Enhancing cell activities through integration of polyanionic alginate or hyaluronic acid derivatives with triboelectric nanogenerators.

The impact of electrical stimulation has been widely investigated on the wound healing process; however, its practicality is still challenging. This study explores the effect of electrical stimulation on fibroblasts in a culture medium containing different electrically-charged polysaccharide derivatives including alginate, hyaluronate, and chitosan derivatives. For this aim, an electrical stimulation, provided by a zigzag triboelectric nanogenerator (TENG), was exerted on fibroblasts in the presence of polysaccharides' solutions. The analyses showed a significant increase in cell proliferation and an improvement in wound closure (160 % and 90 %, respectively) for the hyaluronate-containing medium by a potential of 3 V after 48 h. In the next step, a photo-crosslinkable hydrogel was prepared based on hyaluronic acid methacrylate (HAMA). Then, the cells were cultured on HAMA hydrogel and treated by an electrical stimulation. Surprisingly, the results showed a remarkable increase in cell growth (280 %) and migration (82 %) after 24 h. Attributed to the electroosmosis phenomenon and an amplified transfer of soluble growth factors, a dramatic promotion was underscored in cell activities. These findings highlight the role of electroosmosis in wound healing, where TENG-based electrical stimulation is combined with bioactive polysaccharide-based hydrogels to promote wound healing.

Sulfated polysaccharide as biomimetic biopolymers for tissue engineering scaffolds fabrication: Challenges and opportunities.

Sulfated polysaccharides play important roles in tissue engineering applications because of their high growth factor preservation ability and their native-like biological features. There are different sulfated polysaccharides based on different repeating units in the carbohydrate backbone, the position of the sulfate group, and the sulfation degree of the polysaccharide. These led to various sulfated polymers with different negative charge densities and resultant structure-property relationships. Since numerous reports are presented related to sulfated polysaccharide applications in tissue engineering, it is crucial to review the role of effective physicochemical and biological parameters in their usage; as well as their structure-property relationships. Within this review, we focused on the effect of naturally occurring and synthetic sulfated polysaccharides in tissue engineering applications reported in the last years, highlighting the challenges of the scaffold fabrication process, the position, and the degree of sulfate on biomedical activity. Additionally, we discussed their use in numerous in vitro and in vivo model systems.

Fabrication of affinity-based drug delivery systems based on electrospun chitosan sulfate/poly(vinyl alcohol) nanofibrous mats.

Benign electrospinning of chitosan in aqueous medium is an open challenge mainly due to its insolubility in neutral pH and inter- and intramolecular hydrogen bonding interactions. Here, we developed a simple and widely-used methodology to improve the chitosan electrospinnability through the sulfation of chitosan and its further mixing with poly(vinyl alcohol) for the first time. The FTIR, 1H NMR and elemental analyses showed the successful sulfation of chitosan. Furthermore, the viscosity and electrical conductivity measurements revealed the high solubility of chitosan sulfate (CS) in aqueous media. In the next step, a uniform electrospun nanofibrous mat of CS/PVA was fabricated with a fiber diameter ranging from 90 to 340 nm. The crosslinked CS/PVA (50/50) nanofibrous mat as the optimum sample showed a swelling ratio of 290 ± 4 % and a high Young's modulus of 3.75 ± 0.10 GPa. Finally, malachite green (MG) as a cationic drug model was loaded into different samples of chitosan film, CS film, and CS/PVA (50/50) nanofibrous mat and its release behavior was studied. The results of these analyses revealed that the CS/PVA (50/50) nanofibrous mat can successfully load higher contents of the MG and also release it in a sustained manner.

Fabrication of Cell-Laden AME-Loaded Collagen-Based Hydrogel Promotes Fibroblast Proliferation and Wound Healing In Vitro.

