The microRNA in tissue regeneration

I found a very interesting article that provides a concise literature review with 80 papers about original research in translational medicine .

The polymeric molecules that are the subject of discussion today are the microRNAs ( miRNA ). They are perfect complements to the target messengerRNA ( mRNA ) in plant cells , but they slightly mismatch in base pairing inside animal cells . The gene regulation using the complex micro RNA - m RNA triggers a wide variety of cellular mechanisms .

The cellular mechanisms that achieve organ regeneration , in a spatiotemporally controlled manner , are the differentiation , the dedifferentiation , the transdifferentiation , and the cellular proliferation . They alter the cellular plasticity .

The researchers learn from the invertebrates , the lower vertebrates , and the mammals how to activate the pluripotent stem cells in order to regrow parts or the entire organism . The trigger for the chain of events in the regeneration process is either the under - or the over - expression for the specific microRNA . The article provides a list for the microRNAs that regulate the human mesenchymal , and the human pluripotent ( the human embryonic , the human induced pluripotent ) stem cells .

The modulation of the regeneration process using microRNAs may be done by varying the modulators , the delivery method , or the cellular strategy ( figure 1 ) . The target cells are either the implanted or the endogeneous cells . The scientist may apply a modulation strategy on the endogenous ( stem ) cells , on the implanted cells prior to implantation , or on both the endogenous cells and the implanted cells . The modulators are either the cells themselves , the synthetic oligonucleotides , or the level of expression . The paper presents four delivery methods for the microRNAs : through direct injection , with viruses , with synthetic nanoparticles , and with cells as delivery vehicles .

Figure 1 . The technique variations for the micro - RNA signaling in the process of tissue regeneration .
Copyright ( 2014 ) AlphaMed Press

The heart has a limited potential for endogenous regeneration . The neonatal heart has a different response to myocardial infarction than the adult heart . The underexpressed microRNAs that trigger the proliferation of the cardiomyocytes are the miR - 15 family , the miR - 17 - 92 cluster , the miR - 199a and the miR - 590 families . They may help in the differentiation for the stem cells , for the cellular lineage commitment , and in the direct reprogramming process of the fibroblasts to generate cardiomyocytes . They are critical components in the signalling network for the adult cellular proliferation .

The microRNAs have a complex behaviour that is still not yet fully understood . The modulation strategy varies with the target organ that requires regeneration , and the research application .

References :

Frith J . E . , Porrello E . R . , Cooper - White J . J . ( 2014 )" Concise review : new frontiers in MicroRNA - based tissue regeneration " , Stem Cells Translational Medicine , 3 ( 8 ) , 969 - 976 .

Hydrogels in regenerative medicine

The muscle tissue of the heart is remodelled following the myocardial infarction . The ventricular wall is thinning and the chamber dilates . The adult cardiomyocytes regenerate at a lower rate than the heart requires to function at the required capacity , without any treatment . The clinical strategies are to either use synthetic implants , or to deliver cells directly into the tissue . A solution to avoid their shortcomings is to use hydrophilic hydrogels as a three dimensional structure to deliver cells into the body . The review in Nature presents the hydrogels that demonstrated the ability to enhance vascularisation , or to promote the differentiation of the delivered stem cells .

The mechanical properties of the synthetic hydrogels ( Poly – ethylene glycol , Poly – N – isopropylacrylamide , Poly - 2 – hydroxyethyl methacrylate ) have the ability to control the behaviour of the cells in the damaged area of the myocardium . The NIPAAm - co - AAc - co – HEMAPTMC is a thermosensitive hydrogel that reduces the dimensions of the dilated left ventricle , while implanted in the rat with myocardial infarction . Figure 1 presents the rat myocardium eight weeks after the implantation of the hydrogel . Natural hydrogels ( Matrigel , Fibrin , hydrogel from porcine myocardial extracellular matrix ) increase the cell viability .

Figure 1 . The morphology and the experimental outcomes of the NIPAAm - co - AAc - co – HEMAPTMC thermosensitive hydrogel . ( a ) The composition of the hydrogel . ( b ) The elastic properties of the hydrogel . ( c ) The control experiment with phosphate buffered saline ( PBS ) injection . ( d ) The rat heart after hydrogel injection . ( e ) The histology analysis with hematoxylin / eosin ( H & E ) of the control experiment . ( f ) The H & E histology analysis of the rat cardiac tissue with the injected hydrogel . ( g ) Immunohistochemistry for the tissue grown into the injected hydrogel . Copyright ( 2014 ) Nature Publishing Group .

