Veins and Lymphatics 2018; volume 7:7196

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Nanobiomaterials for vascularbiology and wound manage-ment: A reviewAjay Vikram Singh,1 Donato Gemmati,2Anurag Kanase,3 Ishan Pandey,4Vatsala Misra,5 Vimal Kishore,1Timotheus Jahnke,6 Joachim Bill61Max Planck Institute for IntelligentSystems, Stuttgart, Germany;2Hemostasis & Thrombosis Center,University Hospital of Ferrara, Ferrara,Italy; 3Molecules in Motion Pvt. Ltd,Pune, India; 4Department ofMicrobiology, Motilal Nehru MedicalCollege, Allahabad, India; 5Departmentof Pathology, Motilal Nehru MedicalCollege, Allahabad, India; 6Institute forMaterials Science, University ofStuttgart, Stuttgart, Germany

AbstractNanobiomaterials application into tissue

repair and ulcer management is experiencingits golden age due to spurring diversity oftranslational opportunity to clinics. Over thepast years, research in clinical science hasseen a dramatic increase in medicinalmaterials at nanoscale those significantlycontributed to tissue repair. This chapteroutlines the new biomaterials at nanoscalethose contribute state of the art clinicalpractices in ulcer management and woundhealing due to their superior properties overtraditional dressing materials. Designingnew recipes for nanobiomaterials for tissueengineering practices spanning from microto nano-dimension provided an edge overtraditional wound care materials those mimictissue in vivo. Clinical science stepped intodesign of artificial skin and extracellularmatrix components emulating the innatestructures with higher degree of precision.Advances in materials sciences polymerchemistry have yielded an entire class ofnew nanobiomaterials ranging fromdendrimer to novel electrospun polymer withbiodegradable chemistries and controlledmolecular compositions assisting woundhealing adhesives, bandages and controlledof therapeutics in specialized wound care.Moreover, supportive regenerative medicineis transforming into rational, real andsuccessful component of modern clinicsproviding viable cell therapy of tissueremodeling. Soft nanotechnology involvinghydrogel scaffold revolutionized the woundmanagement supplementing physico-biochemical and mechanical considerations

of tissue regeneration. Moreover, thischapter also reviews the current challengesand opportunities in specializednanobiomaterials formulations those aredesirable for optimal localized wound careconsidering their in situ physiologicalmicroenvironment.

IntroductionNanomedicine is providing explosive

development to complement and augmentclinical and biomedical practices in woundhealing and ulcer management due to itsbetter therapeutic outcome.1 Atomic levelcontrol of nanobiomaterials exhibit quantumcharacteristics compared to their bulkmaterials and when combined withbiological properties, biomolecules exhibitentire new phenomenon in this magicdimension. This is the main reason thatnanomedicine is preferred over traditionalbiomedical practices in recent years. ByNational Institute of Health (NIH) definition,nanomedicine describes use ofnanotechnology in biology and medicineranging from biomedical applications tomolecular nanotechnology andnanoelectronics in developing biosensors fordiagnosis and therapeutics.2,3 Lately,nanoparticles (NPs) have been variouslyused in targeted drug delivery to improvebioavailability and systemic circulation ofdrug and an insight into this is reflected bythe fact that annual investment innanobiomaterials has increased to 3.8 billionUSD.4 In past, targeted drug delivery wasone of most sought nightmare ofpharmaceutical industry. Dendrimer polymerand lipid based drug delivery system hadcontributed immensely in this regard.Dimensional benefits of NPs provide anopportunity to adapt in systemic circulationand move through the cell membranes andare concentrated and accumulated insidecytoplasm. Design of biodegradable NPs,e.g. nanoliposomes, nanospheres,nanocapsule, hollow nanostructures, ceramicNPs, dendrimers and polymeric micelleshave their own advantages over traditionaldrug delivery system5,6 (Figure 1). Theyprovide sustained release of drug at targetsites and can be maintained for longer periodin systemic circulations.7

Most important characteristic thatattracted these nanomaterials to ambitiousresearchers for drug delivery, emerges fromtheir easy and reliable surface modificationfor tissue/organ specific targeting.8 In thiscontext, to catalyze the pharmaceuticalinnovation in vascular biology, a strategicand legislative effort was made via

interviewing patients by the consultingphysicians to highlight most transformativemedicine in last 25 years. Out of 941 patient,513 voted the drugs with superior efficiencyas most transformative medicine. This couldbe future logic for vascular nanotherapeuticsdesign as well since nanotechnology hasprovided tremendous efficacy via vascularnanomedicine9 (Table 1).

Question related with the efficacy: Thisdrug had a huge impact on our ability to treatosteoporosis; Spectacular impact on B cellmalignancies, and on autoimmune diseases;The most potent class of agents to lowerintraocular pressure; Actually modified thecourse of multiple sclerosis, Althoughdifficult to use, has proven life-saving formany patients with arrhythmias.

Novel mechanism of action: Introduceda new set of compounds for treatment ofpsychosis; Prototype of an antibody directedto a growth factor receptor that re-energizedbreast cancer tumour biology as well asproved highly curative as a treatment ofselected subsets.

TNF blockers; rheumatology: Thesedrugs first established the paradigm ofbiological intervention, represented the firstclass of biological in rheumatology and havemade a significant difference in the treatmentof rheumatoid arthritis and spondyloarthro -pathies; Medication that had significantimpact on treatment of insulin resistance andhelped elucidate underlying mechanism.

Impact on practice in field:Remarkable... for a development strategy

Correspondence: Ajay Vikram Singh, MaxPlanck Institute for Intelligent Systems,Heisenbergstr. 3, 70569 Stuttgart, Germany.E-mail: avsingh@is.mpg.de

Key words: Venous leg ulcer; nanomaterials;hydrogel; dendrimers; organ-on-chip.

Acknowledgments: AVS thanks Max PlanckSociety for the grass root project grant 2017(M10335) and 2018 (M10338).

Conflict of interests: the authors declare nopotential conflict of interests.

Received for publication: 16 November 2017.Revision received: 28 January 2018.Accepted for publication: 29 January 2018.

This work is licensed under a CreativeCommons Attribution 4.0 License (by-nc 4.0).

©Copyright A.V. Singh et al., 2018Licensee PAGEPress, ItalyVeins and Lymphatics 2018; 7:7196doi:10.4081/vl.2018.7196

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featuring molecular biomarkers forstratification (BCR-ABL rearrangements)and for pharmacodynamics (receptorautophosphorylation).

