/EcoR1 vector. Colony PCR. Colony PCR was performed by touching a standard 10 ul pipet tip into the center of the bacteria colony and transferred to the bottom of a 96-well PCR plate well. NEB Quick load 2X Taq PCR 5165 Article pubs.acs.org/journal/abseba master mix was added to each well on a cooling block and once the denaturation temperature of 95 °C was reached the samples were added to the thermocycler. The initial denaturation of the PCR was conducted for 2 min. NEB Quick load 2X Taq polymerase manufacture recommendations were followed. Standard PCR. Standard PCR employed either the NEB Quick load 2X Taq polymerase protocol for routine usage or the NEB HiFi Q5 polymerase protocol for cloning and assembly efforts in this study. Flow Cytometry Analysis. Following growth and glucocorticoid treatment, E. coli strains were diluted 1:40 in phosphate buffered saline (pH 7.4) with 20mM Hoechst 33342 DNA stain (Fisher Cat. No. H21492) and incubated at 37 °C for 10 min. Following Hoechst DNA staining, all cells were stored on ice until flow cytometry data acquisition. Samples were analyzed on a 5-laser BD FACSAria II cell sorter (BD Biosciences, San Jose, CA) with instrument setup performed using Cytometer Setup and Tracking Software (BD Biosciences). Hoechst was excited with a 355 nm laser and detected with a 450/50 filter. GFP was excited with a 488 nm laser and detected with a 525/50 nm filter. E2 crimson was excited with 561 nM laser and detected with a 670/30 nm filter. During acquisition, all parameters were collected in log mode. Data were collected and analyzed using FACSDiva 8.0 software (BD Biosciences). To reduce noise, the bacteria were cultured in sterile-filtered media and diluted in sterile-filtered PBS. Prior to acquisition, the background noise of the instrument was evaluated by analyzing water-only, sheath fluid-only, and PBS-only blank samples. Forward and side scatter gates were set to exclude noise and debris; the bacterial population, identified as Hoechst- positive cells, was further analyzed to identify single cells (FSC-height vs FSC-width, followed by SSC-height vs SSC-width). Positivity gates for the different fluorescent parameters were set after analyzing multiple controls (unstained/untreated culture, unstained/treated culture, stained/ untreated culture). Flow cytometry analyses are in the supplementary. 53 Gene Knockout Generation. Ato/C:CAT Knockout in E. coli Nissle 1917. A knockout strain was constructed using a traditional Red Recombination protocol by Datsenko and Wanner. The chloramphenicol acetyltransferase, CAT, gene in pKD3 was utilized as template to generate a CAT flanking 50 bp tolC homology PCR cassette (See Figure 4) using oligos C1 and C2. Electrocompetent EN1917 cells expressing the red recombinase helper plasmid, pKD46, were transformed with the resultant PCR product targeting the tolC gene. Cells recovered for 1 h and selection was performed on LB chloramphenicol plates (15 ug/mL). AtolC:Ap' Knockout in E. coli Nissle 1917. The apramycin resistance gene, Ap', in pMDIAI was utilized as a PCR template primed with oligos A1 and A2 to generate an Ap¹ PCR fragment with flanking 50 bp homology to tolC. According to a published protocol by Yang et al.,52 electrocompetent EcN cells expressing the pREDTKI plasmid were transformed with the PCR product targeting the tolC gene. Cells were recovered in 1 h and plated on LB apramycin selection plates (50 ug/mL) and individual colonies were selected for further analysis. 52 Targeted Gene Integration. Integration of donor cassettes from lysRsf GFP, lysRE2Crimson, and lysRtdc-1 into the tolC locus was accomplished according to published protocols by Yang et al.5² The intermediate strain AtolC:Ap¹ EcN was used as a recipient strain for targeted integration into the tolC locus. See Figures S11, S12, and S13 for workflow and an example of the screening and validation. Tryptamine Production. Early log (OD600 = 0.02) strains were grown in M9 media with 1% LB media and 2% glucose with 6 mM L- tryptophan and were maintained under selection of chloramphenicol 30ug/mL. Following an OD600 = 0.2, cultures were induced with the stated quantities of cortisol. Cultured samples were collected at the stated times and centrifuged at 16 0000g for 1 min to remove biomass and were subsequently prepared for chemical analysis. 