The hydrogels that demonstrate elastic behaviour while exposed to mechanical cyclic loading have the potential to repair the cardiac tissue . The hybrid of resilin – like polypeptide and Poly – ethylene glycol promotes cell differentiation after seven days of culture . The properties of Methacrylated tropoelastin ( MeTro ) are summarised in figure 2 . The micropatterned surface promotes the alignment and maturation for the cardiac cells , in the day 8 of the experiment. The frequency of the spontaneous beating rates of the cardiomyocytes was measured during two weeks of culture .

Figure 2 . The markers expressed by the primary rat cardiomyocytes . ( a ) The troponin I ( green ) and the nuclei ( blue ) on the surface patterned with The Methacrylated Tropoelastin ( MeTro ) hydrogel . ( b ) The sarcomeric &alpha - actinin ( green ) , the connexin - 43 ( red ) , and the nuclei ( blue ) on the patterned surface . ( c ) Thr troponin I ( green ) and the nuclei ( blue ) on the unpatterned surface . ( d ) The sarcomeric &alpha - actinin ( green ) , the connexin - 43 ( red ) , and the nuclei ( blue ) on the unpatterned surface . ( e ) The beating behaviour of the cardiomyocytes on the unpatterned surface . ( f ) The beating behaviour of the cardiomyocytes on the patterned surface . ( g ) Spontaneous beating rates of cardiomyocytes on the unpatterned surface . Copyright ( 2014 ) Nature Publishing Group .

The conductive hydrogels ( Chitosan , Polypyrrole ) connect areas to propagate the electrical signal into the entire cardiac tissue . The carbon nanotubes in gelatin methacrylate are actuators in the system ( figure 3 ) . The direction of motion varies as a function of the tubular thickness .

Figure 3 . The actuators were designed by incorporating carbon nanotubes ( CNTs ) in gelatin methacrylate ( GelMA) . ( a ) The tightly and the loosely rolled – up constructs were designed . ( b ) The displacement of the beating constructs are a function of frequency and electrical source . ( c ) The linear displacement of a triangular swimmer as a function of time . Copyright ( 2014 ) Nature Publishing Group .

The cardiac progenitor cells , the mesenchymal stem cells , and the pluripotent stem cells have increased potential for differentiation . The stem cell differentiation direction into the cardiac lineage is indicated by the hydrogel matrix in figure 4 . The immunostaining in the 2D hydrogels demonstrates that the sarcomere has diminished length in comparison to the one in the 3D structure .

Figure 4 . The cardiac patch was designed as cardiomyocytes derived from the human embryonic stem cells ( hESCs ) in the hydrogel matrix . ( a ) The result of 2 weeks of in vitro culture . ( b ) The gels microfacricated with elliptical pores . ( c ) The alignment of the hESCs in the hydrogel . The immunostaining on 3D hydrogel of ( d ) troponin T ( red ) , ( e ) myosin heavy chain ( red ) , ( f ) connexin - 43 ( red ) and sarcomeric α – actinin ( green ) . ( g ) The immunostaining on 2D hydrogel of sarcomeric α – actinin ( green ) . Copyright ( 2014 ) Nature Publishing Group .

The growth factors delivered with cells in the hydrogel may increase the efficiency of the process . Figure 5 presents the effect the basic Fibroblast growth factor ( bFGF ) has in a porcine model . The arteriolar density , the left ventricular ejection fraction , and the myocardial perfusion are increased at 4 weeks in culture , when the bFGF is used .

Figure 5 . The influence of the released rate of basic Fibroblast growth factor ( bFGF ) on restoring the cardiac function in a porcine model of the myocardial infarction . ( a ) The presence of the sarcomeric α – actin , ( b ) the arteriolar density , ( c ) the myocardial perfusion , and ( d ) the left ventricular ejection fraction ( LVEF ) were measured under control and the bFGF included conditions . Copyright ( 2014 ) Nature Publishing Group .

Only 10 % of the cells delivered directly in the cardiac tissue survive in vivo . The direct exposure to oxygen , free radicals , and inflammatory cytokines reduces the survival rate of the injected cells . A suitable 3D microenvironment is required . The hydrogel that mimics the cardiac tissue both handles the cyclic loading forces , and maintains the elastic , in addition to conductive , properties . The oxygen – releasing properties of a porous hydrogel , with or without the use of growth factors , increase the control of the system on the cellular environment .

Hybrid bioprosthetic heart valve

The peer - reviewed publication Engineering of a bio – functionalized hybrid off – the – shelf heart valve caught my interest . The growth factor signalling mechanism inside the bioprosthetic heart valve is different from the native valve . The Young ' s modulus quantifies the remodeling behaviour after implantation .