Imatinib; oncology: First drug to modifyimmune response by enhancing it. This is asentinel therapeutic approach.

Cinacalcet; nephrology: A uniqueaddition to the armamentarium of drugs forrenal bone disease. Grew directly out ofknowledge gained in basic science researchand shows how transformative such workcan be.

Nitisinone; medical genetics: Firstexample of substrate depletion as atherapeutic mechanism for an inborn error ofmetabolism [Disease].

Improved safety: When introduced in2001, overcame many of the severetoxicities associated with combination ARTregimens before this (tenofovir; infectiousdiseases).

Temozolomide; neurology:Although theeffects are modest, it is clear that the abilityto have access to a tolerable treatment forgliomas is important and driving future drug

Table 1. Justifications used by study participants to indicate a drug as transformative.

Justification Freq. (N = 941); n: %* Illustrative remarks

Improved efficacy 513; 55% Alendronate; endocrinology Rituximab; rheumatology and oncology Latanoprost; ophthalmology Interferon beta-1a; neurology Amiodarone; cardiologyNovel mechanism of action 345; 37% Clozapine; psychiatry Sildenafil; urology Trastuzumab; oncology TNF blockers; rheumatology Metformin; endocrinologyImpact on practice in field 143; 15% Imatinib; oncology Imiquimod; dermatology Cinacalcet; nephrology Nitisinone; medical geneticsImproved safety 139; 15% Tenofovir; infectious diseases Temozolomide; neurology Fluoxetine; psychiatry Propofol; anaesthesiologyWidespread use and impact 110; 12% Fluoxetine; psychiatry Enalapril; nephrology Sildenafil; urology Omeprazole; gastroenterologyEase of patient use 109; 12% Etanercept; rheumatology Combination fluticasone/salmeterol; Pulmonary medicine Zithromax; infectious diseases Tamsulosin; urologyApplication to multiple diseases 63; 7% Infliximab; rheumatology OnabotulinumtoxinA; neurology Rituximab; rheumatology/oncology Leuprolide; endocrinologyACEI, angiotensin-converting enzyme inhibitor; ANCA, anti-neutrophil cytoplasmic antibody; ART, antiretroviral therapy; CKD, chronic kidney disease; PDE5, phosphodiesterase 5; SSRI, selective serotonin reuptakeinhibitor; TNF, tumor necrosis factor. *Round 1 responses only. Answers do not sum to 100% because they were not mutually exclusive. Reproduced from Kesselheim and Avorn, 20139 with permission.

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Figure 1. Nanotechnology based translational quest for next generation biomaterials forvascular biology applications (reproduced with permission from ref.6).

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development.Fluoxetine; psychiatry: Transformed

treatment of depression providing effectiveagent with far fewer side effects and toxicityrisks in overdose than tricyclicantidepressants and monoamine oxidaseinhibitors. More rapid recovery with lessdelirium and less postoperative nausea.

Fluoxetine; psychiatry:Widespread useand impact: First [SSRI] to have a majorimpact worldwide. Ushered in a new era ofnovel antidepressants.

Enalapril; nephrology: The primaryagent that has spawned the widespread useof ACEI in CKD over 25 years.

Sildenafil; urology: It is obvious that thiswas the number one transforming drug notonly in urology but in all of medicine andsociety in last 25 years.

Omeprazole; gastroenterology: Perhapsthe most important proton pump inhibitor;more than 60 million Americans havegastroesophageal reflux disease, many on adaily basis.

Ease of patient use: Huge impactbecause of the ability to administer the drugat home (in addition to its efficacy).

Combination fluticasone/salmeterol;pulmonary medicine: Inhaled steroidsmodify natural history but have poorcompliance. Adding salmeterol improvescompliance.

Zithromax; infectious diseases: The firstantibiotic to have both massive use and oncedaily dosing. First specific alpha-blocker; nodose titration needed.

Infliximab; rheumatology: Applicationto multiple diseases: First anti-TNFmonoclonal antibody approved for multipleclinical indications, including inflammatorybowel disease.

OnabotulinumtoxinA; neurology: Thisdrug completely altered the treatment forfocal dystonia. It has also been useful formany conditions outside of dystonia.

Rituximab; rheumatology/oncology:[Established] the central pathogenic role ofB cells in multiple autoimmune diseasesincluding some (rheumatoid arthritis, ANCAvasculitis, type 1 diabetes, multiple sclerosis)not typically considered B cell diseasesdespite the expression of autoantibodies.

Vascular biology and nanomedicineA recent development in material

synthesis has given an edge to synthesizebiological structures in vitro under ambientlab conditions. These developments coupledwith tissue engineering have put forthenormous capabilities of these materials invascular biology and regenerative medicine.

In biomedicine, vascular biology is one ofvery advance field that seldom needstechnical expertise since this field comprisesvast cardiovascular premises surrounded byblood vessel and associated structures.10Advances in nanomedicine have given alanguishing hope to future promises in thisfield due to ground level cooperationbetween various fields of basic sciencescomprising engineering, medicine, physics,chemistry and biology. An in vitro study hadrevealed that biocompatible nanosurfacespromote growth and proliferation of vascularcells viz. endothelial and smooth musclecells that paved the way for future vascularstents applications in designing endothelialmonolayer.11,12

Application of nanomedicine invascular biology

Advent and increased sophistication ofnanotechnological tools provided us anunderstanding to develop in vitro biologicalcircuits using smart carbon materials that canbe used for neuronal prosthesis in vivo. With

the discovery of conductive carbonnanotubes and nanofibres with the excellentbiocompatibility, a new hope in muscle andbrain neuro-vascular biology has arousedspecially in neurodegenerative disorderssuch as Parkinson (PD), Alzheimer’s (AD)and multiple sclerosis (MS),13,14 where suchdevelopments will bring million dollarsmile.15 Major problem with neuronalvascular biology is their poor capability toproliferate and their nasty requirement forgrowth and interaction with the surroundingcells maintaining their neuronal plasticity.16Nanofibres-polycarbonate urethanes (PU)composite have provided a novel solution togliotic scar formation in tissues that was amajor challenge in neuronal prosthesis andfunctionality. This composite also providespositive interaction and cooperation amongcells in vitro.17 Lately, researchers haveshown that neuronal and bone forming stemcell associated with clinical disorder hadshown reversal in terms ofneuronal/osteopathic anatomy andfunctionality when cultured in biocompatibleenvironment of composite carbonnanofibers18,19 (Figure 2).

