Comparison of the proliferation rate for human chondrocytes obtained using different culture systems. The proliferation rate was determined relative to the initial number of cells that were seeded
\r\n\tIn the book the theory and practice of microwave heating are discussed. The intended scope covers the results of recent research related to the generation, transmission and reception of microwave energy, its application in the field of organic and inorganic chemistry, physics of plasma processes, industrial microwave drying and sintering, as well as in medicine for therapeutic effects on internal organs and tissues of the human body and microbiology. Both theoretical and experimental studies are anticipated.
\r\n\r\n\tThe book aims to be of interest not only for specialists in the field of theory and practice of microwave heating but also for readers of non-specialists in the field of microwave technology and those who want to study in general terms the problem of interaction of the electromagnetic field with objects of living and nonliving nature.
",isbn:"978-1-83968-227-8",printIsbn:"978-1-83968-226-1",pdfIsbn:"978-1-83968-228-5",doi:null,price:0,priceEur:0,priceUsd:0,slug:null,numberOfPages:0,isOpenForSubmission:!1,hash:"8f6a41e4f5ce0e9c48628516d7c92050",bookSignature:"Prof. Gennadiy Churyumov",publishedDate:null,coverURL:"https://cdn.intechopen.com/books/images_new/10089.jpg",keywords:"Electromagnetic Wave, Microwave Energy Application, Electromagnetic Energy Generation, Intelligent Microwave Heating, Microwave Organic Chemistry, Microwave Reactor, Microwave Discharge, Microwave Plasma, Microwave Drying System, Tissue Microwave Heating, Measurement Automation, Industrial Microwave Process",numberOfDownloads:224,numberOfWosCitations:0,numberOfCrossrefCitations:0,numberOfDimensionsCitations:0,numberOfTotalCitations:0,isAvailableForWebshopOrdering:!0,dateEndFirstStepPublish:"July 3rd 2020",dateEndSecondStepPublish:"July 24th 2020",dateEndThirdStepPublish:"September 22nd 2020",dateEndFourthStepPublish:"December 11th 2020",dateEndFifthStepPublish:"February 9th 2021",remainingDaysToSecondStep:"7 months",secondStepPassed:!0,currentStepOfPublishingProcess:5,editedByType:null,kuFlag:!1,biosketch:"Prof. Gennadiy I. Churyumov is a professor at two universities: Kharkiv National University of Radio Electronics, and Harbin Institute of Technology and a senior IEEE member.",coeditorOneBiosketch:null,coeditorTwoBiosketch:null,coeditorThreeBiosketch:null,coeditorFourBiosketch:null,coeditorFiveBiosketch:null,editors:[{id:"216155",title:"Prof.",name:"Gennadiy",middleName:null,surname:"Churyumov",slug:"gennadiy-churyumov",fullName:"Gennadiy Churyumov",profilePictureURL:"https://mts.intechopen.com/storage/users/216155/images/system/216155.jfif",biography:"Gennadiy I. Churyumov (M’96–SM’00) received the Dipl.-Ing. degree in Electronics Engineering and his Ph.D. degree from the Kharkiv Institute of Radio Electronics, Kharkiv, Ukraine, in 1974 and 1981, respectively, as well as the D.Sc. degree from the Institute of Radio Physics and Electronics, National Academy of Sciences of Ukraine, Kharkiv, Ukraine, in 1997. \n\nHe is a professor at two universities: Kharkiv National University of Radio Electronics, and Harbin Institute of Technology. \n\nHe is currently the Head of a Microwave & Optoelectronics Lab at the Department of Electronics Engineering at the Kharkiv National University of Radio Electronics. \n\nHis general research interests lie in the area of 2-D and 3-D computer modeling of electron-wave processes in vacuum tubes (magnetrons and TWTs), simulation techniques of electromagnetic problems and nonlinear phenomena, as well as high-power microwaves, including electromagnetic compatibility and survivability. \n\nHis current activity concentrates on the practical aspects of the application of microwave technologies.",institutionString:"Kharkiv National University of Radio Electronics (NURE)",position:null,outsideEditionCount:0,totalCites:0,totalAuthoredChapters:"2",totalChapterViews:"0",totalEditedBooks:"0",institution:null}],coeditorOne:null,coeditorTwo:null,coeditorThree:null,coeditorFour:null,coeditorFive:null,topics:[{id:"24",title:"Technology",slug:"technology"}],chapters:[{id:"74623",title:"Influence of the Microwaves on the Sol-Gel Syntheses and on the Properties of the Resulting Oxide Nanostructures",slug:"influence-of-the-microwaves-on-the-sol-gel-syntheses-and-on-the-properties-of-the-resulting-oxide-na",totalDownloads:94,totalCrossrefCites:0,authors:[null]},{id:"75284",title:"Microwave-Assisted Extraction of Bioactive Compounds (Review)",slug:"microwave-assisted-extraction-of-bioactive-compounds-review",totalDownloads:12,totalCrossrefCites:0,authors:[null]},{id:"75087",title:"Experimental Investigation on the Effect of Microwave Heating on Rock Cracking and Their Mechanical Properties",slug:"experimental-investigation-on-the-effect-of-microwave-heating-on-rock-cracking-and-their-mechanical-",totalDownloads:28,totalCrossrefCites:0,authors:[null]},{id:"74338",title:"Microwave Synthesized Functional Dyes",slug:"microwave-synthesized-functional-dyes",totalDownloads:21,totalCrossrefCites:0,authors:[null]},{id:"74744",title:"Doping of Semiconductors at Nanoscale with Microwave Heating (Overview)",slug:"doping-of-semiconductors-at-nanoscale-with-microwave-heating-overview",totalDownloads:45,totalCrossrefCites:0,authors:[null]},{id:"74664",title:"Microwave-Assisted Solid Extraction from Natural Matrices",slug:"microwave-assisted-solid-extraction-from-natural-matrices",totalDownloads:25,totalCrossrefCites:0,authors:[null]}],productType:{id:"1",title:"Edited Volume",chapterContentType:"chapter",authoredCaption:"Edited by"},personalPublishingAssistant:{id:"252211",firstName:"Sara",lastName:"Debeuc",middleName:null,title:"Ms.",imageUrl:"https://mts.intechopen.com/storage/users/252211/images/7239_n.png",email:"sara.d@intechopen.com",biography:"As an Author Service Manager my responsibilities include monitoring and facilitating all publishing activities for authors and editors. From chapter submission and review, to approval and revision, copyediting and design, until final publication, I work closely with authors and editors to ensure a simple and easy publishing process. I maintain constant and effective communication with authors, editors and reviewers, which allows for a level of personal support that enables contributors to fully commit and concentrate on the chapters they are writing, editing, or reviewing. I assist authors in the preparation of their full chapter submissions and track important deadlines and ensure they are met. I help to coordinate internal processes such as linguistic review, and monitor the technical aspects of the process. As an ASM I am also involved in the acquisition of editors. 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by",editors:[{id:"4782",title:"Prof.",name:"Bishnu",surname:"Pal",slug:"bishnu-pal",fullName:"Bishnu Pal"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}}]},chapter:{item:{type:"chapter",id:"19025",title:"Direct Use of Resorbable Collagen-Based Beads for Cell Delivery in Tissue Engineering and Cell Therapy Applications",doi:"10.5772/24097",slug:"direct-use-of-resorbable-collagen-based-beads-for-cell-delivery-in-tissue-engineering-and-cell-thera",body:'Tissue engineering and cell therapy represent significant emerging technologies in medicine. Tissue engineering is‚ "the persuasion of the body to heal itself, through the delivery to the appropriate sites of molecular signals, cells and supporting structures" (Williams, 1999). Various approaches have been described which are labelled tissue engineering, which form a continuum from complex regeneration of scaffold-based tissues to more simple cell-based therapies, where these supporting structures are usually absent or are present in the form of gels simply to aid cell delivery or encapsulation. In tissue engineering the implant to be delivered may frequently be a near mature functional product or a cell-seeded tissue scaffold, both derived in a bioreactor (Haycock, 2011). For cell-based therapies, a bioreactor is not necessary but may be used to amplify the cell numbers for the therapy. Indeed, a key element for both approaches is that they normally require significant expansion of the replacement cells, which have typically been derived in low numbers from small biopsy samples (Brittberg et al., 1994; Chiang et al., 2005; Thissen et al., 2005). A consequence of this expansion is that there can be a significant loss in the quality of cells available for the procedure.
In many cases, monolayer cell culture has been the method of choice for cell expansion (Brittberg, 1999; Brittberg et al., 1994; Henderson et al., 2007). However, while this method is well established and simple it has problems. One of these is that certain cells may de-differentiate and lose their phenotype and biochemical characteristics (Arterburn et al., 1995; Mallein-Gerin et al., 1991). For example, in extended culture, chondrocytes lose their rounded morphology (Fig. 1A) and develop an extended, fibroblast like appearance (Fig. 1B). Also, accompanying this morphological change, matrix biosynthesis switches from type II collagen to type I collagen (Aulthouse et al., 1989; Lefebvre et al., 1990; von der Mark et al., 1977). It is clear that the collagen produced changes from type II collagen (Fig. 2A), which is the characteristic collagen of articular, hyaline cartilage (Mayne, 1989; Miller & Matukas, 1969) to type I collagen (Fig. 2B), which is a mechanically-inferior fibrocartilage (Chiang et al., 2005; Wegener et al., 2009). Type I collagen is the principal collagen of many tissues, including skin, bone, ligament and tendons. It is also present in poorly functioning fibrocartilage which can be observed in some arthritic conditions (Aulthouse et al., 1989; Lefebvre et al., 1990; von der Mark et al., 1977). Depending on the cell type, variations in media or other conditions can potentially be used to extend the control on phenotype and biochemical characteristics. For example, lowering of the amount of oxygen has been suggested for control of chondrocyte phenotype in monolayer cultures (Malda et al., 2003), but the positive effect may be limited, possibly to around 10 days (Malda et al., 2003, Oesser & Seifert, 2003), which is less time than would typically be needed for cell expansion for cell therapy applications (Brittberg et al., 1994). Stem cells, especially embryonic stem cells have particular difficulties in maintaining stemness in culture and current trends are aimed at developing specialised media or scaffold-based bioreactors to expand these types of cells.