1. RESULTS AND DISCUSSION 2.1. Designing a Novel Cortisol Biosensor. To design a real-time probiotic stress sensor, the bacterial chassis, E. coli https://doi.org/10.1021/acsbiomaterials.2c01300 ACS Biomater. Sci. Eng. 2023, 9, 5163-5175 ACS Biomaterials Science & Engineering 45-47 Nissle 1917 was employed, due to the probiotic's ability to survive the challenging gastrointestinal environment and its reported use as a framework for synthetic biology. The use of a whole cell approach affords the ability to utilize a transcription factor responsive to cortisol that can activate a downstream transcriptional response (i.e., reporter gene or stress-reducing factor). Published RNA-seq data for C. scindens ATCC 35704 demonstrated an effect for its neighboring regulon upon exposure to cortisol (Figure S1).³ LysR-type transcriptional regulators (LTTRs) are the largest prokaryotic class of transcription regulators. In C. scindens, LysR regulates its own transcription as well as its native divergently transcribed gene product. When LysR disassociates from its own promoter, lysR transcription is active, whereas when bound to the coinducer (i.e., cortisol), promoter activation results in transcription of the divergently located gene (Figure 1). Based on this design, the lysR nucleotide sequence was LysR lysR LysR LysR I Co LysR Co lysR Figure 1. Schematic of the LysR regulation of transcription in Clostridium sp. placed under native control elements for expression in E. coli K-12 and EcN strains and evaluated activation of various LysR responsive reporter genes upon cortisol induction. 2.2. Employing a Novel Cortisol Biological Recog- nition Element, BRE. In Clostridium sp. Ridlon et al. demonstrated that the lysR and its divergently transcribed gene product is transcriptionally active in the presence of cortisol, based on RNA-seq data analysis.³ Preliminary analysis was performed in order to determine if the lysR operon had the ability to function as a glucocorticoid transcriptional driven sensor, by employing the Clostridium scindens lysR native control elements driving the bidirectional expression of the E2 Crimson fluorescent reporter protein. The E2 Crimson fluorescent protein has an excitation maximum at 611 nm and emission maximum at 646 nm, is nontoxic to bacteria and performs well with live animal imaging.* The pLysE2Cr plasmid bears the kanamycin resistance gene and the pMB1 origin of replication. Chemical transformed E. coli K-12 and EcN bearing the plasmid pLysE2Cr following mid log growth were evaluated for their dose response to cortisol by measuring fluorescence with a plate reader and flow cytometry. In Figure 2, the schematic demonstrates that the LysR negatively regulates its own transcription (gold) and positively regulates the divergent reporter gene (green) with putative LysR responsive promoters (blue). Exposing the EcN probiotic bearing the pLysE2Cr to varying doses of cortisol (coinducer in Figure 2) at mid log demonstrated a definitive dose response 48 5166 pubs.acs.org/journal/abseba curve with an EC50 of 158 µM and is supra-physiological, whereas the normal human physiological cortisol concen- trations in plasma observe a diurnal variation and range between 80 and 700 nM, and the water solubility of cortisol is 772 μM. Multiparametric flow cytometry analysis was used in this study, since it affords reproducible and accurate functioning at the single cell level and fluorescent reporter proteins afford accurate representations of isogenic cultures. In the EcN wild type strain bearing the pLysE2Cr plasmid, E2 Crimson fluorescent protein expression was evaluated via flow cytometry following incubation with varying concentrations of cortisol. In Figure 2, the top panel/density plots, show a dose- dependent shift of the cell population into the E2 Crimson positive gate, indicating an increased percentage of cells expressing E2 Crimson in response to cortisol. In Figure 2, the bottom panel histograms show a dose-dependent increase in the population's median fluorescence intensity with increasing cortisol concentration. Evaluation of additional reporters such as sfGFP and iLuX (Figure S3) gave similar results. 2.3. Cortisol BRE Selectivity for a-THF, an Active Cortisol Metabolite. In an effort to determine the glucocorticoid specificity of the sensor, varying concentrations of cortisol and its associated metabolites were evaluated on early growth phase EcN bearing the engineered pLysE2Cr plasmid. Both cortisol and its active metabolite 5-alpha- tetrahydrocortisol (THF) were able to transactivate LysR and drive E2Cr reporter expression in similar percentage of the cell population (73% and 59%, respectively) as demonstrated in the density and histogram plot in Figure 3B. Furthermore, cortisol and THF demonstrated robust dose responses with similar EC50 values (213 µM and 150 μM respectively, Figure 3C). The inactive cortisol metabolite of