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Figure 2. Schematic illustration of nanomaterials used for skin wound therapy: FromNanoparticles to Nanoparticulate Composites. Abbreviations: FMs, films and membranes;HGs, hydrogels; MCs, multicomposites; NFs, nanofibers; NPs, nanoparticles (reproducedwith permission from ref.18 with free use Creative Commons Attribution 4.0International License).

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Nanobiomaterials: New beginning in wound healing and ulcer management

As updated convention, biomaterials can bedefined as substances composed of biologicallyderived moieties (other than drugs and food)those can be successfully used for therapeutic ordiagnostic purposes irrespective of theirapplications.20 In recent years, biomaterialsresearch has gained momentum due to theirsignificant contribution in biology andmedicine.21,22 Use of biomaterials in tissueengineering is revolutionary rather thanevolutionary due to their capabilities tosynthesize artificial connective, epithelial, orneuronal tissues. One of major breakthroughsupporting nanobiomaterials in medicine is theirdimensional versatility that ranges fromequiaxed symmetrical gold,7,23,24 Platinum,Titanium nanoparticles (NPs), quantum dots(QDs) to one dimensional fibrous forms (carbonnanotubes/fibers)25 that make them a suitablechoice for wound dressing materials. Thisdimensional profitability makes them materialsof choice in various implants and prostheticapplications where dimensional stability is mostimportant features. For examplenanobiomaterials have been used in Zirconiumbased Joints (hip, knee, shoulder),Cochlear/dental and breast implants, ear andglaucoma drainage tube, mechanical heart valveand articular graft, intra ocular lens,26-33 etc.Nanoclay and nanohdyroxyapatite are used tofillers and reinforce agents to strengthen themechanical stability of polymers in variousprosthetic processes.27,34 The smart aspect ofnanobiomaterials can be evaluated by theirsmart application in bioelectronics that they notonly can touch, feel or stimulate the biologicalsystem but also can transmit the information assensors, biofuels or circuitry elements forversatile biomedical applications that could beanother promising approach in design ofelectronic skin.28,30 These could be used inwound management with supportedantimicrobial agents as filling materials in deepcuts and burn cases where we need to coverlarge surface areas to protect skin and promoterapid healing.29,30 The biggest advantages ofnanomaterials are their large surface to volumeratio i.e. they can cover large surface areaapplied. This characteristic provides a uniqueopportunity in surface healing process where weneed minimal therapeutic to cover large area.

Challenges in designing biomate-rials for wound healing and ulcermanagement

Challenging aspects that might be

considered during design ofnanobiomaterials for wound healing mustconsider required biodegradability, surfaceproperties, mechanical properties,shapability to sculpt finer details31 (Figure3). Some of the challenges in the designingnanobiomaterials are discussed with

following areas: i) progress and challengesmimicking ECM biologically andstructurally; ii) actively interactionnanostructures, which promote mammaliancell interaction and inhibit bacterialgrowth;32-34 iii) detail list of biomaterialsused for biomedical applications (e.g., soft

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Figure 3. Evolution of resolving versus nonresolving inflammation at a cellular level. (A)Typical features of a normal acute inflammatory response to infection that is detected bypresentation of PAMPs to pattern recognition receptors. Eradication of the pathogeneliminates the stimulus, along with causing some reversible collateral tissue damage, andsets the stage for the resolution/repair phase, leading to restoration of normal tissuehomeostasis. (B) Typical features of a chronic inflammatory disease caused by a nonim-mune pathophysiologic process that in one way or another triggers an initial sterileinflammatory response, often indolent and likely through production of damage-associ-ated molecular patterns (DAMPs). This initial response then becomes amplified bycytokines and chemokines. Because this response does not eradicate the initial stimulus,persistent nonresolving inflammation occurs, ultimately resulting in tissue damage. Theinflammatory response itself may positively influence the production of DAMPS, whichprovides an additional positive feedback loop. For example, in the case of atherosclerosis,reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI) may mod-ify subendothelial lipoproteins in a manner that amplifies their ability to promoteinflammation. Adapted with permission from ref.36 Tabas et al.

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nanomaterials, hydrogels, fiber scaffoldmimicking ECM, dendrimers and electrospun polymers and their advantages);35 iv)corneal wound healing, Bladder wall andvascular biology applications: a special useof biomaterials is required due to complexinflammatory cascade depending uponchronic and acute cases;36 v) details ofadvantageous factors those give an edge tonanomaterials for use over traditional onee.g., surface modifications to prohibitnonspecific protein adsorption, pinpoint andaccurate immobilization of signalingmolecules over required surface, biologicallyinspired nanofibres to mimic natural ECMstructures, arduous and vociferous design of3-D architecture to develop in vitro bloodvessels with supported angiogenesis;15 vi)unique surface energy of nanomaterials:protein-mediated cell interactions.

Artificial skin: emerging conceptfor regenerative medicine

Designing materials for wound healingand ulcer management had taken one-stepforward from traditional materials dissectingthe conventional thinking to design dressingsand antimicrobial supplement.37 The conceptand realization of initial steps of designingartificial skin had given a great hope toregain price and prejudice of aesthetic valuein burn cases where often patients has to losea lot in terms of social values due to lost skintextures that leave a scar after treatment.Natural skin transplant will be the firstchoice for clinicians and surgeons to replacescarred skin in burn cases, nonethelessdesigning artificial skin will enhance thescope of regenerative, and transplantmedicine to underscore the conundrum ofsuccess of manmade organ over natural one.