Chondrocytes have been a cell type of particular interest as they are the cell used in a major cell therapy application, autologous chondrocyte implantation (ACI). The development of ACI has proved to be a particularly useful innovation for certain types of cartilage defects (Brittberg et al., 1994; Brittberg, 1999), and is now an established cell-based therapy for regeneration of certain types of articular cartilage damage. Defects of articular cartilage in the knee do not have the capacity to self repair (Hippocrates, cited by Hunter, 1742-1743). A range of surgical techniques have been proposed for repair of damage and pain relief. These include debridement, abrasion, mosaicplasty and microfracture (Bedi et al., 2010). A range of grafting options have also recently been developed, including osteochondral autografts and allografts and periosteal grafting (Hunziker, 2001; Shah et al., 2007). In ACI a cartilage biopsy sample is taken from the patient that yields low numbers of chondrocytes, which then must be expanded several fold to give sufficient cells for the regeneration procedure. In ACI, the cell expansion takes place in monolayer culture and may take 21 days or longer (Brittberg et al., 1994; Chiang et al., 2005; Henderson et al., 2005). As a consequence, the majority, if not all, of the implanted cells are likely to have lost their chondrocyte characteristics. This compromises the regeneration of a true hyaline cartilage after implant (Brittberg, 1999; Hunziker, 2001; Temenoff & Mikos, 2000). Several other variants on this chondrocyte-based techniques have subsequently been proposed to give tissue regeneration constructs that are placed and secured in the defect (Bartlett et al., 2005; Marlovits et al., 2005). These methods, including ACI, provide methods for pain relief and extend the time period before a total knee reconstruction is required.
There are clear deficiences in using monolayer culture for cell expansion. An alternative culture system that has been developed is based on spinner culture for cells in suspension (McLimans et al., 1957), which was subsequently adapted to use beads as cell microcarriers (van Wezel, 1967). Microcarriers have have been shown to provide clear advantages for cell expansion, as the rate of cell expansion can be considerably greater, and for chondrocytes, the cell phenotype, including the biochemical characteristics, is substantially, if not fully retained (Frondosa et al., 1996; Malda et al., 2003, 2006). Compared to monolayer culture, the mechanical effects that are present in spinner culture may lead to positive effects on certain cell types (Freeman et al., 1994). A wide range of microcarriers have been developed, predominantly based on synthetic materials, which provide a wide range of surface chemistries, such as negatively and positively charged and capable of further modification to introduce biological functions (Jacobson & Ryan, 1982).
The benefits of using spinner culture for chondrocyte expansion for potential application to ACI were initially demonstrated using non-resorbable beads (Frondoza et al., 1996; Lahiji et al., 2000), and spinner culture was shown to be significantly superior to monolayer culture, with excellent proliferation rates achieved while the cells retained their phenotype (Frondosa et al., 1996). In addition, chondrocytes cultured on a variety of bead types in spinner culture systems have a greater ability to re-differentiate to the differentiated phenotype (Lee et al., 2011; Malda et al., 2003).
De-differentiation of chondrocytes in monolayer culture to fibroblast-like phenotype. (A) Cells after 3 days in culture, starting to show some elongated characteristics, (B) Cells after 6 days in culture, showing a predominately elongated, fibroblast-like shape
De-differentiation of chondrocytes in monolayer culture to fibroblast-like phenotype, with collagen production identified by immune-histological staining. (A) Immunostaining for type II collagen production by cells after 3 days in culture. (B) Immunostaining for type I collagen production by cells after 6 days in culture
Various approaches are available for making beads that would potentially be suitable for cell therapy, and several types, for example Cytodex™ beads (Fig. 3A), are commercially available for cell culture. In some cases the beads are biodegradable, for example, resorbable synthetic polymeric (PLGA) beads (Fig. 3B) have been described, including those with surface modifications to enhance cell attachment (Chen et al., 2006; Hong et al., 2008; Thissen et al., 2005).
Chondrocytes in culture on synthetic beads. (A) Culture for 6 days on Cytodex™ beads coated by absorption with type II collagen, showing retention of a round cell morphology. (B) Culture for 12 days on PLGA beads (Thissen et al., 2005) coated by absorption with type II collagen, showing proliferation of cells with rounded morphology
Natural, extracellular matrix tissues can also be used to make beads for spinner culture, and some, for example highly crosslinked gelatin, Cultispher™, are commercially available. Natural tissue-based beads have a range of advantages compared with synthetic beads, including the presence of natural cell binding and growth factor binding sites, a three dimensional environment including variation in surface topography, in vivo resorption using normal biochemical pathways, and when crosslinking is introduced, control of the resorption rate can be achieved (Glattauer et al., 2010). These beads can often be readily processed for cell isolation; for example, mildly crosslinked gelatin beads can be readily dispersed using trypsin or collagenase allowing easy separation of cells by filtration. A wide range of possible beads has been described, along with various approaches for manufacture. Beads can be made of natural tissue components (Glattauer et al., 2010), including those based on powdered acellular dermis (Cymetra™) (Maloney et al., 2004), porous collagen beads that are formed after removal of an alginate carrier (Tebb et al., 2007), or mildly, <0.5% (Glattauer et al., 2010) or heavily, >>1%, cross-linked gelatin (Tao et al., 2003).
If cells are expanded for cell therapy using an in vivo biodegradable, resorbable bead, then there is no need to isolate the cells from the beads prior to administration (Glattauer et al., 2010; Werkmeister et al., 2006). Inclusion of the resorbable beads as an integral part of the therapy procedure provides the advantage of minimizing the extent of cell handling and eliminating a final trypsin treatment to detach cells from the beads. Delivery of a cell/bead construct is also preferably performed using a gel system so that an appropriate distribution of the material is obtained and the cell/bead construct can be readily administered. A gel delivery system could be a synthetic polymer, preferably one that could be cured in situ to prevent migration after delivery of the cell/bead materials, while maintaining sufficient porosity to allow cell respiration and growth (Adhikari et al., 2010; Gunatillake et al., 2006; Werkmeister et al., 2010). Another good material for a delivery system is collagen. When cold solutions of soluble collagen are warmed to body temperature, 37 °C, the soluble collagen forms into fibrils and a gel then forms (Gross and Kirk, 1958). This gel is biocompatible, and has been the basis for soft tissue augmentation technology (Knapp et al., 1978; Kaplan et al., 1983). Hence, with use of cell/bead constructs, where the cells do not require final removal after the expansion phase and can be implanted directly as a cell/bead/gel delivery system, the "curing" of the collagen delivery gel will hold the cell/bead constructs in place. In addition, the potential presence of some extracellular matrix (ECM) that had accumulated during culture (Frondosa et al., 1996; Tebb et al., 2007; Thissen et al., 2005) could assist in retaining the cells at the desired implant location. When resorbable beads are used for cell expansion and then direct delivery of cells, it is possible to design suitable carrier beads with a range of stabilities that matches the implant requirements.
Solid gelatin-based beads with cross-linking throughout the bead were produced as previously described (Glattauer et al., 2010). Briefly, 20% w/v porcine A-type gelatin, 270-300 g Bloom (Sigma, St Louis, MO) in 50 mM acetic acid was heated to 50 °C and dispersed at 10% v/v in olive oil at 37 °C by rapid stirring. After 90 min, the emulsion was transferred to 4 °C and stirred for 30 min. Beads were separated from oil by 3 extractions with 0.2% Triton X-100 in phosphate buffered saline, and then cross-linked by addition of selected concentrations of glutaraldehyde (GA) ranging from 0.005% to 0.1%. Cross-linked beads were further extracted with 0.2% Triton X-100 in phosphate buffered saline, water and finally EtOH. Beads were collected by filtration and freeze dried.
For gelatin/collagen beads containing type I or II collagen, collagen was added to cooled, 37 °C, gelatin to give a 10% w/w collagen to gelatin mixture immediately prior to emulsion formation, as for gelatin only beads. Soluble bovine hide type I collagen and soluble bovine articular cartilage type II collagens were prepared using pepsin digestion (see below).
Collagen beads, based on co-formation with alginate followed by alginate removal were prepared as previously described (Tebb et al., 2007).
Collagen beads can also be formed by an emulsion method, similar to that used to prepare gelatin beads (see above). Type I collagen in phosphate buffered saline, pH 7.3, at 8 mg/ml and 4 °C was dispersed at 10% v/v in olive oil at 12 °C by rapid stirring. After 15 min, the emulsion was heated quickly to 37 °C and stirred very gently. The collagen was allowed to gel for 3 h at 37 °C. The collagen gel beads were then stabilised by addition of 0.1% w/v glutaraldehyde and were crosslinked overnight. Beads were washed, as above for the gelatin beads, collected and freeze dried, followed by ethylene oxide sterilisation prior to use. The typical size range prior to fractionation, eg: by sieving, was 100-200 µm.