OBJECTIVE: The biological factors secreted from cells and cell-based products stimulate growth, proliferation, and migration of the cells in their microenvironment, and play vital roles in promoting wound healing. The amniotic membrane extract (AME), which is rich in growth factors (GFs), can be loaded into a cell-laden hydrogel and released to a wound site to promote the healing of the wound. The present study was conducted to optimize the concentration of the loaded AME that induces secretion of GFs and structural collagen protein from cell-laden AME-loaded collagen-based hydrogels, to promote wound healing in vitro. MATERIALS AND METHODS: In this experimental study, fibroblast-laden collagen-based hydrogel loaded with different concentrations of AME (0.1, 0.5, 1, and 1.5 mg/mL, as test groups) and without AME (as control group), were incubated for 7 days. The total proteins secreted by the cells from the cell-laden hydrogel loaded with different concentrations of AME were collected and the levels of GFs and type I collagen were assessed using ELISA method. Cell proliferation and scratch assay were done to evaluate the function of the construct. RESULTS: The results of ELISA showed that the concentrations of GFs in the conditioned medium (CM) secreted from the cell-laden AME-loaded hydrogel were significantly higher than those secreted by only the fibroblast group. Interestingly, the metabolic activity of fibroblasts and the ability of the cells to migrate in scratch assay significantly increased in the CM3-treated fibroblast culture compared to other groups. The concentrations of the cells and the AME for preparation of CM3 group were 106 cell/mL and 1 mg/mL, respectively. CONCLUSION: We showed that 1 mg/ml of AME loaded in fibroblast-laden collagen hydrogel significantly enhanced the secretion of EGF, KGF, VEGF, HGF, and type I collagen. The CM3 secreted from the cell-laden AME-loaded hydrogel promoted proliferation and scratch area reduction in vitro.

Assessment of the Efficacy of an LL-37-Encapsulated Keratin Hydrogel for the Treatment of Full-Thickness Wounds.

Wound healing remains a burdensome healthcare problem due to moisture loss and bacterial infection. Advanced hydrogel dressings can help to resolve these issues by assisting and accelerating regenerative processes such as cell migration and angiogenesis because of the similarities between their composition and structure with natural skin. In this study, we aimed to develop a keratin-based hydrogel dressing and investigate the impact of the delivery of LL-37 antimicrobial peptide using this hydrogel in treating full-thickness rat wounds. Therefore, oxidized (keratose) and reduced (kerateine) keratins were utilized to prepare 10% (w/v) hydrogels with different ratios of keratose and kerateine. The mechanical properties of these hydrogels with compressive modulus of 6-32 kPa and tan δ <1 render them suitable for wound healing applications. Also, sustained release of LL-37 from the keratin hydrogel was achieved, which can lead to superior wound healing. In vitro studies confirmed that LL-37 containing 25:75% of keratose/kerateine (L-KO25:KN75) would result in significant fibroblast proliferation (∼85% on day 7), adhesion (∼90 cells/HPF), and migration (73% scratch closure after 12 h and complete closure after 24 h). Also, L-KO25:KN75 is capable of eradicating both Gram-negative and Gram-positive bacteria after 18 h. According to in vivo assessment of L-KO25:KN75, wound closure at day 21 was >98% and microvessel density (>30 vessels/HPF at day 14) was significantly superior in comparison to other treatment groups. The mRNA expression of VEGF and IL-6 was also increased in the L-KO25:KN75-treated group and contributed to proper wound healing. Therefore, the LL-37-containing keratin hydrogel ameliorated wound closure, and also angiogenesis was enhanced as a result of LL-37 delivery. These results suggested that the L-KO25:KN75 hydrogel could be a sustainable substitute for skin tissue regeneration in medical applications.

Partial sulfation of gellan gum produces cytocompatible, body temperature-responsive hydrogels.

Gellan gum (GG) is a biodegradable polysaccharide and forms thermosensitive hydrogels by a helix-mediated mechanism. Unfortunately, the wide use of GG in tissue engineering has been restricted due to its dramatically higher gelation temperature than normal body temperature. Here, we show that partial sulfation of GG affords a cytocompatible body temperature-responsive hydrogel with an interesting thermoreversibility at 42 °C. The partial sulfation of GG was confirmed by FTIR, EDX and elemental analyses. The sulfated GGs (SGGs) had a higher swelling ratio and degradation in PBS compared to the neat GG. Based on the results of rheometry analysis, the SGG with a degree of sulfation of 0.27 (H3 sample) showed a gelation temperature close to the physiological temperature. In addition, the drop in mechanical properties of SGGs was compensated by a further calcium-mediated ionic crosslinking, where Young's modulus of H3 increased from 10.6 ± 1.9 kPa up to 38.4 ± 5.5 kPa. Finally, we showed that the partial sulfation reaction of GG is a simple and mild strategy to modify chemical structure of GG, and to produce a cytocompatible, body temperature-responsive hydrogel compared to other modifying reactions such as oxidation reaction.