The chosen method to generate the scaffold is the electrospinning of either poly - lactide acid ( PLA ) or equal amounts of it and poly - ethylene glycol dimethacrylate ( PEGdma ) . The performance of the polymeric scaffold is tested under physiological conditions . The blood is assumed to interact with the heart valve leaflet at large amplitude , in a laminar flow , under oscillatory shear stress . The nonlinear flow properties affect the response of the heart valve .

Valvular endothelial and interstitial cells are seeded in the scaffold . The protein configuration is visualised on the cell – seeded polymeric scaffolds using immunofluorescence staining . The cell viability is assessed with 3 - ( 4 , 5 - dimethyl – 2 - thiazolyl ) - 2 , 5 - diphenyl - 2 H - tetrazolium bromide ( MTT assay ) . The cells are compatible with both the PLA and the PEGdma – PLA scaffold . The valvular endothelial cells adhere in a greater amount onto the PEGdma – PLA scaffold than on the one with PLA fibres . The valvular interstitial cells adhere similarly on both polymeric scaffolds . The valvular actin cytoskeleton for both the endothelian and the interstitial cells is more visible in the PEGdma – PLA than in the plain PLA scaffold .

Native porcine aortic valve tissue , unseeded and cell – seeded polymeric scaffolds are analysed using the scanning electron microscopy ( SEM ) . The PLA scaffold has 30 times greater fibre diameter and 1160 times greater pore size than the native leaflet .

The atomic composition of the surfaces is determined using the electron spectroscopy for chemical analysis ( ESCA ) . The comparison of the data is made using literature values for single components . The PLA – PEGdma scaffold has both PLA and PEGdma groups on the analysed surface .

The mechanical properties of both native and polymeric scaffolds are using the atomic force microscopy ( AFM ) . The spongiosa is the softest layer of the native porcine leaflet , with a Young ' s modulus twice as small as the one for the ventricularis and fibrosa layer . The Young ' s modulus of the PLA – PEGdma scaffold is 10 times greater than the ventricularis and fibrosa native layer .

Uniaxial tensile testing provides information regarding the Young ' s modulus , the tensile strength and the elongation values . Both the PLA scaffold and the native leaflet is soft , with similar values for the Young ' s modulus . The computed Young ' s modulus for the PLA – PEGdma scaffold is twice the value for the PLA scaffold . The PLA scaffold is 55 times more expansible than the native leaflet . The tensile strength value is similar for both the PLA and PLA – PEGdma scaffold .

The polarity of the molecules on the surface is detected using the contact angle goniometry technique . The PLA scaffold is highly hydrophobic . The PEGdma scaffold is hydrophilic . The PLA – PEGdma scaffold is highly hydrophilic . The PLA – PEGdma is highly hydrophilic .

The capacity of water - holding of both the native and the polymeric scaffolds is compared . The PLA scaffold has 11 % water intake capacity , the PEGdma scaffold has high value , while the native leaflet has 90 % . The PLA – PEGdma scaffold has 5 times more water intake capacity than the PLA scaffold .

The protein expression patterns are revealed in the early development for the human heart tissue . Collagen type 1 is present in the leaflets , Versican in the atria – ventricular valves and in the outflow tract , in the first trimester of the fetal development . Collagen type 1 is present in the fibrosa layer of the aortic valve , Versican in the leaflet tip and close to the arterial wall , in the second semester of the fetal development . The valvular histoarchitecture in the 19 years old adolescent is similar to the one in the second semester of the fetal development .

The bio – functionalised prosthetic leaflet has PLA – PEGdma scaffold , Collagen type 1 gel layer , and Versican tip , in a proof – of – principle experiment . The polymeric scaffold requires further structural modifications to have a biocompatible material with a set of mechanical properties closer to the one of the human heart valve .

References :

Hinderer S . , Seifert J . , Votteler M . , Shen N . , Rheinlaender J . , Schaffer T . E . , Schenke – Layland K . ( 2014 ) “ Engineering of a bio – functionalized hybrid off – the – shelf heart valve ” , Biomaterials , 35 ( 7 ) , 2130 – 2139 .

Hyun K . , Wilhelm M . , Klein C . O . , Cho K . S . , Nam J . G . , Ahn H. , Lee S . J . , Ewoldt R . H . , McKinley G . H . ( 2011 ) “ Progress in Polymer Science ” , 36 ( 12 ) , 1697 – 1753 .