Powdered bovine bone particles were obtained from Waitaki Biosciences (Christchurch, NZ) and were sieved to give a 70 to 150 m fraction. For production of demineralised bone (DMB) particles, the bovine bone particles were suspended in either 0.6 M HCl, or 0.5 M EDTA at pH 7.4, and stirred for 16 h at 4°C. After settling, solutions were removed by decanting. Particles were then further extracted each day for 6 days using the same conditions, before being washed and freeze dried (Glattauer et al., 2010). A 70 to 150 m fraction was collected by sieving. The loss of Ca was confirmed as previously described (Glattauer et al., 2010).
Endosteal particles from bovine bone marrow were prepared from fresh sternum from young calves, ~4 weeks old (Nigro et al., 2010). Briefly, dissected pieces of sternum were crushed in liquid nitrogen and the frozen bone marrow powder was freeze-dried. To decellularise the dried particles, they were suspended in water and mixed vigorously for 30 min, and then allowed to settle. The water was replaced with 4% ethanol/0.1% peracetic acid and mixed vigorously for 2 h, and then allowed to settle. The solution was then replaced and the particles mixed with phosphate buffered saline for 15 min, and then allowed to settle. This wash was repeated and then the settled particles were treated with a cocktail of DNase I (50 U/ml) and RNase A (1 U/ml) in 10 mM Tris-HCl/2.5 mM MgCl2/0.5 mM CaCl2, pH 7.6 for 24 h at 37 °C, with gentle agitation. The particles were washed with phosphate buffered saline and then water, and then exchanged into 80% v/v ethanol and sieved to provide a fraction between 40 - 230 µm. To remove any residual calcification, particles were treated with 0.6 N HCl for 16 h at 4 °C, washed three times in sterile water and then stored in 80% (v/v) ethanol.
Fresh bovine testis was obtained from an abattoir. After removal of the external membranes, the tissue comprising the seminiferous tubules was sliced into approximately 5 x 5 x 5 mm pieces and was macerated on a stainless steel wire mesh to break the tissue and release cells with continual irrigation by phosphate buffered saline, including a protease inhibitor cocktail. The tubules were retained on the mesh. Samples were suspended in excess phosphate buffered saline containing protease inhibitor cocktail, and washed and collected by settling 3 times. Washed samples were examined by immunohistology to examine the presence of basement membrane components using antibodies to laminin (Sigma) and collagen type IV (Biodesign, ME) following the methods previously described (Glattauer et al., 2007). Basement membrane samples were fragmented further using Ultra Turrax blender (IKA Werke, Germany). The resulting suspension was examined by microscopy to determine the particle size that was present. The particles were observed to have a maximal dimension of 500 µm. The basement membrane particles can be fractionated if required to give particles of the desired size by use of mesh sieves of appropriate size.
All collagen-based particle types were sieved to collect 70 to 150 m fractions and were sterilised by ethylene oxide, except the seminiferous tubule-derived particles which were sanitised by soaking in 80% EtOH. Bead size distributions were verified by microscopy and image analysis. Unmodified Cytodex™ beads (Amersham Biosciences, Sweden) were used as a synthetic control bead and were sterilised prior to use by autoclaving.
Ovine chondrocytes were isolated from fresh articular cartilage by digestion of 1 mm3 pieces with 10 % trypsin (2 ml/g) (MP Biomedicals), for 1 h at 37 °C, followed by 300 units/ml bacterial collagenase (354 U/ml Clostridium histolyticum, type IAS, Sigma, St Louis, MO) and 760 units/ml hyaluronidase (2 ml/ g) (760 U/ml bovine testicular type IV-S, Sigma, St Louis, MO), for 16 h at 37 °C. Cells were collected through a 70 m filter, and washed and counted prior to seeding. Human articular chondrocytes were obtained from Edward Keller (Cambrex BioScience, Mount Waverley, Australia), (EK 23-7-02, KN8823) and maintained in complete chondrocyte medium (F12:DMEM, 1:1, containing 10% FCS, plus 1 mM glutamine and 10 mM 2-mercaptoethanol); comparable results were obtained with both the ovine and human cells.
Cells were seeded onto the various microcarrier beads and cultured in spinner bottles, with an initial density of 5 x 105 cells/150 mg particles in 50ml DMEM/F12/10% FBS containing 100 g/ml penicillin and streptomycin, at 37 ºC in 5% CO2. Cell seeding was performed with initial 25 rpm intermittent stirring for 2 min every 30 min. After 3 h, the stirring speed was increased to 45 rpm, intermittently as above, for 1.5 h, followed by continuous stirring at 15 min intervals at 45, 50, 55 and finally 60 rpm. After 24 h in culture, the efficiency of cell attachment onto the beads was monitored.
Mesenchymal stem cells were obtained and seeded as previously described (Nigro et al., 2010). A settled volume (0.45 ml) of particles was exchanged into 50 ml MEM/20% FCS/5 ng/ml FGF-2 before adding 2.5 x 105 mesenchymal stem cells in a spinner flask. The flask was stirred at 25 rpm for 2 min every 30 min at 37 °C with 5%CO2. After 24 h, the spinner flask was set to stir at 25 rpm continuously. After 7 days, a sample of the mesenchymal stem cells/particle construct was taken and cell viability was assessed using Calcein-AM. Images were captured with a fluorescence microscope (Olympus).
Type I bovine collagen was isolated and purified from bovine dermis by pepsin digestion (1 mg/ml) in 50 mM acetic acid at 4 °C as previously described (Ramshaw, 1986). Type I collagen was collected by precipitation by 0.7 M NaCl, and was the further purified by differential salt precipitation at neutral pH (Trelstad et al., 1976). Type II collagen from bovine articular cartilage was isolated in a similar manner. The purified bovine collagens were dialysed exhaustively against 20 mM acetic acid and then freeze dried and stored at -20 °C.
To prepare collagen gels, purified collagen was dissolved at 3 mg/ml in 50 mM acetic acid. Once dissolved, the solution was taken to 1 M acetic acid and sealed in a dialysis bag and held against 1 M acetic acid for 24 h. It was then dialysed exhaustively against water followed by 20 mM acetic acid and then taken to about 25 mg/ml by air evaporation at room temperature. Finally, while still maintaining the seal of the dialysis tube, it was taken to 4 °C and dialysed against water and then phosphate buffered saline, prior to removal from the dialysis bag under sterile conditions in a biohazard cabinet.
Cells were taken after culture on beads in a spinner culture system. Typically, 14 x 106 cells on 0.5 ml beads were used. This was to mimic the concentration of cells in natural hyaline cartilage. The beads/cells were incorporated by mixing with 0.4 ml of 20 mg/ml type I collagen gel in phosphate buffered saline containing 0.1 ml of 10x concentration cell culture medium. The mixture was then taken into a syringe and centrifuged briefly at low speed to remove any air bubbles and 200 l aliquots dispensed into 96 well tissue culture plate, which was then incubated at 37 °C for 1 h to allow collagen fibril formation and collagen gelation to occur (Gross and Kirk, 1958). The cell-bead-collagen plugs were then transferred into 24 well culture plates, covered with cell culture medium, and kept in culture for up to 21 days. Ascorbate was added daily to a final concentration 50 µg/ml.
Samples were processed for conventional histology to examine the extent of bead and particle degradation as well as the extent of cellular infiltration into the collagen/bead (particle) plugs. Stains used were standard Haematoxylin and Eosin and Alcian Blue at pH 2.5 and pH 5.8.
Immunohistology staining used specific antibodies against collagen types I, II, VI and other extracellular matrix components. Prior to staining with antibodies, cultured cells on beads were washed in warm phosphate buffered saline, and then pre-fixed in ice cold methanol. A range of antibodies was used to assess the type of matrix: goat anti-collagen type II (Southern Biotechnology, Birmingham, AL), goat anti-collagen type I (Southern Biotechnology, Birmingham, AL), mouse monoclonal anti-collagen type VI (1D8-F8/Col6) (Werkmeister et al., 1993) and mouse monoclonal anti-keratan sulphate (5D4, Seikagaku Corp., Tokyo, Japan). Beads were incubated with antibodies for 1 h, washed and then reacted with corresponding FITC-conjugated secondary antibodies, rabbit anti-goat IgG (Southern Biotechnology, Birmingham, AL) or sheep anti-mouse Ig (Silenus, Melbourne, Australia). As controls in all studies, primary antibodies were omitted and replicate slides were stained with only secondary FITC-labeled antibodies.
We have successfully prepared a wide range of biological, collagen-based beads from a variety of starting tissues. These include beads based solely on gelatin, the denatured form of collagen, and on gelatin mixed with non-denatured collagen (Fig. 4) which are readily formed by an emulsion method. Further stabilisation by crosslinking is then used, as otherwise the gelatin beads would not be stable at the 37 °C required for the subsequent cell culture. In the present case, glutaraldehyde has been used as the crosslinking agent. Variation in the extent of the glutaraldehyde crosslinking allows control of the rate of turnover of the beads (Glattauer et al., 2010). A wide range of other crosslinking agents are also possible (Ramshaw et al., 2000) which have also been used successfully in biomedical materials for clinical applications. The gelatin beads formed regular spheres with a reasonably smooth surface (Fig. 4A), although at high power, using SEM, some texture is present (Glattauer et al., 2000) The gelatin beads containing either 10% w/w collagen type I or type II were again uniform spheres, but in this case both showed some surface texture (Fig. 4C,\n\t\t\t\t\tFig. 4E). When the beads were dehydrated in EtOH, the gelatin beads retained their spherical shape (Fig. 4B), while the collagen containing beads became irregular in shape (Fig. 4D,\n\t\t\t\t\tFig. 4F). However, when these beads were re-hydrated for cell culture they generally returned to their previous spherical shape.