Advances in mechanically robust and biomimetic polysaccharide-based constructs for cartilage tissue engineering.

The purpose of cartilage tissue engineering is to provide artificial constructs with biological functions and mechanical features that resemble native tissue to improve tissue regeneration. Biochemical characteristics of the cartilage extracellular matrix (ECM) microenvironment provide a platform for researchers to develop biomimetic materials for optimal tissue repair. Due to the structural similarity of polysaccharides into physicochemical characteristics of cartilage ECM, these natural polymers capture special attention for developing biomimetic materials. The mechanical properties of constructs play a crucial influence in load-bearing cartilage tissues. Moreover, the addition of appropriate bioactive molecules to these constructs can promote chondrogenesis. Here, we discuss polysaccharide-based constructs that can be used to create substitutes for cartilage regeneration. We intend to focus on newly developed bioinspired materials, fine-tuning the mechanical properties of constructs, the design of carriers loaded by chondroinductive agents, and development of appropriate bioinks as a bioprinting approach for cartilage regeneration.

Hybrid gelatin-sulfated alginate scaffolds as dermal substitutes can dramatically accelerate healing of full-thickness diabetic wounds.

Diabetic foot ulcers (DFUs) are defined as chronic and non-healing wounds that cause skin disorders. Here, we introduce a novel biodegradable gelatin/sulfated alginate hybrid scaffold as a dermal substitute to accelerate the healing of full-thickness diabetic ulcers in a diabetic mouse model. The hybrid scaffold possessing different weight ratios of sulfated alginate, from 10 % up to 50 %, were prepared through chemical crosslinking by carbodiimide chemistry and further freeze-drying. Based on the in vitro cytotoxicity experiments, the hybrid scaffolds not only showed no cytotoxicity, but the cell growth also dramatically increased by increasing the sulfated alginate content. Finally, the pathology of hybrid scaffolds as the dermal substitutes for healing of full-thickness diabetic wounds showed the more appropriate formation of epidermal layer, more homogeneous distribution of collagenous tissue and lower penetration of immune cells for the hybrid scaffolds-treated wounds.

Antibacterial Host-Guest Intercalated LDH-Adorned Polyurethane for Accelerated Dermal Wound Healing.

Curcumin has a limited clinical application because of its extremely poor accessibility. In the present study, improved curcumin bioavailability within a castor oil polyurethane/layered double hydroxide (LDH) wound cover was achieved by preparing a curcumin p-sulfonic acid calix[4]arene (SC4A) inclusion complex. Then, it was utilized to intercalate MgAl-layered double hydroxide (MgAl-LDH) nanosheets. The incorporation of the nanostructure into a PU/Cur-SC4A-LDH film provided bacteria-killing performance against both Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli bacteria. This finding is due to an increase in curcumin bioavailability in the PU matrix. Furthermore, all PU nanocomposites exhibited appropriate cytocompatibility based on an MTT assay. Mainly, the proliferation of L929 fibroblast cells in contact with the PU/Cur-SC4A-LDH sample was significantly further enhanced than that for other nanocomposites within 7 days. This observation can be related to the better availability of curcumin on the film's surface, which causes an improvement in the proliferation rate of cells. Regarding the histological results, the hematoxylin and eosin (H&E) images showed faster epidermal layer formation and a larger quantity of matured hair follicles for PU/Cur-SC4A-LDH-healed wounds in comparison with those for the negative control over a period of 28 days. Thus, this practical healing ability of the PU/Cur-SC4A-LDH nanocomposite makes it a promising candidate as a wound dressing film.

A supramolecular injectable hydrogel based onß-cyclodextrin-grafted alginate and pluronic-amine loaded with kartogenin for chondrogenic differentiation of mesenchymal stem cells.