cortisone, 5-beta- tetrahydrocortisone (THE), was unable to initiate the E2Cr reporter in a dose dependent fashion (Figure S4), further indicating the selectivity of the pLysE2Cr sensor for active glucocorticoids. Article 49 The lumen of the gut is a harsh environment with additional cortisol analogs present, such as bile salts. Bile salts are amphipathic steroid molecules found exclusively in the G.I. tract and bile salts are critical for the absorption of dietary lipophilic foodstuffs in the lumen of the small intestine because of their capacity to form micelles spontaneously in a concentration dependent manner. Deoxycholate (DOC), a secondary bile salt (typically present at 20 µM to 100 µM in the small intestines) was investigated to see if DOC would elicit a response with the LysR sensor. DOC afforded a dose response (Figure S5) with an EC50 of 1.5 mM, although these supra-physiological (>1 mM) quantities of DOC proved deleterious to ECN growth (Figure S5).5⁰ 2.4. Targeted Ablation of Glucocorticoid Efflux in E. coli Nissle 1917 to Improve Glucocorticoid Sensor Sensitivity. Although the LysR sensor is able to selectively recognize cortisol (Figure 3), the limit of cortisol detection by the LysR sensor in the wild type EcN host is not physiological relevant (EC50 = 158 µM) and requires further improvements. Available acrAB and tolC mutants (Keio collection mutants JW0451 and JW5503) generated in the wild type parental strain E. coli BW25113 were tested for cortisol sensitivity. Both acrAB and tolC mutants demonstrated a 2-log improvement in cortisol sensitivity (Figures S6 and S7). Following this, the tolC locus was ablated in EcN by directing the chloramphenicol acetyltransferase gene (CAT) gene to the tolC locus using Red homologous recombination (data not shown). PCR- https://doi.org/10.1021/acsbiomaterials.2c01300 ACS Biomater. Sci. Eng. 2023, 9, 5163-5175 ACS Biomaterials Science & Engineering A. B. C. 530/30 Blue-A 10² 10³ 104 D. LysR family TF Regulator -57 plysE2 Crimson lysR ΟμΜ Singlets 0 10 -81 %E2Cr+, Normalized Response 10 0 10 10 105 E2Crimson 670/30 YG-A Singlets 0% E2Crimson+ E2Crimson MdFI 10⁹ 10 10° E2Crimson 670/30 YG-A 100 50 10⁰ 530/30 Blue-A -65 -65 10² 62.5 μΜ Singlets 0 10² 10 Singlets pubs.acs.org/journal/abseba 10 10 E2Crimson 670/30 YG-A verified AtolC:CAT ECN mutants (data not shown) were transformed with the pLysE2Cr plasmid (Figure 4A) and 10 0.6% E2Crimson MdFI 0 10² 10ª 105 E2Crimson 670/30 YG-A E2Crimson+ 5167 reporter/response E2 Crimson 530/30 Blue-A 10² 10³ 104 105 106 Count 500 1.000 1.500 2,000 46 pMB1 ori 0 125 μM Singlets TIMME TIITING. ITIINI 10 10ª 10$ E2Crimson 670/30 YG-A 28% E2Crimson+ Singlets E2Crimson MdFI 10⁰ E2Crimson 670/30 YG-A 10² 10ª 10⁰ 158μM = EC50 104 [Cortisol nM] Figure 2. A novel cortisol responsive transcription factor drives reporter expression. (A) Schematic of the LysR operational unit, where LysR negatively regulates its own promoter and lysR (gold) and drives the expression of the reporter or response gene (green). (B) Plasmid map of pLysE2 Crimson. (C) Flow cytometric analysis of pLysE2 Crimson in EcN with 0, 62.5, and 125 uM of cortisol. Top row, density plots: the gates are indicative of positive E2 Crimson expression. Bottom row, histograms: the interval gates measure the MdFI, median fluorescence intensity. (D) Cortisol dose response curve (EC50 = 158 uM) of pLys E2Crimson in wild type EcN. Article evaluated in mid log cultures for a cortisol dose response (Figure 4C). The AtolC:CAT mutation in EcN enhanced https://doi.org/10.1021/acsbiomaterials.2c01300 ACS Biomater. Sci. Eng. 2023, 9, 5163-5175/nSee Answer
- Q15: Review Films for Wound Healing Fabricated Using a Solvent Casting Technique pharmaceutics Fabiola V. Borbolla-Jiménez ¹,2D, Sheila I. Peña-Corona Emiliano Pineda-Pérez ¹,2D, Alejandra Romero-Montero María Luisa Del Prado-Audelo ², Sergio Alberto Bernal-Chávez 4, Jonathan J. Magaña ¹,²,* and Gerardo Leyva-Gómez ³,*D I Citation: Borbolla-Jiménez, F.V.; Peña-Corona, S.I.; Farah, S.J.; Jiménez-Valdés, M.T.; Pineda-Pérez, E.; Romero-Montero, A.; Del Prado-Audelo, M.L.; Bernal-Chávez, S.A.; Magaña, J.J.; Leyva-Gómez, G. Films for Wound Healing Fabricated Using a Solvent Casting Technique. Pharmaceutics 2023, 15, 1914. https://doi.org/10.3390/ pharmaceutics15071914 check for updates Academic Editors: Giyoong Tae, Martin Federico Desimone and Gorka Orive CC Received: 18 May 2023 Revised: 10 June 2023 Accepted: 27 June 2023 Published: 9 July 2023 4.0/). BY Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 1 2 3 4 * 3 D, Sonia J. Farah ¹,2, María Teresa Jiménez-Valdés ¹,2, 3 Laboratorio de Medicina Genómica, Departamento de Genómica, Instituto Nacional de Rehabilitación Luis Guillermo Ibarra Ibarra, Ciudad de México 14389, Mexico; fvbj@hotmail.com (F.V.B.