Beads can also be made from collagen alone, without the gelatin component, by various approaches. For example, the emulsion approach used for gelatin (Fig. 4A) can be adapted for use with collagen. In this case the collagen solution at neutral pH must be kept cold during the emulsification stage, so as to prevent fibril formation. Once an emulsion has been formed, it is then warmed to 37 °C which causes the collagen to form fibrils (Gross & Kirk, 1958) and then stable gel particles. Like the gelatin beads, these can then be further stabilised by crosslinking using glutaraldehyde or other reagents (Fig. 5).
Alternatively, collagen beads can be formed by the use of a carrier during the bead forming stage, which is subsequently removed. For example (Fig. 6) alginate has been used as an easy and effective carrier system. In this case a mixture of alginate with collagen that contains a high proportion, ~30-40%, of collagen can be formed, and beads are produced by dropwise (aerosol) addition to 1.5% CaCl2 (Tebb et al., 2007). The CaCl2 leads to the precipitation of the alginate to form stable beads, and the collagen can be stabilised by fibril formation and crosslinking as required. The alginate phase can be removed by treatment with Na citrate, giving a collagen only bead (Tebb et al., 2007). An advantage of this method is that the collagen beads are quite porous, and cell infiltration readily occurs (Tebb et al., 2007). Other materials could be used as carriers, including gelatin and other proteins. The collagen triple-helix structure is generally highly stable to proteolytic action, and pepsin treatment is often used during its preparation (Miller & Rhodes, 1982). So a protein carrier, such as gelatin, can be removed by proteolysis, leaving porous collagen beads.
Collagen beads can also be formed directly from tissue. For example, Cymetra™ is prepared from acellular human dermis by fragmentation (Maloney et al., 2004) and is used clinically for tissue augmentation and equivalent procedures. The particles can also be used readily as a cell growth substrate.
An alternate source of collagen beads or particles is from natural collagen containing tissues. The principle protein component of bone is type I collagen. Hence bone particles themselves may present surface collagen to cells to enhance binding (Fig. 7A). Bone particles can be refined using demineralisation, for example with EDTA or HCl, to produce beads enriched of collagen (Glattauer et al., 2010) (Fig. 6B). There are also low levels of growth factors and other biological macromolecules that can add to the effectiveness of the collagen particles in promoting cell adhesion and proliferation. The type and quantity of these molecules may vary depending on the method used to demineralise the tissue and may vary between batches (Wildermann et al., 2007). However, when chondrocytes were grown on particles prepared by both these methods no significant difference was seen in the proliferation rate for the cells (Glattauer et al., 2010). The bone and demineralised bone particles differ significantly from the previous gelatin and collagen beads in that they are irregular in shape, have an irregular surface topography, and do not need additional crosslinking as they are already fully stabilised by the naturally occuring collagen crosslinks. Any soluble collagen would be removed during the demineralisation process. The demineralised bone particles are preferred for spinner culture as they are more bouyant and hence easier to use. Whereas the area of smooth spherical beads in an aliquot, such as synthetic beads or gelatin beads, can be readily calculated from the size distribution of the beads, this is more difficult for irregular shaped beads, and beads with variations in surface topology.
Production of gelatin and gelatin/collagen beads. Beads in phosphate buffered saline (A, C, E) and after dehydration in EtOH (B, D, F). After rehydration, these beads returned to their original size and shape. (A, B) Gelatin beads, average hydrated bead size prior to fractionation, 106 ± 20m. (C, D) Gelatin/10% w/w type I collagen beads, average hydrated bead size prior to fractionation, 169 ± 53m. (E, F) Gelatin/10% w/w type II collagen beads, average hydrated bead size prior to fractionation, 159 ± 53m
Collagen beads prepared by an emulsion method
Production of type II collagen beads using an alginate carrier (Tebb et al., 2007), with type II collagen at 6.6 mg/ ml and alginate at 0.3% during initial bead formation. Bead size ~ 0.7 mm
Examples of (A) bone particles, and (B) demineralised bone particles
The gelatin, collagen and bone particles are all based principally on type I collagen. Other collagens are abundant in different tissues. For example, type II collagen is the collagen found in normal hyaline cartilage (Mayne, 1989; Miller and Rhodes, 1982) and which could be used to make type II collagen particles by tissue fragmentation and further processing, if required, to remove other components such as proteoglycan. Similarly, type III collagen is a major component of blood vessels, along with type I collagen and elastin (Hanson & Bentley, 1983; McCloskey and Cleary, 1974). Hence, particles rich in type III collagen could be prepared by processing arterial tissue. Type IV collagen is the major collagen component of basement membranes. Basement membranes are unique, highly organised supportive sheet-like structures in the extracellular matrix that are formed at the interface between parenchymal cells and their surrounding tissues (Yurchenco et al., 2004). There are different types of basement membranes in the body. Some act as a tissue boundary where certain cells can attach; some can act as filters with selective permeability, and some support very selective cellular differentiation of a number of different cell types, including stem cells. The basement membrane comprises a range of specific macromolecular components. As well as type IV collagen, which may be important for mechanical properties (Yurchenco et al., 2004), the other components are laminin, nidogen and heparan sulphate proteoglycans, such as perlecan, which are believed to interact in a highly ordered manner with type IV collagen (Yurchenco et al., 2004).
Thus, we have shown that by using natural tissues from different origins, it is also possible to make other tissue derived particles. By having different protein and other macromolecules present, these have the potential to provide different interactions with various cell types. An example is the production of particles from the endosteum of bone (Fig. 8) (Nigro et al., 2010). This tissue is proximal to the location of haematopoetic stem cells (Haylock et al., 2007) and so may be useful in the proliferation of this cell type. The particle composition is distinct; examination of the composition using immunohistology showed that they contain perlecan and some laminin, suggesting similarity to a basement membrane, but collagen type IV was not detected (Nigro et al., 2010).
Decalcified endosteal particles
Basement membrane particles can also be formed from a range of other tissues, including placenta, muscle and kidney (Werkmeister et al., 2006). In the present example, particles have been prepared from seminiferous tubules. The tubules are readily isolated from testis tissue (Fig. 9A), and immunohistological analysis has shown that the major components of collagen IV (data not shown) and laminin (Fig. 9B) have been retained during the processing. Further fragmentation can be used to prepare particles (Fig. 9C). The composition of this basement membrane is distinct (Glattauer et al., 2007) showing the presence of various laminin types based on the positive staining for the α3, α4, α5 and α6 chain of collagen type IV and the positive staining of various laminin chains, including the α2 and β2 chains. This composition is distinct from that of the commercial basement membrane material, Matrigel™, which is based on (α1)2α2 type IV collagen and α1β1γ1 laminin (laminin 111)(Kleinman & Martin, 2005). Matrigel™ has been used extensively for addressing cell attachment so a different composition basement membrane may provide a useful alternative option for cell culture.
Seminiferous tubule-based particles. (A) Fragments of isolated seminiferous tubules. (B) Immunostaining of isolated seminiferous tubules, showing the retention of laminin during the processing. (C) Seminiferous tubules particles after further fragmentation of isolated tubules
To be useful, any bead must clearly not be cytotoxic and must allow ready attachment and growth of cells. All the beads and particles described above (Figs. 3-9) meet these criteria. There was no evidence of any cytotoxicity due to residual glutaraldehyde that had been used to crosslink some of the beads.
Gelatin-based beads with chondrocytes after 3 days in spinner culture, showing good attachment and retention of the round chondrogenic phenotype. (A) Gelatin beads, (B) Gelatin/Type I collagen beads, (C) Gelatin/Type II collagen beads
As an example, chondrocytes readily attach to gelatin and gelatin/collagen beads and are viable and starting to proliferate after 3 days in culture (Fig. 10). Similarly, cells readily attach and start to proliferate on bone (Fig. 11A) and demineralised bone particles (Fig. 11B). Attachment and growth of chondrocytes was good on the seminiferous tubule particles, even after only 2 days in culture (Fig. 12).
Chondrocytes growing on bone particles. (A) Sheep chondrocytes on bone particles after 3 days in spinner culture, (B) Human chondrocytes on demineralised bone particles after 4 days in spinner culture
Human chondrocytes growing on bovine seminiferous tubule particles for 2 days
Previous studies (Frondoza et al., 1996) had suggested that cell proliferation of chondrocytes on synthetic microcarrier beads was superior to that found in monolayer culture. The present work using biologically based beads supports this finding (Fig. 13). Thus, over a 21 day period, with cell collection and re-seeding after 7 and 14 days, the spinner culture using both gelatin and demineralised bone particles was significantly better than that found using monolayer cultures. In all cases, the bead experiments were established so that the surface area of the carriers was equivalent to that used in the monolayer cultures. The cell yield for the gelatin beads was almost one order of magnitude greater than with monolayer cell culture (Fig. 13). The performance of demineralised bone particles was even better, being better than 2 orders of magnitude greater than for monolayer culture. As similar culture conditions were used for both the gelatin and demineralised bone particles, this reflects the differences in surface compositions and the surface topography of the two bead types as well as the potential for the demineralised bone particles to contain some active growth factors that may boost cell proliferation (Wildemann et al., 2007). Comparisons in an additional experiment including other bead systems (Table 1) showed that proliferation rates were consistently better for biological, collagen-based beads when compared to monolayer culture and a synthetic bead.