Owing to the similarity of hydrogels to cartilage extracellular matrix, they have been extensively utilized in the chondral lesions. Moreover, their tunable administration properties are desirable for reducing injuries in lesion sites. Generally, injectable hydrogels are mechanically weak, requiring some modifications for being used as a cell carrier in place of articular cartilage. In this study, a combination ofß-cyclodextrin-grafted alginate (Alg-ß-CD) and pluronic-amine with multiple physical crosslinking was used for the first time. Supramolecular interactions, including electrostatic forces, host-guest interaction, and hydrophobic interaction with increasing temperature maintain injectability of hydrogels while these interactions boost mechanical properties to the extent that shear modulus surpassed 40 kPa. Vacantß-CD cavities in conjunction with gel network were exploited for kartogenin (KGN) loading. All groups had gel time of less than one minute and gel temperature was 28 °C. No toxic effect of hydrogels on encapsulated cells was observed. While the optimum combination of polymers provided a sustainable release for KGN, it also extended thein vitrodegradation time of hydrogels from six days to two weeks. KGN facilitated encapsulated mesenchymal stem cells differentiation towards chondrocytes. Taken together, the synthesized hydrogel proved to be a promising candidate for being utilized in cartilage regeneration.

Regioselective sulfated chitosan produces a biocompatible and antibacterial wound dressing with low inflammatory response.

The aim of current study is to tailor chitosan derivate which is water-soluble while presents original biological features of chitosan. For this purpose, the 6-O chitosan sulfate (CS) with naked amine groups was synthesized via regioselective modification of chitosan (C) during which both crosslinking capacity and antibacterial properties of the C were remained intact. This was achieved by sulfation the C under controlled acidic conditions using chlorosulfonic acid/sulfuric acid mixture. Subsequently, a chemically crosslinked hydrogel of the CS was used as a wound dressing substrate. The modified sulfate groups retained the biocompatibility of C and showed antibacterial effects against gram-positive and gram-negative bacteria. In addition, the presence of sulfate groups in the CS chemical structure improved its anticoagulant activity compared to the unmodified C. Both in vitro and in vivo enzyme-linked immunosorbent assay (ELISA) measurements showed that CS had a higher potential to bind and scavenger anti-inflammatory cytokines, including IL-6 and transforming growth factor-ß (TGF-ß), both of which play critical roles in the early stage of the wound healing process. After treatment of full-thickness wounds with CS hydrogels, the macrophage cells (c.a. 6 × 104 cells) expressed significantly more M2 phenotype markers compared to the C group (4.2 × 104 cells). Furthermore, the CS hydrogel induced better re-epithelialization and vascularization of full-thickness wounds in mice compared to the C hydrogel during 30 days.

Micro- and nanotechnology in biomedical engineering for cartilage tissue regeneration in osteoarthritis.

Osteoarthritis, which typically arises from aging, traumatic injury, or obesity, is the most common form of arthritis, which usually leads to malfunction of the joints and requires medical interventions due to the poor self-healing capacity of articular cartilage. However, currently used medical treatment modalities have reported, at least in part, disappointing and frustrating results for patients with osteoarthritis. Recent progress in the design and fabrication of tissue-engineered microscale/nanoscale platforms, which arises from the convergence of stem cell research and nanotechnology methods, has shown promising results in the administration of new and efficient options for treating osteochondral lesions. This paper presents an overview of the recent advances in osteochondral tissue engineering resulting from the application of micro- and nanotechnology approaches in the structure of biomaterials, including biological and microscale/nanoscale topographical cues, microspheres, nanoparticles, nanofibers, and nanotubes.

Anti-fatigue, highly resilient photocrosslinkable gellan gum hydrogels reinforced by flexible nanoparticulate polyurethane multi-crosslinkers.

In this study, multifunctional polyurethane nanoparticles (MPUNs) were embedded into the methacrylated gellan gum (MGG) to prepare stimuli-responsive hydrogels with improved mechanical properties including remarkable fatigue resistance and excellent self-recoverability. The photocurable MPUNs/MGG nanocomposite hydrogels with different formulations were synthesized through a facile and green solution mixing method. The result obtained from mechanical analysis displayed an excellent improvement in compression strength (120 6 ± 83.7 kPa) and ultimate strain (94.2 ± 2.7%) in the optimized formulation. Furthermore, the optimized formulation could restore approximately its original shape after continuous loading-unloading compression tests over 100 cycles which might result from its favorable crosslinked structure. These reinforced hydrogels exhibited a dual physical and chemical crosslinking mechanism based on the hydrogen bonding formation and photocrosslinking of methacrylate functional groups, respectively. Interestingly, the nanocomposite hydrogels exhibited no significant cytotoxicity to human dermal fibroblast cells which made them suitable as the appropriate biomaterials for the engineering of soft tissues.