-J.); soniafarah1d@gmail.com (S.J.F.); teresajimenez00@gmail.com (M.T.J.-V.); emilianopineda_perez@hotmail.com (E.P.-P.) Tecnologico de Monterrey, Campus Ciudad de México, Ciudad de México 14380, Mexico; luisa.delprado@tec.mx Departamento de Farmacia, Facultad de Química, Universidad Nacional Autónoma de México, Ciudad de México 04510, Mexico; sheilairaispc@gmail.com (S.I.P.-C.); alejandra.romero.montero@outlook.com (A.R.-M.) MDPI Departamento de Ciencias Químico-Biológicas, Universidad de las Américas Puebla, Ex-Hda. de Sta. Catarina Mártir, Cholula 72820, Puebla, Mexico; q901108@hotmail.com Correspondence: magana.jj@tec.mx (J.J.M.); leyva@quimica.unam.mx (G.L.-G.) Abstract: Wound healing is a complex process that involves restoring the structure of damaged tissues through four phases: hemostasis, inflammation, proliferation, and remodeling. Wound dressings are the most common treatment used to cover wounds, reduce infection risk and the loss of physiological fluids, and enhance wound healing. Despite there being several types of wound dressings based on different materials and fabricated through various techniques, polymeric films have been widely employed due to their biocompatibility and low immunogenicity. Furthermore, they are non-invasive, easy to apply, allow gas exchange, and can be transparent. Among different methods for designing polymeric films, solvent casting represents a reliable, preferable, and highly used technique due to its easygoing and relatively low-cost procedure compared to sophisticated methods such as spin coating, microfluidic spinning, or 3D printing. Therefore, this review focuses on the polymeric dressings obtained using this technique, emphasizing the critical manufacturing factors related to pharmaceuticals, specifically discussing the formulation variables necessary to create wound dressings that demonstrate effective performance. Keywords: skin; wound; wound healing; wound dressings; polymers; films; solvent casting 1. Introduction A wound is a disruption of the continuity of body tissue caused by physical or chemical damage. Usually, a wound is disinfected using a typical medical procedure, and antibiotic treatment is initiated to start the healing process [1]. However, a possible lack of response to antibiotics or an uncontrolled inflammatory phase can trigger the generation of chronic or infected wounds, which represents a clinical challenge [2]. In this sense, developing minimally-invasive smart dressings that integrate drug release with different therapeutic targets (such as antibiotics, anti-inflammatory agents, and analgesics) represents a desirable alternative [3,4]. Based on understanding the physiological process of wound healing, the ideal dress- ing should be biocompatible, acting as a physical barrier against microorganisms while allowing gas permeation to keep the wound hydrated and remove excess exudate [5,6]. Additionally, desirable properties include good mechanical strength and flexibility. Non- toxicity, biocompatibility, and biodegradability are also important criteria for materials used Pharmaceutics 2023, 15, 1914. https://doi.org/10.3390/pharmaceutics15071914 https://www.mdpi.com/journal/pharmaceutics Pharmaceutics 2023, 15, 1914 in dressings [7]. Hydrogels, polymer films, foams, gauzes, and hydrocolloids are among the most extensively studied dressings, depending on the wound type and therapeutic needs [8,9]. Films serve as convenient physical barriers to bacteria, maintain gas permeability, and enable in situ drug release. Furthermore, their flexibility can be tailored to accommodate individual morphology [10]. Consequently, research focuses on utilizing film dressings as drug carriers to control infections and inflammatory processes [11]. By providing a moist environment, removing wound exudates, and accelerating cellular and tissue regeneration, dressings can maintain optimal conditions for wound repair [12]. However, it is important to acknowledge the limitations of these products. For example, they encounter difficulties in simultaneous application to both external and internal wounds. Additionally, their application can exert external pressure, leading to secondary injuries [13]. This study aims to analyze the solvent casting procedure, the most widely used method for film production. Solvent casting is preferred over other methods, such as salt leaching, spin coating, microfluidic spinning, and 3D printing, due to its