Proliferation of human chondrocytes in monolayer culture and in spinner culture using different microcarrier systems. The initial loading was 0.25 x 106 cells on beads that gave equivalent surface areas of ~125 cm2 (equivalent to 5 T25 monolayer flasks. ● monolayer culture, ♦ spinner culture using gelatin beads, ▲ spinner culture using demineralised bone particles
Culture system | Bead/particle type | Proliferation rate after 21 days |
Monolayer | n/a | 5.5 |
Spinner Culture | Cytodex | 6.5 |
Spinner Culture | Gelatin | 38.4 |
Spinner Culture | Bone | 37.2 |
Spinner Culture | Demineralised bone | 47.2 |
Comparison of the proliferation rate for human chondrocytes obtained using different culture systems. The proliferation rate was determined relative to the initial number of cells that were seeded
In the present study we have focussed principally on chondrocytes. In these cases, with a variety of different beads in spinner culture, newly formed extracellular matrix was found to accumulate around the beads and cells, causing clumps of tissue-like material to form in the cultures. This was clearly evident by 7 days, for example, with gelatin and gelatin/collagen beads (Fig. 14), where a matrix of cells and collagen can be seen holding most beads in clumps.
Growth of chondrocytes on various beads after 7 days in spinner culture, showing the clumping of beads due to the formation of new extracellular matrix. (A) Gelatin beads, (B) Gelatin/Type I collagen beads, (C) Gelatin/Type II collagen beads
Histology, using Haematoxylin and Eosin staining (Fig. 15) shows the presence of this new connective tissue with cells throughout. Not all cells have migrated from the beads into this new matrix and some are still stained on the bead surfaces. A similar result is seen for chondrocytes grown in spinner culture on bone particles (Fig. 15C).
Haematoxylin and Eosin staining of the growth of chondrocytes on various beads after 7 days in spinner culture, showing the new extracellular matrix that is formed (A) Gelatin beads, (B) Gelatin/Type I collagen beads, (C) Bone particles
With the porous collagen beads, the initial phase of cell proliferation was mainly infiltration of the collagen bead and less accumulation of cells and extracellular matrix external to the particles and hence less clumping was observed than with the gelatin beads (Fig. 16). On the other hand, a small amount of clumping of particles was also observed when human mesenchymal stem cells were grown on decalcified endosteal particles for 7 days. Cell attachment was good and proliferation had commenced, but was not as advanced as seen for chondrocytes on other bead systems (Fig. 17).
Growth of chondrocytes for 7 days on porous collagen beads prepared through alginate removal (Tebb et al., 2007), showing infiltration of the beads by cells
Growth of human MSCs for 7 days on decalcified endosteal particles. (A) Cells attached to particles, (B) staining with Calcein-AM, which stains cell cytoplasm, and (C) Merging of the previous 2 images
Haematoxylin and Eosin staining of the growth of chondrocytes on various beads after 21 days in spinner culture, showing the formation of new extracellular matrix. (A) Gelatin beads, (B) Gelatin/Type I collagen beads, (C) Gelatin/Type II collagen beads
After longer periods of culture, 14-21 days, continued extracellular matrix production was seen when ascorbate was included, and cell proliferation also occured. After 21 days, Haematoxylin and Eosin staining of materials containing gelatin and gelatin/collagen beads showed extensive matrix between particles (Fig. 18), which were now more separated than in the 7 day clusters (Figs. 14 & 15). In addition, the particles containing collagens were now showing erosion and degradation (Fig. 18B,\n\t\t\t\t\tFig. 18C), while the gelatin alone particles were still substantially intact (Fig. 18A). Alcian Blue staining of these samples confirmed that the gelatin alone beads were more stable than those containing collagen (Fig. 19) and showed that the extracellular matrix contained proteoglycans/glycosaminoglycans which are key products of chondrocytes and are indicators of proper cell function and cartilage formation. The staining was strongest around the periphery of the particles where cells were still attached. The chondrocyte cell phenotype was also examined by immunohistology (Fig. 20). This showed that after 14 days in spinner culture the chondrocytes were still producing type II collagen, the correct collagen for articular (hyaline) cartilage formation, and the amount of type I collagen being produced was very low (Fig. 20).
Alcian Blue staining of the growth of chondrocytes on various beads after 21 days in spinner culture, showing the formation of proteoglycans within the new extracellular matrix. (A) Gelatin beads, (B) Gelatin/Type I collagen beads, (C) Gelatin/Type II collagen beads
Immunohistological staining of chondrocytes grown on gelatin beads in spinner culture for 14 days, showing continued production of type II collagen and little, if any type I collagen production. (A) Immunostaining for type II collagen, (B) Immunostaining for type I collagen
It was noted earlier, in the Introduction, that if cells are expanded for cell therapy on biodegradable, resorbable beads, then there is no need to isolate the cells from the beads prior to administration (Glattauer et al., 2010; Werkmeister et al., 2006), and that this has the advantage of minimizing the extent of cell handling that is required. In such a system, delivery of the cells/bead in a gel or gel forming material has further advantages. The presence of the gel stabilises the cell implant, and aids in the delivery of the cell/bead constructs.
Collagen type I gel is an ideal FDA approved protein that can be used as the delivery vehicle. When mixed with the cell/bead constructs the mixture can still be readily delivered through a 22-gauge needle, while the particles with bound cells remain uniformly dispersed and do not settle out rapidly (see Fig. 21A, where the particles have been stained to highlight the distribution in the gel). On warming to 37 °C for 1 h, the collagen formed a fibrous, solid gel that was able to be picked up, if required (Fig. 21B). This would not be necessary for in situ application of the mixture by injection into tissue. Indeed, we have injected a solution of cell/bead constructs in collagen gels and found that the mixture gelled in situ and gel contraction was markedly reduced by the presence of the beads (data not shown).
The cell/bead constructs embedded in a collagen gel can be maintained in culture for greater than 3 weeks. During the initial stages of culture, within 3 days, if synthetic, unmodified Cytodex™ beads are used, the cells show a greater affinity for the collagen of the gel than the bead and migrate from the beads into the surrounding gel. Cell migration is much slower from gelatin and other biologically–seeded beads where there seems a possible better affinity for the bead.
Delivery of cell/bead constructs using a collagen gel. (A) Stable dispersion of gelatin beads, after staining, in a 1% collagen type I gel. (B) Collagen gel with beads, after heating to 37 °C forms a mechanically stable gel
After 3 weeks in static culture, the cell/gelatin beads/collagen gel construct had a tissue-like appearance (Fig. 22), and the loose structure of the original gel construct had formed a firmer, more compact structure, which showed very minimal contraction. When cells are seeded into collagen gels alone, the gel contracts significantly, by ~50% or more, within the first 7 days of culture (Ramshaw et al., 1991) and again forms a more compact tissue-like format.
A collagen type I gel with cells/gelatin beads after 3 weeks in static culture, showing the tissue–like material that is formed
The presence of the beads appears to retard any contraction of the collagen gel, while at the same time the synthesis of extracellular matrix augments the structure. Chondrocytes in culture can produce matrix metalloproteinases (Lefebvre et al., 1990) which could degrade the construct. The presence of the beads, where the cells have formed an established matrix prior to incorporation in the collagen gel may minimise the production of these proteinases. However, after 2 to 3 weeks of culture some erosion of gelatin beads was seen (Fig. 23A,\n\t\t\t\t\tFig. 23B). The extent of this erosion was greater than when the collagen delivery gel was absent (Fig. 18A).
Histological examination of cell-bead constructs in a collagen gel after 2-3 weeks in culture. (A) Haematoxylin and eosin staining after 2 weeks of culture, (B) Alcian Blue staining after 3 weeks in culture, and (C) Immunostaining of Type II collagen after 2 weeks in culture
The presence of new extracelluar matrix can be seen by histochemical and specific immunohistological staining. Heamatoxylin and Eosin staining after 2 weeks of culture (Fig. 23A) shows extensive cell infiltration of the collagen gel, and new collagen synthesis around the beads and within the gel. Alcian Blue staining after 3 weeks of culture (Fig. 23B) shows significant deposition of proteoglycan/glycosaminoglycan within the gel surrounding the beads, as well as very strong staining around the beads themselves. This staining was not present in the original collagen gel and is therefore indicative of new matrix production. This is also the case for type II collagen production. Immunohistology after 2 weeks of culture (Fig. 23C) shows that there is type II collagen accumulation, possibly at low levels, throughout the matrix, while there is strong staining around the beads.
The data presented in this Chapter show that a wide variety of collagen-based beads and particles can be made which are useful for spinner culture. The beads can be made with a range of tissue-like conformations, ranging from denatured collagen (gelatin) through to beads that are based around basement membrane and collagen type IV structures. The beads, including those that have been further crosslinked using glutaraldehyde, were not cytotoxic and readily supported cell growth. The cell growth in spinner culture was excellent and within 7 days there was normally accumulation of new extracellular matrix. For cell therapy applications, the proliferation rates of chondrocytes were excellent, several fold better than monolayer cultures and synthetic beads. While most biological tissue based particles or biologically fabricated beads were excellent cell carriers, demineralised bone particles and gelatin beads were particularly good, the former possibly augmented by residual growth factors. In a therapeutic application the demineralised bone-based beads could have a fairly long resorption rate. If a shorter resorption rate was preferred, then gelatin beads are ideal, and the rate of resorption can be controlled by varying the extent of glutaraldehyde stabilisation (Glattauer et al., 2010). After longer times in culture, exceeding 21 days, the chondrocytes maintained their phenotype and were still producing macromolecules characteristic of native articular cartilage, such as proteoglycans and type II collagen. For therapeutic applications a superior approach is to deliver the cell/bead constructs in a delivery gel; in the present study a gel of type I collagen has been used. In this case the choice and design of the beads can be selected to match the requirements with respect to cell type and bead resorption rate. These gel constructs were shown to be stable, retaining their size and becoming more robust with new matrix accumulation. Again the chondrocytes maintained their phenotype over 21 days, as shown by proteoglycan and collagen type II production.