Highly tough and ultrafast self-healable dual physically crosslinked sulfated alginate-based polyurethane elastomers for vascular tissue engineering.

Since vascular diseases are regarded as a major cause of death worldwide, developing engineered biomimetic elastomers with physicochemical and biological properties resembling those of the natural vascular tissues, is vital for vascular tissue engineering (VTE). This study reports synthesis of highly tough supramolecular biologically active alginate-based supramolecular polyurethane (BASPU) elastomers that benefit from the presence of two physical networks with different strength of soft tertiary ammonium-soft sulfate pairs, as strong ionic bonds, and soft tertiary ammonium-hard carboxylate groups, as the weak bonds. The presence of sulfate groups resulted in low Young's modulus, high toughness and stretchability, proper energy dissipation, ultrafast self-healing and complete healing efficiency of BASPU. In vitro studies showed higher endothelial cells attachment, higher anticoagulation ability and significantly less platelet adhesion for BASPUs compared to the commercial vascular prosthesis. The histological studies of subcutaneously implanted scaffolds confirmed their low fibrosis and gradual biodegradation during 2 months of following.

How does counter-cation substitution influence inter- and intramolecular hydrogen bonding and electrospinnability of alginates.

Despite numerous applications of nanofibrous alginate (Alg) mat, its facile fabrication via electrospinning is still challenging. The low alginate content compared to the carrier polymer and existence of impurities are the main drawbacks of existing approaches. The purpose of this research is both to study and improve alginate electrospinnability by focusing on the effect of inter- and intramolecular hydrogen bonding. Based on hard and soft acids and bases (HSAB) theory, the Na+ cations (carboxylate counter-cation) were substituted with a harder acid, Li+ cation, to increase the strength of ionic interaction and decrease the density of hydrogen bonding. Viscosity and electrical conductivity measurements as well as FTIR and 1H NMR revealed a lower intramolecular hydrogen bonding density in Li-Alg. SEM images showed improvement of alginate electrospinnability for Li-Alg compared to the salts of Na-Alg and K-Alg. This study sheds more light on underlying reasons hindering alginate electrospinning and introduces a simple method for fabrication of nanofibers with high alginate content.

Co-delivery of minocycline and paclitaxel from injectable hydrogel for treatment of spinal cord injury.

Spinal cord injury (SCI) induces pathological and inflammatory responses that create an inhibitory environment at the site of trauma, resulting in axonal degeneration and functional disability. Combination therapies targeting multiple aspects of the injury, will likely be more effective than single therapies to facilitate tissue regeneration after SCI. In this study, we designed a dual-delivery system consisting of a neuroprotective drug, minocycline hydrochloride (MH), and a neuroregenerative drug, paclitaxel (PTX), to enhance tissue regeneration in a rat hemisection model of SCI. For this purpose, PTX-encapsulated poly (lactic-co-glycolic acid) PLGA microspheres along with MH were incorporated into the alginate hydrogel. A prolonged and sustained release of MH and PTX from the alginate hydrogel was obtained over eight weeks. The obtained hydrogels loaded with a combination of both drugs or each of them alone, along with the blank hydrogel (devoid of any drugs) were injected into the lesion site after SCI (at the acute phase). Histological assessments showed that the dual-drug treatment reduced inflammation after seven days. Moreover, a decrease in the scar tissue, as well as an increase in neuronal regeneration was observed after 28 days in rats treated with dual-drug delivery system. Over time, a fast and sustained functional improvement was achieved in animals that received dual-drug treatment compared with other experimental groups. This study provides a novel dual-drug delivery system that can be developed to test for a variety of SCI models or neurological disorders.

Assessment of the Efficacy of Tributylammonium Alginate Surface-Modified Polyurethane as an Antibacterial Elastomeric Wound Dressing for both Noninfected and Infected Full-Thickness Wounds.