cost-effectiveness, simplicity, practicality, and ability to generate robust films with appropriate mechanical properties and homogeneity. However, the properties of materials obtained through solvent casting can vary significantly between production batches, influenced by environmental conditions, which can also slow the production process. Moreover, maintaining sterility throughout all manufacturing steps poses a challenge and can result in batch contamination. Thus, this study comprehensively reviews the objectives of developing polymer films and highlights the key considerations to ensure optimal final properties during the solvent casting process. Despite the method's considerable potential for industrial scaling and clinical application, there is a noticeable lack of standardized commercial products [7]. Therefore, this review identifies areas for improvement and underscores the main advan- tages of utilizing this type of dressing in wound treatment. 2. Wound Healing The wound healing process is a natural physiological reaction to tissue injury that consists of four highly integrated phases: hemostasis, inflammation, proliferation, and remodeling (Figure 1). These phases must occur in a specific sequence, at a specific time, and for a particular duration, in order that the physiologically involved functions fulfill their expected role [14]. Otherwise, this leads to improper or impaired tissue repair [15]. Epidermis Dermis Fibrin clot Erythrocyte- Blood_ vessel 1) Hemostasis Proliferating fibroblasts Epithelial cells monolayer New blood vessels -Injury 3) Proliferation Dobago -Platelet -Eschar Type III Collagen fibers Granulation tissue (2) Inflammation Necrotic tissue or bacterias Macrophage- Neutrophil- Monocyte 2 of 27 4) Remodeling Scar tissue Fibroblast. Type I Collagen fibers Figure 1. Four significant phases represent the wound healing process: (1) hemostasis, the formation of a platelet seal that prevents blood loss and a fibrin clot; (2) inflammation, where neutrophils and macrophages remove debris and prevent infection; (3) proliferation, where blood vessels reform Pharmaceutics 2023, 15, 1914 3 of 27 through angiogenesis, and fibroblasts replace the fibrin clot with granulation tissue; (4) remodeling, where the matrix is remodeled replacing type III collagen with type I, maturing to a scar. 2.1. Hemostasis The hemostasis phase begins minutes to hours after an injury through a cascade of serine protease activation, resulting in platelet activation. This activation also facili- tates the release of growth factors, such as PDGF (platelet-derived growth factor), VEGF (vascular endothelial growth factor), and TGF-α (transforming growth factor x), as well as immune mediators, contributing to the transition into the inflammatory phase [16]. PDGF and TGF-ß (transforming growth factor ß) recruit neutrophils and monocytes to initiate the inflammatory response [17]. Furthermore, this leads to the formation of a fibrin clot, referred to as an eschar, which acts as a plug for the wound, preventing blood loss, providing a scaffold for incoming immune cells, serving as a reservoir for cytokines and growth factors during the early stages of repair, and offering protection against bacterial invasion [15,16,18]. The extrinsic clotting cascade is initiated upon tissue damage and blood leakage, releasing molecules such as serotonin that induce localized vasoconstriction [19]. Sub- sequently, platelets aggregate and become activated upon contact with subendothelial collagen, forming a hemostatic plug. This plug mitigates hemorrhage and serves as a provisional matrix for cell migration by releasing scaffold proteins such as fibronectin, vitronectin, and thrombospondins, facilitating the migration of keratinocytes, immune cells, and fibroblasts [16,20]. Once a fibrin clot is formed, the coagulation process is switched off to prevent excessive thrombosis [17]. 2.2. Inflammation The inflammatory phase, which overlaps with hemostasis, occurs during the first 72 h [16]. During this phase, both humoral and cellular inflammatory responses are activated to establish an immune barrier against invading microorganisms [21]. Neu- trophils and monocytes infiltrate the wound bed to prevent further damage and eliminate pathogenic organisms and foreign debris [16,22]. The inflammation phase can be divided into early and late inflammatory phases. In the early phase, the complement cascade is activated, leading to the infiltration of neutrophils into the wound. Their primary role is to phagocytose bacteria, foreign particles, and damaged tissue [21,23]. Various chemoattractive agents attract neutrophils