Together, these data show the utility of expanding cells on biological, collagen-based beads in spinner culture for tissue engineering and cell therapy applications. Further, these data also show that an ideal approach is to use resorbable beads for cell expansion and then direct delivery of cells still attached to the beads, where it is possible to design carrier beads with a range of stabilities that matches the implant requirements. An ideal way of achieving this implant is to combine the cells/beads together with a delivery gel. This proposed approach has been used in an animal trial for articular cartliage repair, where it was compared to a standard ACI approach (Chiang et al., 2005). The results strongly confirmed the validity of the proposed method, with an independent multifactor scoring system showing that the cell/bead/gel approach was significanly better than a standard ACI procedure and a non-cell control in a mini-pig model (Chiang et al., 2005). The approach can be further improved by examining delivery systems that allow the delivery and stabilisation of the cell/bead construct by arthroscopy, removing the need for open knee surgery. Equally, although the present techniques use cartilage biopsies obtained from non-load bearing cartilage, recent developments in cell biology, for example the ready acquisition of mesenchymal stem cells which can be differentiated into chondrocytes, opens up further opportunities that may minimise invasive surgery for the patient.
Since the turn of the twentieth century, the air temperature has risen, expected to proceed to rise as a result of climatic variability. These rises in temperatures may trigger high-temperature stress (HTS): serious damage to plants [1, 2]. As a result, food and feed security have become a crucial challenge under current prevailing agro-climatic conditions [3, 4, 5]. Climate modeling has indicated that high temperature during the day and night is threatening global agriculture production system [6]. The result is that maize crop yield is reduced globally [7, 8]. Maize is one of the important crops being cultivated globally with a wide range of uses, and it is an important food crop in the world [9, 10, 11], it has been primarily aimed for increasing yield, quality, and stability under different environments [12, 13, 14, 15]. Maize is an important component of human food, animal feed, and biofuel industries [5]. It ranks top among cereal crops globally and becomes raw material of numerous food and feed industries. Among growth limiting factors, heat stress has a major effect on maize growth and nutrient composition at different developmental stages. Since several abiotic stresses occur simultaneously, such as drought stress and heat stress, the development of improved breeding procedures is essential for increasing the maize productivity and quality [16]. There is a crucial need for further research to develop maize genotypes tolerant to high temperature and drought stress.
Various physiological and biochemical processes govern plant growth and yield. Stomatal conductance, for example, regulates water loss as transpiration as well as an influx of CO2 for its fixation in the Calvin cycle. Several researchers had suggested that the stomatal conductance is an important indirect heat-tolerant selection criterion in crops [17]. Similarly, osmoprotectants and chaperone proteins got an important part in the adaptive reaction of maize to heat stress and combined stresses. Moreover, leaf senescence-related proteins enhance maize tolerance to combined heat and drought stress [18]. Introgression of these traits in locally acclimated maize hybrids through potential donor hybrids helps in developing maize hybrids tolerant to heat and drought stress. Moreover, identification of donor genotypes possessing favorable traits is important in heat stress breeding programs [19]. Therefore, the present review aimed to evaluate the updates on the effect of heat stress on different plant developmental stages, some physiological and biochemical traits, yield and yield traits of maize. Moreover, this review included updates on various strategies used to improve crop tolerance against heat stress including, conventional breeding strategies, management practices, shotgun approaches, and molecular biology-based strategies. Given the critical analysis of success and limitations for improving maize crop productivity under heat stress, future directions for research are also suggested.
Temperature above 350C for a prolonged period is considered unfavorable for crop growth and development and, particularly 400C during flowering and grain filling have severe negative impacts on grain yield [5]. Plants under heat stress exhibited significantly reduced stomatal conductance resulting in a reduced rate of photosynthesis. Excessive heat also causes a reduction in net photosynthesis, leaf area, reduced biomass accumulation and seed weight [20]. However, heat-tolerant maize varieties that produced the highest metabolites are not usually high yielding varieties. The heat-tolerant maize varieties are usually characterized by the reduced plant height, leaves plant−1, and leaf area index ultimately reduced the yield. Therefore, several factors should be put into consideration when selecting for heat tolerance in maize. At the cellular level, HTS triggers the appearance of certain genes and increases the accumulation of certain metabolites that may enhance the heat enduring ability of plants [21]. Generally, remarkable genotypic variations in the stomatal conductance were observed [22, 23]. Stomatal conductance, which is a key trait of the photosynthetic leaf, was significantly influenced by abiotic stresses [24]. Delay canopy senescence due to various light interceptions by green leaf area has been reported to be necessary for high productivity of hybrid maize under normal watering and drought stress [16]. The impinging of high-intensity light to plants can lead to permanent damage to membrane structure [20]. The cell membrane is considered the first physiologically sensitive structure to the high temperature and becomes functionally inactive at heat stress [25]. Membrane function and cell wall stretch have inverse relation [26, 27]. Continuous damage in the biological membrane may downregulate the mobility of water, ions, and soluble organic solid molecules within plant cell membranes; hence carbon of production, transport, and accumulation may be affected by these factors. Membrane stability could be used as an assessment of high-temperature tolerance of plants. It is the most appropriate and convenient test; leakages of electrolytes at a high temperature can be measured by this test [28].
Soil plant analyses development (SPAD) value and grain yield have a significant relationship after anthesis, but no positive association has been noticed during the middle and later grain-filling stages [29, 30]. During HTS, the chlorophyll biosynthesis gene gets downregulated [31]. Experimental observation has suggested that the differences among net photosynthetic ratio after exposure to high temperatures were related to the conversion of the chlorophyll “a” into chlorophyll b ratio; due to low chlorophyll “a” and rapid leaf senescence, the photosynthetic rate is negatively affected [32]. HTS induces several metabolic events at the cellular and subcellular levels. The heat stress influences the production of ROS and oxidative stress as well [33, 34, 35]. The antioxidative defense system includes both enzymatic and nonenzymatic antioxidants that are shown to participate in response to the development of oxidative stress influenced by heat stress [21].
Scientists showed that rather extreme heat intensity could cause serious tissue damage as well as mortality may arise in a matter of minutes and could ultimately be due to a massive collapse of cell organization [36]. Damages can occur just after deep-term exposures at moderate to maximum heat stress. Informal and gradual damages caused by high temperatures include chlorophyll and mitochondrial destruction of enzymatic activity, protein catabolism impairment, protein deterioration, and cell turgidity looseness [37]. As can be seen in studies, with either the introduction of heat-shocked proteins, plants and animals react to high-temperature pressure [38, 39]. These are intended to avoid species from the harmful impacts of heat stress as well as other sources of pressure [40]. A simple reaction to high-temperature stress is a reduction in regular cellular metabolism. This drop is especially marked at 45°C. The fall in the natural production of protein also goes hand in hand with increased expression and transcription of a fresh set of molecules identified as heat-shock proteins (HSPs) [41]. Previous studies demonstrated that in Zea mays, high-temperature stress reduced the protein production and changes the chemical structure of these proteins [42]. Heat stress at the reproduction phase negatively affects the physiology of plants like flower initiation, source-sink relationship, and falling of pods, which ultimately decreases the number of seeds [43]. High-temperature stress is most crucial for the physiological traits of crop plants. High temperature reduced the number of ears, number of kernels, chlorophyll efficiency, firing of leaf, and blasting of the tassel [44]. Climatic stress like high-temperature stress severely reduces the growth and yield of several crops belongs to Leguminosae (Fabaceae). Heat stress severely reduced the physiological growth development and production of Vigna radiata. Heat stress reduced dry matter production and other yield attributes [45].
HTS hampers the plant growth; particularly germination and seedling emergence are more sensitive [46]. Stressful environment severely reduces the germination and early seedling growth in several crop plants [47, 48]. However, seeds of sensitive crops exposed to 24 and 48 h moderate heat stress exhibited a higher germination rate. Such an increase in seed germination rate due to short-term exposure to moderate heat stress was attributed to the altered expression of gibberellin and abscisic acid biosynthesis genes [49]. The seedling stage is generally considered as the most sensitive stage to stress in maize development [50]. However, the detrimental impact of water deficit stress on the initial phase of growth and seedling establishment of maize plants cannot be underestimated [51, 52, 53].
The appropriate sowing date is important for seed germination and seedling establishment to physiological maturity. The heat-tolerant maize varieties germinated earlier than the non-drought tolerant maize varieties under the critical level of watering. During germination, HTS is associated with an impaired emergency, and a reduced plant stand and plant density [54]. Biochemical components such as soluble sugar and proline increased with increased stress, while starch content and relative water content reduced with increased water deficit [55]. Fluctuations in mean daily temperature (either it is maximum or minimum) disturb seed germination ability [56]. High-temperature stress is the main cause of the reduction in plant yield due to poor germination. [57, 58] studied the impact of high temperature on various developmental phases, especially at seedling emergence in various crop genotypes. Critical periods of stress in maize include seedling establishment stages, rapid growth period, pollination and grain-filling stage. It is proven that in the maize plant with the implementation of stress, not only the leaf area is reduced, but also its growth rate is affected and the appearance of each leaf is delayed [59].
HTS at the grain-filling stage in spring maize is the main obstacle [60]. Temperature beyond 40°C, mainly during flowering and grain filling has a severe impact on plant grain productivity [5]. Grain filling is highly sensitive to drought and heat, due to the involvement of the array of diverse enzymes and transporters, located in the leaves and seeds [45]. During HTS, the stability of the thylakoid membrane structure is reduced, resulting in degrading chlorophyll, which reduces light energy absorption, transfer, and photosynthetic carbon assimilation, and ultimately photosynthesis is reduced. Inhibited photosynthesis decreases the supply of photosynthates to the grain, leading to a serious reduction of kernel weight and grain yield [60, 61, 62]. Delay in the development of reproductive organs might be the result of the reduced cell division and cell elongation processes due to reduced supply of photosynthates and carbohydrate metabolism during the active vegetative growth stages [63].