Fundamentals of designing artifi-cial skin

Skin with integument system makesprimary defense barrier against microbes andkeeps body surface in tune with the externalenvironment. Histology of skin comprisesepidermis composed of stratified squamousepithelium, a dermis having denseconnective tissue and fibroblasts withunderlying hypodermis with adipose andconnective tissue fibers.38Among above two,epidermis is home of ECM formingkeratinocyte, melanin producing melancholyand epithelial cells. Considering design offully functional artificial skin, graft materialsshould adhere with wounded skin and should

be porous enough to allow diffusion ofgases, water, nutrients, waste and mostimportantly prevent dehydration ofsurrounding wounded skin. Graft materialsshould allow migration and relocation of cellcomponents and should be comparable withnatural skin in mechanical and electronicproperties. Finally yet importantly, artificialgraft must support regeneration ofunderlying dermal layer that in turn willsupport the regeneration of epidermal layer.These advances on one hand will preventmicrobial invasion to wound site, on theother hand give an opportunity to fasthealing.39 Material design for artificial skingraft must fulfill nutrient and growth factorrequirement, taking care of immunologicalaspect to avoid foreign body and preventrejection of its own. Further, they must haveinherent ability to integrate artificial tissuewith supporting innate vasculature and mustbe supplemented with cultured skin cells thatmakes it truly bioactive and helps inestablishing connection with the underlyingnatural tissue.40 Moreover, we need to put thebasics on designing skin withmechanoreceptors, pressure and tactilereceptors with impregnated hair follicles andnerve branching. One fascinating work hadreveled construction of nanotransistorstagged with large area, flexible pressuresensor matrix that eventually work aselectronic skin for futuristic nanorobotsaimed to surgical procedures in vitro.37,41 Sofor progress made in this area adapteddiscriminative approaches supplying graftswith cultured epidermal cells in one case,cultured dermal in another case and asupplement of duo in third case. Researchersuggested that grafts with dermal layer afterregeneration supports a subsequent autograft and provide best opportunity in burnedtissue regeneration.42

Basic design of artificial skin mustinclude the vide-infra scope of chemicalcomposition that leads to the fundamentalsuccess of the story. Studies hypothesizingimportance of chemical compositions havegiven insight that collagen-glycosaminoglycan (GAG) membranescross linked with glutaraldehyde (as crosslinking agents), used as artificial skin, hadshown to have capacity to escape fluid lossand infection over longer period of time(Figure 4).

Most important they prevent graftrejection and contraction of wounds that isof primary importance for cosmetic purposesin facial wounds and functional importancein corneal wounds since contraction incorneal wounds produces astigmatism.43 Asan evidence, chemically crosslinkedglycosaminoglycan (GAG) with hyaluronan(HA) and chondroitin sulfate (CS) as anactive ingredients have been shown toperform better wound dressing materialsthan the traditional one.44 Nanobiomaterialshave numerous advantages over thetraditional one due to their various propertiesas discussed next.

In terms of thermal sensitivity,traditional biomaterials are moderate due tomeso-micro scale modifications incomparison to the nanobiomaterials showinghigher thermal sensitivity due to nano scalemodification.45 The hydrophilicity ofnanobiomaterials due to surroundingtemperature control46 is more than thetraditional biomaterials. Thenanobiomaterials have higher surface-volume ratio,47 which helps in cellattachments and grafting more than thetraditional ones, which have the low proteininteraction surface energies. Due to thebiological origin and bio-polymers,48nanobiomaterials are superior, in terms of

Figure 4. Schematic plan showing hyaluronan-GAG-core protein cross-linking strategy todesign hydrogel based artificial skin-ECM analogue. Fundamental design includes HA-GAGs backbone that supports nanoscale protein-carbohydrate monomer buildingsblocks pegged meticulously to mimic biostructure (n: repeating units; authors personalcommunication).

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biocompatibility and biodegradability, totraditional biomaterials. Nanobiomaterialsalso has preferences in terms of hydroretention due to use of hydrogel-baseddressing49 rather than traditional gauze baseddressing materials used in traditionalbiomaterials. Also nanobiomaterials aremore bioadhesive and antimicrobial due tothe impregnated antimicrobial supplementinto its nanoporous membrane50 which issupplied from outside in the conventionalbiomaterials. However, traditionalbiomaterials have an advantage concerningthe tissue abrasion over nanobiomaterialsdue to foam-based airfluidized.51 Withreference to mechanical properties, the usualbiomaterials show sufficient strength but lessthan the nanobiomaterials which havenanopolymer layer.52

Designing of biomaterial mimicking ECM using nanoscaletissue engineering

ECM defines extra cellular part of cellthat often provides structural supports to celland more precisely defines connective tissue.ECM regulates cell’s dynamic behavior interms of intercellular communication anddeporting a number of growth factors thosehelps in cell signaling and cell anchorage invarious biological phenomenons.53 Itbecomes an important issue to considerECM morphology and function whenconsidering design of nanobiomaterials forwound healing and reparative tissuemanagement, as formation of ECM is thefundamental process in morphogenesis,wound healing, growth and fibrosis. Deepunderstanding ECM components such asproteoglycan and non-proteoglycan (GAGs)that form a matrix by interlocking with thefibrous proteins will help to assemblebiological moieties to form artificial ECMmembrane for wound healing and burnedskin.54 ECM contains cell-binding domainsthose interact with cell receptors andtransmit cellular signals to cell-cell/cell-ECM adhesion and binding. ECM mediatedcell signaling also helps in sequesteringvarious bioactive molecules such as fibrousproteins, GAGs, growth factors andcytokines which is important steps in tissueremodeling at wound sites.55

In vitro ECM synthetic analogueArtificial design must sketch structural

and functional relationship between ECMand biomaterials that can sustain andrespond pharmacological action at livingtissue and engineered interface.56 Somestudies had shown nanofibre scaffold

mimicking ECM designed by tissueengineering.12,57

Dynamic interaction among cellcomponents

Nanobiomaterials must exhibit adynamic microenvironment at wound site tofacilitate cross talk among soluble (growthfactors, cytokines, morphogens) andinsoluble components (cells, ECMcomponents) under ambient chemicalenvironment (pH, O2)58 to establish a vitalconnection between the living tissue andnonliving biomaterials.12

Tissue regenerationMost sought requirement of designing

ECM replica should support tissueregeneration at wounded tissue site. Thiscould be achieved by meticulous surfacemodification of proteins and cell bindingdomains and incorporation of such ligands(RGD, IKVAV) in molecular design. Thescaffold must provide a guided mechanicalplatform for new skin growth.59

Active cellular/tissue responsesBiomaterials design so for give

nonspecific responses towards biologicalsystem due to inappropriate design ofcellular components. This is major challengeto today’s material research to pinpoint thetarget specific receptor to respond cellularfunctions viz. adhesion, proliferation anddifferentiation in artificial design ofmolecular components. This needscontrolled incorporation of cell binding andenzymatic sequences sites in bioactivedesign.