to the wound site, including TGF-ß, complement components such as C3a and C5a, and formyl- methionyl peptides produced by bacteria and platelet products [16,19]. During their activity, neutrophils release proteolytic enzymes, oxygen-derived free radical species, and inflammatory mediators such as TNF-α and interleukin (IL)-1 [17,21]. Once their role is fulfilled, neutrophils undergo apoptosis and are cleared from the wound typically within 2-3 days, making way for the influx of monocytes [18]. Monocytes, stimulated by cytokines, chemokines, growth factors, and soluble frag- ments of the extracellular matrix (ECM), differentiate into activated macrophages. These macrophages patrol the wound area, ingesting and killing bacteria and removing devi- talized tissue through the action of secreted matrix metalloproteinase and elastase [15]. Additionally, macrophages play a crucial role in transitioning to the proliferative phase by releasing various growth factors and cytokines, including PDGF, TGF-α, TGF-ß, insulin-like growth factor-1 (IGF-1), fibroblast growth factor (FGF), tumor necrosis factor-a (TNF-α), IL-1, and IL-6. These soluble mediators promote cell proliferation and the synthesis of ECM molecules, and activate fibroblasts for subsequent phases [19,24]. A decrease in macrophage presence within the wound indicates the inflammatory phase's conclusion and the proliferative phase's initiation [15]. Pharmaceutics 2023, 15, 1914 4 of 27 2.3. Proliferation The proliferative phase of wound healing involves several interconnected processes that restore tissue structure and function. It begins on the third day after the injury and lasts approximately two weeks [21]. One of the critical aspects of this phase is the replacement of the provisional fibrin matrix with a new matrix composed of collagen fibers, fibronectins, and proteoglycans, which are synthesized by fibroblasts. During this phase, angiogenesis, the generation of granulation tissue, collagen deposi- tion, re-epithelialization, and wound contraction occur [16,25]. Angiogenesis is stimulated by local conditions such as low oxygen tension, low pH, and high lactate levels. It involves the migration and proliferation of endothelial cells to form new blood vessels, which is cru- cial for tissue viability [17]. Macrophages are vital in promoting angiogenesis by producing VEGF [16]. Granulation tissue formation occurs concurrently with angiogenesis and primarily comprises type III collagen, fibroblasts, and new vessels. Fibroblasts are the main cells involved in granulation tissue formation. Their proliferation, and synthesis of extracellular matrix components, contributes to tissue restoration [21]. Additionally, the interaction between fibroblasts and keratinocytes plays a significant role in re-epithelialization. Factors like EGF, KGF, and TGF-α, produced by platelets, keratinocytes, and anti-inflammatory macrophages, promote keratinocyte migration and proliferation [25-27]. The process is further facilitated by the production of fibronectin, tenascin C, and laminin 332 by keratinocytes [16]. As re-epithelialization progresses, the stratified layers of the epidermis are re-established, and the maturation of the epidermis begins to restore its barrier function. TGF-ß can accelerate this maturation process [17]. The proliferation and differentiation of keratinocytes are essential for the reestablishment of a functional epidermis [25,27]. 2.4. Remodeling The remodeling phase, which occurs several weeks after the initial wound, marks the transition from granulation tissue to scar formation. This phase may last up to 1-2 years [21]. During this phase, angiogenesis slows down, and type III collagen in the granulation tissue is replaced by stronger type I collagen [16]. Myofibroblasts play a crucial role in driving the remodeling phase. They originate from fibroblasts and develop in response to mechanical tension and TGF-ß signaling. Myofibroblasts are responsible for wound contraction, and their expression of smooth muscle actin (SMA) generates the contractile force exhibited by these cells [28,29]. It is worth noting that myofibroblasts are considered terminally differentiated and undergo apoptosis once the remodeling process is complete [16]. During the remodeling phase, the granulation tissue matures into a scar, and the tensile strength of the tissue increases. This maturation is characterized by a reduction in the number of capillaries, an aggregation into larger vessels, and a decrease in the number of glycosaminoglycans. The cell density and metabolic activity in the granulation tissue also increase during this