A projection based on the increased daily maximum temperatures concluded that to increase the maize yields by 12% for the period 2016–2035, improved technologies would be needed [64]. Maize plant can face moderate to high temperature, but temperature above 35°C for a long duration is considered unfavorable for crop growth and development, and temperature beyond 40°C, mainly during flowering and grain filling will have a severe impact on plant grain productivity [5]. Meanwhile, early season temperature increases have induced the maize reproductive period to start earlier, developing the risk of water and heat stress. Declines in time to maturation of maize shown of independence of effects to availability of water, the potential of yield which becoming increasingly limited by warming itself [65]. Irrigation regimes were the major determinant of grain yield during the grain-filling stage in maize while significant differences in the number of kernels per row were obtained among irrigation regimes [66]. A large difference in grain yield is caused due to HTS, which is shown in Figure 1. Tissue injuries inversely influence the photosynthetic rate during heat stress, which can cause leaf damaging and increase the rate of leaf senescence that largely results in decreasing photosynthetic efficiency [44]. Reduced chlorophyll content, including grain yields and oxidative damages, possibly had a direct correlation under heat stress [5, 67]. Previous research studies indicate that high temperature has a severe effect on the cob growth rate as well as biomass partitioning [68]. Many factors including duration of pollen viability, increased kernel abortion rate, lower the rate of cell division in storage tissue (endosperm), decrease in starch synthesis, downregulate the sink capacity of developing kernel, increased rate of sugar accumulation, kernel development, and less/higher enzyme activities could be responsible for the reduction in kernel per row under heat stress [44, 67]. Stress environment leads to a severe reduction in yield of crop plants probably by disrupting leaf gas exchange properties, which not only limit the size of the source and sink tissues, but the phloem loading, assimilate translocation, and dry matter partitioning are also impaired [46]. Unsuccessful fertilization reduces the seed size and increases flower abortion rate owing to high temperature and it has negative effects on plant reproductive phase [69, 70]. Temperature range 0–35oC, is considered suitable for leaf growth, the temperature range 35–40oC has an inverse relation with leaf growth. Temperature beyond 35-40oC is responsible for lower net photosynthetic rate, which further leads to protein aggregation, enzyme inactivation, inhibition of protein synthesis leading to the degradation of protein synthesis [69, 71]. Eventually, an increase in temperature beyond its critical value leads to generating a heat stress that harms the morphological growth, grain yield, and yield-related attributes of two maize cultivars “Xida 319” and “Xida 889” [72].
Differences in total leaf collars, cumulative leaf area, and grain yield of three corn hybrids grown under normal Ames, Iowa temperatures and normal +4°C temperatures.
Temperatures higher than 35°C negatively affect maize grain quality. Grain quality, which is governed by factors including the duration and rate of grain filling and the availability of assimilates, is negatively influenced under water deficit conditions. Similar negative effects of stress were reported on the grain weight of wheat [15, 73, 74, 75]. Variations in flour quality in a hard-grained crop could be related to changes in protein composition due to heat stress during the grain-filling stage [76]. As per the findings of Mousavi et al. [77], heat stress at the flowering stage greatly reduced the starch content due to the reduction in the photosynthetic activities leading to an increase in the grain protein ratio. Usually, maize quality properties are affected by genotypes, environmental factors, and their interactions (Figure 2). Therefore, growth and development of maize are dramatically affected by heat stress leading to reduced grain weight with low starch, crude oil, and protein contents [30]. Grain filling is the most environmentally sensitive phase in maize, which strongly affects grain development quantitatively and qualitatively [7, 15]. Oury and Godin [78] reported a negative correlation between protein contents and grain weight in maize under stress conditions. Association analysis revealed that cob length, thousand-grain weight, and protein contents had a significant relationship with grain yield of maize [79].
Quality of maize is influenced by genotype, environment, and their interaction.
In the previous study, the starch content in waxy maize grain was decreased, whereas protein content was increased, resulting in the change of grain quality [80]. However, the activities of enzymes involved in the synthesis of starch and protein are still lacking [81]. The qualitative and quantitative characteristics of grain productivity are mainly influenced by the environmental fluctuation and these changes inversely influence the development and maturing of seed that affect the seed-filling process and deposition of reserves [80]. Generally, high impinging of light affects negatively in plant productivity by causing premature senescence, decreased seed-filling duration, and enhancing remobilization of photosynthates from source to sink [82]. These factors combined, mainly lowers plant biomass and productivity, and finally lowers the assimilate production and mobilization of the reserve to different developing crops [83]. Generally, it is predicted that gene controlling cell division gets downregulated due to water stress, which could be responsible for the decreased cell number in cotyledons along with endosperm. However, further research is required to find out the actual mechanisms controlling these events. Probably due to low enzyme efficiency or high km carbohydrate gene gets downregulated in developing seedling, resulting in limited availability of sucrose, finally producing reduced seed size [45]. The time of seed filling reduced in pea, soybean, and white lupin, resulting in smaller grains [84]. Heat stress during grain filling markedly decreased starch accumulation in wheat [85] and rice [86].
High-temperature stress decreases the protein concentration in the wheat seeds during seed formation stage [76]. Carbon and nitrogen transmission in the seed is improved with the maximum temperature but C transfer is reduced by the daily temperature fluctuations [87]. Temperature variability effects are more visible on the size of seed than seed N contents [87]. Size of seed and protein concentration in the seed are inversely proportional to each other [88]. High-temperature stress reduces seed production, which ultimately declines the seed protein contents [89]. Protein accumulation in the seeds depends upon high-temperature stress [89]. When high-temperature stress occurs at the seed-filling stage it declines the seed protein contents [89]. When wheat crops are exposed to the high-temperature, glutenin protein production is decreased while gliadins protein production remains stable [90]. Seed protein contents of various crops are decreased after imposing the high-temperature stress, but various amino acid concentrations become low [91]. Heat stress damaged the protective layer of seed and food storage tissues of seed, which is why the quality of seed was deteriorated (Figure 3).
Quality of maize is deteriorated due to heat stress.
Enhancement of the antioxidant defense system is an important strategy to scavenge ROS by antioxidant enzymes [92]. Similar to antioxidant defense, phytohormones such as auxin (indole acetic acid, IAA), cytokinins (CKs), abscisic acid (ABA), ethylene (ET), gibberellins (GAs), salicylic acid (SA), brassinosteroids (BRs), and jasmonates (JAs) have key roles in coordinating various signal transduction pathways during the abiotic-stress response [93]. Many studies have shown that altering cultural practices, such as planting rate [94], planting date [95, 96], the phenological variation of crop cultivars [60, 95] soil management [97], nutrient management [60], and irrigation [60] can positively or negatively modify maize yield response to climate change.
Advancing or delaying the sowing date may be a potent, farmer-friendly and biologically viable strategy to avoid HTS. Earlier findings reported that earlier sowing dates and longer season varieties have overcome the negative effects of climate warming on spring maize yield [95]. Similarly, other findings reported by [98] showed that by changing sowing date from late April to late May, the mean daily temperature decreased 1.7 and 4.3°C whereas the diurnal temperature increased 4.3 and 3.1°C during grain-filling middle stage (16-45 days after silking) and grain-filling late stage (45 days after silking to maturity), respectively.
High air temperatures during the crop growing season can reduce harvestable yields. However, crop varieties with improved heat tolerance traits as well as crop management strategies at the farm scale are thus needed for climate change mitigation. Therefore, to mitigate the negative impact of increased growing season temperatures on crop growth and yield, especially in low latitude regions, heat-tolerant crop varieties, as well as modified farm management practices are needed, especially in the areas when irrigation is needed for crop production and irrigation water depends on the underground aquifers [99]. They also observed that applied irrigation at nighttime through subsurface drip reduced the root-zone soil temperature, which helped plant for improving plant growth and yield of corn. Optimizing irrigation has the potential to improve the water use efficiency of maize leading to enhanced heat tolerance [60]. Soil drought stress and atmospheric high temperature in the vegetative growth period could delay the process of growth of spring maize and shorten the reproductive stage, but those get improved when the soil moisture content in the maize field is maintained 65% field capacity by drip irrigation [100].
Plant growth hormones and exogenous chemicals (e.g., ABA and CaCl2) play important roles in strengthening heat tolerance in maize under HTS [60]. Exogenous ABA induces maize to produce HSPs, strengthening PSII heat tolerance [101]. An exogenous CaCl2 increases the maize cell membrane antioxidant capacity to improve heat tolerance [102]. Phytohormones such as auxin (IAA), cytokinins (CKs), abscisic acid (ABA), ethylene (ET), gibberellins (GAs), salicylic acid (SA), brassinosteroids (BRs), and jasmonates (JAs) have key roles in coordinating various signal transduction pathways during the abiotic-stress response [93].