Specialized application of softnanomaterialsCorneal wound healing

Corneal wounds repair is of paramountimportance as cornea is site of refraction andfocusing light in order to make correctvision. Non-vascular nature and patternedcollagen fibrils provide characteristicstransparency to cornea that makes it specialtissue for normal vision. Corneal wounds arecaused due to a number of reasons includingcorneal ulcer, ocular surgery (intraocularimplantations, incisions for cataract surgery,in situ keratomileusis and transplants) andtrauma caused by lacerations or perforations.Currently, nylon sutures are choice ofsurgical procedures but they achieved coldreception from clinicians due to someundesired properties. Nylon sutures oftencause wound constrictions and asymmetricalhealing that often leads to astigmatism.

Moreover, suture and incision during ocularsurgery inflicts additional trauma to cornealtissue and thus inflammations andvascularization ending into corneal scar thatcontribute significantly to astigmatism. Moreoften, sutures need special attention andtechnical skills by trained ophthalmologistsin order to avoid postoperative loosening andcorneal trauma during in and out of sutureremoval.60 Therefore, technological skills todesign new surgical materials and toolsconstitute competitive race in this field inorder to restore natural vision and patientcare in ophthalmic and corneal woundshealing. Designing new recipes mustconsider technical achievements mentionedabove. Considering above designrequirements, we need a polymer adhesivethat can seal corneal wound rapidly andaccurately restoring correct intra ocularpressure (>80 mmHg) and must comprisesufficient viscosity allowing clinician forprecise placement and workability (viscosity<100 cP).61 Moreover, polymer adhesivemust restore structural integrity of patternedcollagen fibrils to provide native cornealtransparency to focus the lights accuratelyand most important elastic modulus ofpolymer adhesive must be greater thancorneal tissue to negate any possibility ofastigmatism. Solute diffusion properties,biocompatibility, microbial barrier andbiosorption of wound exudate are additionaldesigning essentials where beauty meetsutility for corneal wound care.62Fundamentals of corneal woundmanagement require repairing lacerationsand clear sealing of incisions with securingcorneal transplants. Closings of LASIK flapsare other ophthalmic indicators wherenanoadhesives may prove to be landmarksuccess. Next age materials such aspolymeric dendrimer provide uniquesolutions to special wound care such as incorneal and ophthalmic injuries.Biodendrimers63 made up of polyethyleneglycol (PEG), succinic acid (SA) andpolylactic acid (PLA) are uniquebiocompatible and degradable polymericdendrimers that shows controlledhydrophilicity and cellattachment/detachment properties.64

Further, capability of peptide-ligationbased soft cross-linking avoids complexphotochemistry and procedural risks thatmakes this more amenable for clinicalprocedures. Peptide cross-linking providesadditional support to physico-mechanicalproperties (e.g., modulus and plasticity) andfit for clinical response in situ.65 Peptide lockand cross links in hydrogel and adhesiveused for corneal wound care further givepossibility to peptidases based easy cleavagewhen it comes to integration of nano-domain

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protein or monomer in multigenerationalbiodendrimer synthesis66 (Figure 5).

Nanobiomaterials for synthetic blad-der wall substitute

A large number of populations sufferfrom cancer and disorders related to bladder.In absence of suitable surgical treatment andchemotherapeutic panic, removal of entirebladder wall is considered best strategy toprevent the recurrence of bladder cancer anddisorders. Considering essentials ofdesigning new materials, mimickingtopographic features of bladder wall withstretchability, mechanical properties andoptimal knowledge of cellular and molecularevents in vivo, are the main requisite.67Surface features of bladder gives anunderstanding of designing biologicalnanostructures, such as nano-dimensionextra cellular matrix constituent proteins thatshould be precisely incorporated and taggedwith the biological moieties to give maximalcell response. Researchers have designednano-dimensional poly (latide-co-glycolide)acid (PLGA) and polyurethane (PU) basedartificial structures mimicking bladdertopography in 50-100 nm range those exhibitexcellent in vitro cytocompatibility andenhanced bladder smooth muscle celladhesion and proliferation.68 Designedmaterials show parallelism with in vivofunctionality of natural bladder wall thatopens new avenues for nanobiomaterials fordesigning tissue engineered artificialstructures.69

Nanobiomaterials in artificial bloodvessel replacement: A special case ofintravascular wound healing

Apart from surface wound, deepvascular tissues often meet intra vasculartissue damage due to ischemia and perfusionsuch as in myocardial and endothelialinjuries which need immediate clinicalattention, being part of vital organ.69 Moreoften cardiac procedures are supplementwith bypass surgery of affected blood vesselswith autologous arteries or veins of less than6 mm diameter, in order to reduce proceduraland invasive risk of heterologous graftsrejection.70 Many myocardial infarctionpatients need an alternative to innate bloodvessels in coronary artery bypass graftsurgery (CABG) as a replacement to theirdiseased or sclerosed blood vessels.71 In thisregard, the design strategy involving coretechnology must meet a number of cellconstruct in vitro that can precisely mimicthe vessel architecture and land marks invivo with respect to biochemical andbiomechanical aspect. New age biomaterialsand micro to nanoscale technologies provide

artistic liberty to meet the precision in thisregard by meticulously mountingbiochemical building blocks. For example,artificial endothelial layer production needscross linking of collagen fibers, integratedwith growth factors needed for cellcommunications between different vesselwall layers.72 Lately, in vitro studies shownthat radial stresses induced by cyclicstimulations give better mechanical strengthand histological organizations in which cellsget arranged circumferentially throughoutthe wall thickness like natural blood vesselwall. This mechanical stimulation plays akey role in precise netting and increasingcollagen content in artificial structurecompared to unstrained controls.72 Evidenceto mechanical conditioning that stimulatescell mediated construct remodeling isdemonstrated by over expression of matrixmetalloproteinases (MMPs). These areexpressed in relation with the production ofECM structural proteins (elastin andcollagen) and reinforce the remodeledtissue.73 This is further supported byincreased level of collagen and elastinmRNA content in mechanically stimulatedand cyclically strained cells in vitro.74

Another most important factor that pavessuccess of artificial design of blood vesselsis cell technologies enabling pinpoint cellmanipulation and their controlledfunctionality.75 These features can be furtherregulated by creating extracellularenvironment of embryonic cells (ECs)utilized for making synthetic grafts byencapsulating them in reconstituted collagenmatrix, providing functional vaso-activity inbiological scaffold.76 By inducingendothelialization in synthetic graft, it ispossible to design an artificial blood vessel.