maturation process. The collagen type and organization changes further enhance the tissue's tensile strength [17]. However, the tensile strength of the healed tissue never fully reaches its original strength. A newly epithelialized wound typically has approximately 25% of the tensile strength of normal tissue, and it may take several months for the tensile strength to increase to a maximum of 80% of normal tissue [30]. The enhancement of tissue tensile strength primarily occurs through reorganizing collagen fibers, initially deposited randomly during the granulation phase. After, the enzyme lysyl oxidase, secreted by fibroblasts into the extracellular matrix (ECM), facilitates the increased covalent cross-linking of collagen molecules [17]. Pharmaceutics 2023, 15, 1914 Type of Polymer Natural Synthetic 3. Polymeric Films for Wound Healing Polymeric films (PFs) as dressings for wound healing were first vastly introduced dur- ing the Second World War as a response to the demand for advancements in medicine [31]. Bloom et al. [17] were the first to document semipermeable films in 1945 using cellophane to treat burnt prisoners of war in Italy; they reported “gratifying results” with complete healing nine days after dressing application [32]. Nowadays, PFs are commonly utilized in the medical field for healing as physical barriers for wounds which help to prevent inflammation, control the environment of the wound, and accelerate healing [33]. PFs have gained popularity due to their non-invasive nature, ease of application, biocompatibility, and the potential inclusion of antimicrobial treatments. Moreover, PFs offer flexibility, adherence, gas exchange capabilities, and trans- parency [34]. The material's flexibility enables the film to conform to complex shapes while facilitating gas exchange, which has been proven to promote healing [34]. Additionally, its transparency allows for the close monitoring of the wounded area without removing the film, thereby reducing trauma during dressing changes, minimizing exposure to bacteria, and lowering the risk of infection by up to one week [34,35]. PFs are recommended for treating partial-thickness wounds, minor burns, lacerations, and certain low-exudate ulcers such as ischemic ulcers, diabetic ulcers, venous ulcers, and pressure ulcers [34]. However, it is important to note that due to the occlusive nature of the material, films should not be used on wounds that require significant fluid absorption, as excess exudate can potentially lead to peri-wound maceration. Nevertheless, maintaining a certain level of moisture can be beneficial for the healing process by promoting keratinocyte migration to the affected area [34,36]. The performance of PFs depends on the chemical composition and the type of wound. Polymers used to formulate PFs can be divided into natural, synthetic, and blended. Therefore, the PFs will possess different qualities depending on the type of polymers employed to form the film dressing. 3.1. Natural Polymeric Films Natural polymers, also called biopolymers, offer advantages such as biocompatibility, biodegradability—at present a sought-after quality-healing properties, inertness, and adhesiveness [37]. For this reason, generally, natural polymers are preferred over synthetic ones [37]. However, they are prone to microorganism contamination and lack quality mechanical properties to form an optimal polymeric film. Some examples of natural polymers used for elaborating PFs are chitosan, hyaluronic acid, starch, silk fibroin, sericin, keratin, sodium alginate, gelatin, collagen, zein, and konjac glucomannan, among others (Table 1) [36]. Chitosan is one of the most exploited and abundant natural polymers in wound dressings, with antimicrobial and film-forming properties [38,39]. Moreover, due to their innocuousness, polymers like chitosan, cellulose, gellan gum, alginates, and starches can be used for oral cavity films [40] (Figure 2). Table 1. Types of polymers used in polymeric film casting. Examples Chitosan, hyaluronic acid, starch, silk fibroin, sericin, keratin, sodium alginate, gelatin, collagen, zein, cellulose, and konjac glucomannan Polyvinyl alcohol, polyacrylic acid, polycaprolactone, polyethylene glycol, polyvinylpyrrolidone, polylactic acid, and polydimethylsiloxane. 5 of 27 Properties Biocompatibility, biodegradability, high disponibility, healing properties, permeability, inertness, and bioadhesiveness Resistance, flexibility, structure, high degree of polymerization, thermo-responsiveness, hydrophilicity, and occlusivity Ref. [36,37] [36]See Answer
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