Auxin or indole-3-acetic acid (Aux/IAA) acts as a chemical messenger to communicate cell activities when crops face different environmental stresses, including salinity, drought, waterlogging, extreme temperatures (heat, chilling, and freezing), heavy metals, light (intense and weak), and radiation (UV-A/B) [92, 103, 104]. Cytokinin (CK) is one of them, which functions solely and or with other hormones to mediate different mechanisms within plants in response to environmental fluctuations. During heat stress, protein denaturation and metabolic imbalance are occurred due to the excessive production of ROS. While to survive against heat stress, plants stimulate heat-shock proteins as a protective measure to prevent protein denaturation [105]. For example, the upregulation of heat-shock proteins in tobacco and bentgrass was recorded due to the enhancement of the antioxidant activity as a result of higher CK in plant cells [106]. Besides this, external application of CK inhibits the damage in photosynthesis under heat stress in maize, rice, and passion fruit [107, 108]. Salicylic acid (SA) is a naturally occurring phenolic compound [109] which plays a crucial part in the regulation of growth and development of the plants, and also a defensive mechanism to survive against abiotic stresses [110]. Similar to SA, abscisic acid (ABA) plays a vital role in plants’ physiological adjustments such as against abiotic stresses [111, 112] along with increasing seedling growth, endogenous levels of ABA, and reduced oxidative damage to plants due to heat stress. Similarly, Hasanuzzaman et al. [21] observed that ABA is a signaling molecule and also enhance the number of other signaling molecules such as nitric oxide for thermos-tolerance. Similar to other phytohormones, gibberellic acid (GAs) also interacts with other phytohormones in numerous developmental and stimulus-response processes in plants. GAs have been reported to alleviate the adverse effects of abiotic stress in plants, including rice as reported by Yamaguchi [113]. Brassinosteroids (BRs) is a new group of phytohormones, present in almost every part of the plants [114]. Similar to other phytohormones, BRs have shown tremendous potential against the abiotic stress-induced oxidative stress [103] including high temperature [115].
Inadequate and imbalanced nutrients and impaired soil fertility are associated with mineral-nutrient deficiencies and toxicities [116, 117, 118]. Adequate nutrition is essential for the integrity of plant structure and key physiological processes. For example, nitrogen (N) and magnesium are a structural part of chlorophyll and these are needed for photosynthesis. Nitrogen plays a very crucial role in temperature stress tolerance. At higher temperatures, the intensity of light is also very high. So, high light intensity, as a function of high temperature, which affects the uptake of mineral nutrients, ultimately influences the plant growth negatively. Since N plays a major role in the utilization of absorbed light energy and photosynthetic carbon metabolism [119, 120]. Whereas phosphorus is needed for energy production and storage; it is a structural part of nucleic acids and potassium is needed for osmotic regulation and activation of enzymes [117, 118]. Maize physiological function decreases under abiotic stress but can be compensated by nutritional management, for example, adequate potassium fertilizer improves cell membrane stability, turgor pressure, water potential in maize under water-deficit conditions [60]. Thus, a strategy to improve heat tolerance in maize at the grain-filling stage is to regulate nutrition.
Selection criteria have been proposed in traditional breeding to facilitate the detection of heat-tolerant maize variety. As different varieties respond differently to HTS, breeding heat-tolerant varieties is an effective strategy to improve heat tolerance at the spring maize grain-filling stage [60]. Screening of various cultivars was done to screen the warmness of the plant canopy, stomata behavior of upper most leaf (flag leaf), and photosynthesizing efficiency that are closely related to each other for the production maximum grain production under high-temperature stress conditions [121, 122, 123].
Under HT conditions, plants exhibit various mechanisms for surviving, which include long-term evolutionary phenological and morphological adaptations and short-term avoidance or acclimation mechanisms such as changing the leaf orientation, transpirational cooling, or alteration of membrane lipid compositions [92]. Also, high-temperature stress can be avoided by crop management practices such as selecting proper sowing methods, choice of sowing date, cultivars, irrigation methods, etc. It was discussed that combined hotter and drier climate change scenarios cause a greater maize yield reduction than hotter only scenarios. The incorporating drought and heat tolerance into maize germplasm has the potential to offset predicted yield losses and sustain maize productivity under climate change [19].
Tao and Zhao [60] reported that superoxide dismutase (SOD) increased and malonic dialdehyde (MDA) decreased in maize ear leaf for enhancing the stability of cell membrane, which helps to improve photosynthesis for good grain-filling characteristics (long quickly increase period and high mean rate of grain filling). It also produced high kernel weight under HTS [124, 125] leading to reporting of new origins of genetic engineering which exhibited leakage of electrolytes and MSI are the two basic parameters to screen the temperature stress-tolerant cultivars of various crops [126]. Electrical ions were gathered from the affected plants and were washed out with pure water to measure the membrane stability index MSI [127]. Seed production ability and stability index of the membrane were closely related to each other [3]. Mitochondrial tetrazolium is a very useful indicator of HTS sensitivity. Leaves’ tissues were dipped in triphenyl tetrazolium chloride chemical mixture during HTS. The spectrographic technique was used to quantify the related rates of triphenyl tetrazolium chloride reduction to formazan and tissues viability [128]. Heat tolerance (HT) of the crop is generally defined as the ability of the plant to grow and produce an economic yield under HS. This is a highly specific trait, and closely related to the species, even different organs and tissues of the same plant, may vary significantly in this respect. Plants have evolved various mechanisms for thriving under higher prevailing temperatures. They include short-term avoidance/acclimation mechanism or long-term evolutionary adaptations [92]. Many alternative traits related to heat resistance in Zea mays have been identified, including leaf kinetics, net photosynthesis rate (Pn), leaf anatomy at seedling stage [129] anther emergence [130], pollen grain viability [131], etc. However, the utility of those traits in stress breeding is not well established to date. Furthermore, most of the research focused on the heat stress on temperate maize, whereas only limited information is available on tropical maize [42].
One of the ways to deal with the adverse effects of heat stress may involve exploring some molecules that have the potential to protect the plants from the harmful effects of HT. In recent decades, exogenous application of protectants such as osmoprotectants, phytohormones, signaling molecules, trace elements, etc., have shown a beneficial effect on plants grown under HTS and these protectants have growth-promoting and antioxidant capacity [21, 92]. Exogenous applications of several phytohormones were found to be effective in mitigating heat stress in plants. Accumulation of osmolytes such as proline (Pro), glycine betaine (GB), and trehalose (Tre) is a well-known adaptive mechanism in plants against abiotic stress conditions including HT [92]. Supplementation with Pro and GB considerably reduced the H2O2 production, improved the accumulation of soluble sugars, and protected the developing tissues from heat stress effects. At the field level, managing or manipulating cultural practices, such as the timing and methods for sowing, irrigation management, and selection of cultivars and species, can also considerably decrease the adverse effects of HT stress. In recent decades, exogenous applications of protectants such as osmoprotectants, phytohormones, signaling molecules, trace elements, etc., have shown beneficial effects on plants growing under HT, due to the growth-promoting and antioxidant activities of these compounds [21, 92].
The genetic analytical study depends upon the genetic markers. Information about genetic reproduction aids to identify potential gene markers [132]. To mitigate the harmful effects of high-temperature several gene markers like a random polymorphic amplifier, AFLP (amplifier fragmentation length polymorphism), as well as sequenced simple repeats SSR, were used to increase the crop production under heat-stress [133, 134]. During genetic breeding, the SNP marker was used because of its genetic sequence in legumes to identify resistant genotypes against heat stress [135]. QTL chromosome numbers and their origin were very useful to mitigate the effects of heat stress [132]. Different molecular markers are studied in population genomics across the environment in many individuals to find out novel variation patterns and help to find if the genes have functions in significant ecological traits. Genome-wide association study (GWAS) is a powerful tool for understanding the complete set of genetic variants in different crop cultivars to recognize allelic variant linked with any specific [136]. GWASs generally highlight linkage among SNPs single nucleotide polymorphism marker and traits and based on GWAS design, genotyping tools, statistical models for examination, and results in interpretation [137].
Heat stress disturbed the crop metabolic activities by changing tissue balance. Heat stress directly produced toxic substances in plant tissues call ROS due to which plant suffers from oxidative stress. Moreover, to reduce oxidative damage resulting from heat-induced oxidative stress, plants have developed different adaptive mechanisms, via the biosynthesis of enzymatic and non-enzymatic antioxidants and the sequestering of other materials in crop tissues. Enhancement of antioxidant defense system is an important strategy to scavenge ROS by antioxidant enzymes such as ascorbate peroxidase (APX), ascorbate reductase (AR), catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GPX), and superoxide dismutase (SOD) and with non-enzymatic antioxidants such as ascorbate (AsA), glutathione (GSH), carotenoids, flavanones, and anthocyanins [92]. Furthermore, adaptation to temperature changes, at the molecular level, was accompanied by the degradation of the normal proteins and the synthesis of HSPs involved in the mechanism of defense in plants. Seed germination is the most critical growth stage of the whole plant life cycle because it is the first step to carry out whole-plant growth and development, but heat stress is the main reducing factor of seedling emergence in semiarid areas [138, 139].
Heat stress and unprecedented climate changes have become a major challenge for sustainable crop production globally. Plant growth, development, and productivity get compromised due to heat stress. Elucidating maize hybrid for temperature tolerance could be an indispensable step toward a balanced yield. Tolerance and avoidance of stress could be an easy way to boost crop production under a changing climate; for example photosynthetic rate can be improved by targeting candidate traits and candidate genes involved in photosynthesis at a molecular level. It could lead to high assimilates production, more transportation of sugar to grain; finally, it decreases grain-filling rate, improves kernel size, and could be very useful to improve plant productivity. Heat-insensitive maize hybrids can be developed by gene editing CRISPER-CAS9 system through targeting a gene that is responsible for heat sensitivity. The base of further research should be focused on spring maize crops. Field experiments regarding the sowing date are essential by analyzing the impact of meteorological factors on maize growth and grain yield. Application of osmoprotectants, nanotechnology, and the use of sustainable agriculture agents have become necessary for further research. Further, interdisciplinary studies that include agronomy, animal sciences, and climate modeling are warranted to assess the impact of the feeding of both the HTS-tolerant maize varieties and those grown under heat stress on animal health and production. This review could encourage such interdisciplinary approaches to develop maize hybrids with high nutritional values and are not prone to drastic yield reductions owing to fluctuations in agro-climatic factors (especially temperature) and the outcome may lead to sustainable maize production in the tropics under changing climate.
The authors declare no conflicts of interest.
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