This construct should contain an outeradventitia made of fibroblast and collagen, amiddle layer (media) made of mesenchymalstroma cells (MSCs) and ECs based internalmonolayer (intima), when molded in tubularfashion.

Insight into nanomaterials used forwound healing and tissue repairprocessNano-hydrogel in tissue repair: softNanochemistry in new role

Hydrogels are hydrophilic, insoluble andswallowable materials; made up of natural(alginates/chitosan/agarose/chitosan/fibrin)or synthetic (Poly (acrylic acid) and itsderivatives;77 poly (ethylene oxide) and itscopolymers and polyvinyl pyrrolidine,polypeptides and their derivativespolymers.78 Hydrogel derived frombiopolymers and designed as bioscaffoldprecisely mimic ECM in its chemicalcompositions comprising numerous aminoacids, fibrous proteins, growth factors andsugar. Designed hydrogels also contribute toECM functions by bringing cell junctiontogether, recruiting growth factors for cellcommunications, allowing extracellularmetabolite/nutrient transport, and mostimportant controlling cellular architecture invitro.79 In last few decades, realization ofartificial prosthetic organs ranging fromdentin, cartilage, ligament, tendon, bone tosoft vascular prosthetic implants (arteries,bladder, skin) have been made possible dueto generous hydrogel based scaffolds.80

Lately, uses of hydrogel for woundhealing and tissue repairs process have gainapplause in medicine based on capability todesign and control their physical propertiesas per tissue repair requirement and support

Figure 5. (A) Dendrimer with internal cavity, core and branching units with unique inte-rior and surface chemistry to couple the therapeutic pay-load (colored dots show simul-taneous loading of different drug molecule delivery at wound site). (B) Cartoon showingsequential addition of generations in those has advantages over controlling globular/lin-ear/symmetrical faces of dendrimer to design 3-D architecture. Inset showing interactionof ligand-receptor complex at dendrimer surface (yellow line shows peptide spacer linkedto ligands; Authors personal communication).

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for tissue remodeling.81 Hydrogels have beendevised for occlusive dressing, cartilagetissue repair, and injectable cellular scaffold,axonal regeneration in spinal cord injury andtissue sealant in neurosurgery.82-86 Yet thereare many open prospects those can serve formore advance use of hydrogel as novelnanomaterials for tissue repair. One novelstrategy could be design of hydrogel-basedbioscaffold comprising fibronectinfunctional domains (FNfds) and hyaluronan(HA) for tissue repair. Since FNfds hydrogelmatrix is a tremendous medium forpromoting wound healing by binding withthe platelet derived growth factors (PDGFs),it assists with the fibroblast migration in situthat is a crucial step for tissue induction forremodeling of injured tissue.86,87 Asmentioned above, hydrogel matrices areavailable in a wide range of designs fromnanoporous, fibrous to fine network ofembedded proteins. 3-D hydrogel matricesgive a unique opportunity for designingartificial ECM pegged with growth factorsand therapeutics required for rapid healingas shown in Figure 6.

Such designs, mimicking porous andfibrous ECM will support cell growth andmigration by trapping fibroblast and otherinflammatory cells required for tissueremodeling.88 More ambitious; 3-D hydrogelarchitecture housed with the culturedfibroblast with growth factors, acts as smartnatural skin to establish aesthetic remodelingof lost skin in burn cases and massiveinjuries with severe tissue loss.89

Electrospun polymers scaffold: tis-sue repair at the rate of spinningchemistry

Biomaterial designers have given a newhorizon to biomedicine by developingprocedure to control nanostructure byintroducing biocompatibility, mechanicalstiffness, stability and biodegradability.Electrospinning, electrostatic and gasblowing technologies had given an edge todesign nanowoven fibrous materials fromorganic/inorganic to biological polymers.Simultaneously we have control overporosity and diameter, mesh size, texture andpattern giving larger surface area and highsurface to volume ratio (S/V) for biomedicaland industrial applications.90 Nanofibrousmaterials have secured their application frommedicine, cosmetics, tissue scaffold forimplants to industrial purposes dependingupon nanofiber shape, alignment and crosssection that are exclusive features inselecting the fiber materials for a particularapplication.91 One of biggest advantage thatwe can count over use of electro spunscaffolds for wound healing and tissue repairprocess over native polymers is their

physical resemblance with ECM in nativetissue, which makes them a promisingcandidate for regenerative medicine.Electrospun ECM substratum camouflagegives an opportunity to assess cellularfunction in vitro and applications where suchscaffolds are used as cell delivery vehiclesand viable cell grafts. Compare to traditionalpolymer engineering techniques, electrospunpolymer nanofibers involve controlling avast array of parameters. This characteristicis tremendously important for theirapplication in postoperative surgeries andstimulating tissue regenerations bypromoting adhesion, proliferation, anddifferentiation cellular flora at the woundsite.92 Further, electrospun materials fall inboth i.e. natural and synthetic polymercategories. Clinicians have free option tochoose best for our tissue repair mechanismthat can either support physical(strength/durability) or biological (cellattachment and proliferations)functionalities, or both and indeedresearchers had shown is past such subtleadvantage while combining the two.93 Thus,above findings reveal the potential ofelectrospun polymer applications one-stepahead to develop the artificial skin and smartECM Grafts for tissue repair in severeclinical crisis such as heavy burn and injuriesinvolving massive muscular damage.

Recently polymer colloids have attractedmuch glare in biomedical applications due totheir superior properties of design andfeasibility to introduce branching andbiodegradability.94,95 Polymer colloids are100-400 nm diameters with great softarchitectural diversity. Their ability to easyencapsulation, modifyhydrophilicity/hydrophobicity and sustainedrelease at application site, makes them goodcandidate for their surgical use in arteriesand connective tissue. Moreover, new

categories of polymer colloids can bedesigned based on requisite as perbiomedical applications. Polymer chemistryhas given an opportunity to designresorbable colloids those could be used aspromising wound dressing materials.96 Inaddition, resorbable colloids offer greatcommercial viability for clinical applicationssuch as postoperative anti-adhesionmembranes in trauma. Another benefit theseresorbable colloids offer is that anti-adhesionbiofilms that on one hand offers excellentbiosorption barrier to prevent postoperativecomplications; on the other hand, itundergoes self-degradation when tissueremodeling is completed at augmentedwound site.