Risk factors of nonhealing wounds include persistent bacterial infections and rapid onset of dehydration; therefore, wound dressings should be used to accelerate the healing process by helping to disinfect the wound bed and provide moisture. Herein, we introduce a transparent tributylammonium alginate surface-modified cationic polyurethane (CPU) wound dressing, which is appropriate for full-thickness wounds. We studied the physicochemical properties of the dressing using Fourier transform infrared, 1H NMR, and 13C NMR spectroscopies and scanning electron microscopy, energy-dispersive X-ray, and thermomechanical analyses. The surface-modified polyurethane demonstrated improved hydrophilicity and tensile Young's modulus that approximated natural skin, which was in the range of 1.5-3 MPa. Cell viability and in vitro wound closure, assessed by MTS and the scratch assay, confirmed that the dressing was cytocompatible and possessed fibroblast migratory-promoting activity. The surface-modified CPU had up to 100% antibacterial activity against Staphylococcus aureus and Escherichia coli as Gram-positive and Gram-negative bacteria, respectively. In vivo assessments of both noninfected and infected wounds revealed that the surface-modified CPU dressing resulted in a faster healing rate because it reduced the persistent inflammatory phase, enhanced collagen deposition, and improved the formation of mature blood vessels when compared with CPU and commercial Tegaderm wound dressing.

Improving anti-hemolytic, antibacterial and wound healing properties of alginate fibrous wound dressings by exchanging counter-cation for infected full-thickness skin wounds.

Nowadays, considerable effort is made to overcome bacterial diseases and combat bacterial resistance. In this context, development of safe and efficient antimicrobial wound dressings which can selectively fight against the bacteria and decrease disruption of normal cells such as red blood cells in wound bed is highly required. In this study, a series of ammonium salts of alginate were prepared and the role of different counter-cations including sodium, triethylammonium, tributylammonium and dihexylammonium were examined with respect to antimicrobial efficacy and selectivity as well as fibroblasts viability. We found that many different parameters such as hydrophilicity, linearity and branching structure, molecular weight and charge density can influence the selectivity of ammonium counter-cations. In vitro biological studies showed that tributylammonium alginate (TBA-Alg) possesses optimum anti-hemolytic and antibacterial properties with less cytotoxicity at 1 mg mL-1 compared with other counter-cations. Furthermore, the fibrous mat of TBA-Alg demonstrated higher swelling ratio and better anti-hemolytic and cytotoxic activities against fibroblasts compared to a commercial silver-impregnated calcium alginate wound dressing. Moreover, histopathological analysis of tributylammonium alginate fibrous mat revealed that this dressing accelerates reepithelialization of infected full-thickness skin wounds as well as the commercial silver-impregnated calcium alginate wound dressing.

Fabrication challenges and trends in biomedical applications of alginate electrospun nanofibers.

Alginate as a naturally-derived biomaterial with marine algae sources has gained much attention in both laboratorial and industrial applications due to its structural and chemical resemblance to extracellular matrix (ECM) as well as desirable properties like biocompatibility, biodegradability, processability and low cost. Electrospun alginate nanofibrous scaffolds have found wide applications in biomedical field such as tissue engineering, biomedicine and drug delivery systems. However, electrospinning of alginate is challenging due to the low solubility and high viscosity of high molecular weight alginate, high density of intra- and intermolecular hydrogen bonding, polyelectrolyte nature of aqueous solution and lack of appropriate organic solvent. The aim of this review is to summarize the challenges and obstacles in alginate electrospinning reported in the literature as well as the introduced solutions for them, in order to open new opportunities for more intended and successful investigations in the field.

Facile fabrication of sulfated alginate electrospun nanofibers.

Mass fabrication of sodium alginate nanofibers using single-nuzzle electrospinning process is an open challenge mainly due to its inter- and intramolecular hydrogen bonding, rigid chain conformation and low solubility. In this regards, we synthesized sodium sulfated alginate (SSA) through sulfation of hydroxyl functional groups of alginate. Not only decreases the hydrogen bonding density through the sulfation reaction, but the sulfated alginate also demonstrates more solubility in aqueous media compared to the pristine alginate. Beside the sulfation of alginate, its electrospinnability in combination with polyvinyl alcohol (PVA) significantly improves. In contrast to the neat alginate, concentrated aqueous solutions of sulfated alginate, 10 wt%, can be easily prepared and electrospun to obtain nanofibers of sulfated alginate. In this regards, facile fabrication of electrospun nanofibers of alginate derivatives with 50 wt% content in dry electrospun mat of SSA/PVA using single-nuzzle electrospinning and flow rate of 5 mL h-1 was developed for the first time.