Recently endothelial progenitor cell(EPCs) has been used as smart biomaterialcomposite for the control ofmicroenvironmental cues since they promotedifferentiation, angiogenesis and bonemarrow-derived endothelial progenitor cellmobility via secreting VEGF and VEGFR-2factors. Circulating EPCs from human fetalaorta with strong self-renewal ability expressCD133, CD34, and VEGFR2, which play avital role in cure of diabetic foot in murinemodels. Transplantation of human fetal aorta(HFA)-derived EPCs has recently proved tobe vital for treating microvasculardysfunction prevalent in diabetic vascularbiology, could be useful for innovativetherapeutic strategy for managing othervascular diseases. Easy availability and rapidexpansion of adipose-derived stem cells(ASCs) from autologous adipose tissue withantiapoptotic factor, secretion ofproangiogenesis, and capacity formultilineage differentiation has been usedvascular tissue repair, rescue and vasculargrowth.

Apart from aforementioned applications,electrospun polymer scaffold have been used

Figure 6. Image showing immunostained cells growing (left panel) fine network of hydro-gel mesh (right panel) housing granulated collagen, growth, network of keratinocyte andchondroitin 6-sulphate as viable skin for wound repair (Authors personal work).

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in many clinical emergencies as artificialimplants for tissue repair. Electrospunnanofibres have been developed intodifferent tissue scaffold combining culturedcells with natural and biocompatiblematerials depending upon clinicalrequirement as stated in Table 2.97-107

Limitation, progress andprospect

Development in nanomedicine hadoffered many opportunities to improvewound healing and tissue repair process dueto progress in biomimetic approach to designbioactive nanomaterials with much closer tonatural tissue with respect to structure andfunction. Research in this area haveresponded to long arguments in scientificcommunity by putting forth rationaladvances to develop artificial skin and ECManalogues which can be an innovativesupplant for future clinical emergenciesneeding commendable tissue repair back up.Recent advances in nanotechnology such aselectrospinning have given liberty that ismore artistic to researcher while designingthe materials of clinical as well as aestheticsuperiority.

Present review outlined the principalclinical requirement for wound healing andtheir subsequent challenges and applaudingmeasures provided by recent developmentsin nanomedicine and materials science.Perspective arduous biomedical challenges

and their possible solutions in designingartificial skin and ECM have been reportedprovided with ambitious progress inmaterials sciences. We also reported the useof nanobiomaterials in special clinicalpractices such as corneal wound, bladderwall and vascular biology applications wherewe need to put maximum precautions whiledeveloping new materials. In addition,details regarding state of the artnanobiomaterials such as dendrimer andpolymers (electrospun and hydrogel) thosehave served in tissue repair process arementioned. Artificial 3D cell culture from a

modern neurobiology perspective is atinfancy but provides a great hope fortomorrow, bringing relevance and meaningto cell based assays for differentapplications. Realizing the fact that cells intissue operate in a 3D environment,switching to a 3D cell culture system willinvolve substantial time and cost, in terms oftechnology, and most importantly inthroughput and scalability.108 The currentstate-of-the-art three dimensional culture andimaging in multiple planes involvesdetecting a 3D multicellular body most oftenembedded in a gel type, ECM mimic, or

Table 2. Represents electrospun nanofibers conjugated (using coupling chemistry) withthe mammalian cells, natural and synthetic biocompatible materials for various tissuemanagement clinical practices.

Clinical conditions Biomaterials

Nerve implants Neuronal Stem Cell + Poly(L-lactic acid)97

Vascular grafts Myofibroblasts, Arterial Smooth Muscle Cell Human coronary artery endothelial cells, PLA, Collagen type I&II, Elastin98,99

Bone implant Osteoblast, Mesenchymal stem cells + Silk/polyethylene oxide/hydroxyapatite / bone morphogenetic protein100

Cardiac implants Cradiomyocytes and myoblasts + Polyaniline and gelatin, Polylactide101,102

Human ligament Implants Ligament fibroblast + Polyurethane103

Cartilage implants Chondrocytes + Collagen Type II104

Skin implants Human Fibroblast and Keratinocytes + Collagen type I coated with collagen type I and laminin, Poly(lactide-co-glycolide)12,105,106

Breast implants Human Fibroblast and Keratinocytes + Collagen type I coated with collagen type I and laminin, Poly(lactide-co-glycolide)107

Table 3. Current organs-on-the chip devices with drawbacks in organ blue prints.

Model Cell niches Organ blueprint Drawbacks

Blood brain barrier (BBB) Astrocyte-endothelia Tight Junctions and TEER No human cell lines tested for barrier functionalityRetinal blood barrier (RBB) Ocular vascular cells Epithelial barrier tight junctions Vitreous and mechanotransduction mechanism are and corneal epithelial cells not exploredMammary gland Breast specific endothelial Cancer model for metastasis Cancer molecular markers expression fibroblast and epithelial cells and tumor invasion is not validatedBone-marrow-on-a-chip Viable marrow tissue with Organ level marrow toxicity Interaction between bone components functional hematopoietic responses to radiation and such as lacunae, canaliculi missing niches protective effect of the radiation counter measure drugs Gastro-intestinal-tract (GIT) Gut epithelial cells Intestinal absorption mechanism Model lack mechanical stimuli mimicking screening of molecular markers microvillus in gutKidney Glomerular Screening of molecular markers Glomerular anatomy blueprint missing Network (Renal tubular epithelial cells) Lungs Airway epithelial cells Demonstration of lung inflammation Complex fabrication, live imaging Alveolar epithelial cells and extra pulmonary absorption is challenging, hybrid model lacks Pulmonary microvascular alveolar capillary interface vascular network endothelial cells Liver Hepatocytes Liver zone and sinusoid formation, Model close to regeneration but lack capability as Vascular endothelial cells serum protein synthesis independent implantable device Fibroblasts

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hanging in a drop type culture.109 Thoughexisting systems continue to be plagued withissues of reproducibility and variability, thebandage pharmaceutical industry is focusedon making great strides in this area ofresearch. The challenge of recreatingcomplex CNS functions such as cognition inthe brain cannot be recapitulated via 3Dpatterning. This is due to microscale spatialheterogeneity and macroscopic architectureof the spinal cord and brain. It is howeverpossible to reproduce organ-level functionsand electro-kinetic responses by copyingbarrier functions.110 Adding complexity andfunctionality in the third dimension, furtherposes a hurdle for high-resolution imagingto determine spatiotemporal locations ofcell-cell or tissue-tissue interfaces, similar tovisualize processes in living organs.

In vitro 3D engineered tissue and itsongoing therapeutic applications so far havebeen vastly carried out on collagen-basedscaffolds.111 However, the CNS has minimalfibrillary matrix and collagen in ECMcompositions, rendering it difficult toextrapolate the results from an in-vivoperspective. Nanotemplate materialfabrications, bottom-up nanoengineering andphysicochemical modulations of theneuronal culture environment provides newopportunities forneuropharmacology.20,24,108,112-119 Forexample, self-assembled bioactive peptides,derived from laminin when electrospun asnanofiber scaffolds provide long-termsurvival and differentiation of neuralprogenitors into neurons and astrocytes. Thedensity of the laminin epitope in the thirddimension is crucial for such fastdifferentiation and could be aneurotherapeutic target.120 Nanostructuredsurfaces integrated with micropatterns givequalitative versus quantitative informationfor prosthesis design to minimize bacterialgrowth and promote neuronal like cellgrowth.30,33,34,121 Multipartite micro- andnanocarrier designs could be used fortargeted diagnostics and therapeutics(theranostics) for debilitating diseases likemultiple sclerosis or brain tumors.10,20,23-25,122-127

Organ-on-chip devices for current therapeutic developmentfor human organs

Organs-on-chips are microfabricatedsystems mimicking in vivo organ likefeatures replicated onto PDMS or elasticsilicone. The devices imitate key functionalcomponents of in-vivo organs to reiterateintegrated organ-level physiology in vitrowith microfluidic channels.128 The

microdevices could be the future forabsorption, distribution, metabolism,excretion, and toxicity (ADMET) testing ofneurotherapeutic pharmacokinetics.Synthetic organ-on-chip systems could befurther useful to create in vitro models ofhuman neurodegenerative disorders tounderstand pathophysiological modulationunder drug trials. Drug toxicities based onpreclinical animal trials often fail tocategorize the drug uptake between thehuman and animal models owing to species-specific molecular differences (e.g.,membrane transports). In suchcircumstances, tissues or human cells arenecessary to draw definite conclusions.Therefore, miniature-human-brain likedevices, designed via integrating 3Dpatterning and microfluidics, may replacethe conventional animal models forneuropharmaceutical and neurochemicalapplications, reducing the cost of drug trialsin the future. With advances in microfluidicsand microfabrication technology, it ispossible to spatially pattern and connectmultiple tissue types on single chip, creatingfunctional humanoid model with connectedorgans like features on chip. The rise of bio-engineered neuronal tissue network, it ispossible to predict the outcome of drug-organ interactions in humans using organ onchip platform, negating shortcoming ofgenetic differences of animal models.129Particularly, microengineering recipes forthe assembly and operation of themicrofluidically lined blood brain barrier-on-chip systems have attracted more attentionof the neuropharmacology industry. Forexample, microengineering principles of the

silicone industry could be used to design amultilayered microfluidic device, lined withhuman endothelial cells separated by a thinporous flexible membrane from astrocytes,into two parallel elastomeric microchannel(Figure 7). BBB specific cells cultured underphysiological flow and apico-basal polarityinto these microdevices can be easilyadapted to develop BBB-on-chip using facileexperimental techniques to quantify BBBspecific functionality on CHIP.110,130 Thebiohybrid engineered CHIP lined with self-organizing pluripotent cells, which couldeventually segregate into distinct brainregions, further provides a tool forneuropharmacology as a prototype ofneurodevelopmental disorders.130,131 Inpreliminary reports, hydrophobic andhydrophilic drug transport demonstrated onchip across the artificial blood brain barriercorroborates in vivo findings, supportsunderstanding gene-gene interactions andnanoparticle-guided diagnostics.131,132

Many in vitro cerebral organoidsproduced via 3D dynamic culture thatresemble miniature brains, faithfully exhibitCNS like characteristics.110 However, in vivoneurons surrounded by the extremelycomplex system of heterogeneous tissues arephysiologically different. Therefore, BBB-on-chip models lined with network of glialcells may put forth a low cost non-animalresearch initiative to test stress signaling inneurons and neurotransmitter uptake andrecycling. In addition, axonal myelination,nourishment, and trophic support of glialcells could be shown as a minimal, syntheticfunctional unit to recapitulate a part of brainon CHIP module, refer to Table 3.

Figure 7. Concept of all human organ-on-a-chip. Advances in biomimetic micro engi-neering enable different organs features could be replicated on CHIP and to be integratedinto a single micro device. Each further connected with each other via microfluidic cir-culatory system mimicking physiologically relevant functions as an in vitro model withcomplex, dynamic process into human body. Reproduced from ref.131 with free useCreative Commons Attribution 4.0 International License.

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Furthermore, combining the miniature brainon chip with 3D electrode array, intracellulartransport, and axonal targeting may yieldnovel insight about calcium signaling andsynaptogenesis.25

In lieu of organ-on-chip development,not only is there a paradigm shift in approachto research in the pharma industry but alsopublic outreach of technology; especially indeveloping countries. With clinical trials,countless animal lives are lost amidstgrowing ethical concerns. In some instances,animal models used fail to predict humanresponses, as they are not physiologicallyrelevant. Cutting edge research topics, suchas the BBB-on-chip proposed herein couldtremendously lower research budget for drugdiscovery and may boost sustainabledevelopment of therapeutics for poor ruralpopulations of developing countries byhelping researchers and scientists elucidatehow tissues respond to new drug candidates.The BBB-on-chip model probably providemore understanding about BBBneurovascular unit, which will help toprevent current trend of 90% drug failurewhen switching from clinical trial fromanimal model to humans due to genericdifference between the two. Furthermore, theBBB-on-chip could spearhead thedevelopment of in vitro therapeutic vaccinesand drugs to counter infectious disease likecholera, diarrhea and tuberculosis, whichcontribute to high infant mortality in therural parts developing countries.

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124.Khare M, Singh AV, Zamboni P.Prospect of brain machine interface inmotor disabilities: the future support formultiple sclerosis patient to improvequality of life. Ann Med Health Sci Res.2014;4:305-12.

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