Heliopsis longipes (Asteraceae) accumulates in root a C10 alkamide called affinin in its roots, but not in leaves. The closely related species Heliopsis annua does not produce alkamides. To elucidate the biosynthetic pathway of the alkamides acyl chain, a comparative global gene expression analysis contrasting roots and leaves of both species was performed.Alkamides are plant-specific bioactive molecules. They are low molecular weight N-substituted α-unsaturated acyl amides that display biological explicit activities in different organisms from bacteria, fungi, insects to mammals and plants. The acyl chain has been proposed to be biosynthesized from a fatty acid; however, this has not been demonstrated yet. H. longipes roots, but not in tissues and species that do not accumulate alkamides. The first domain searched was the Ketosynthase (KS) domain. The phylogenetic analysis using sequences of the KS domain of FAS and PKS from different organisms, revealed that KS domains of the differentially expressed transcripts in H. longipes roots and the KS domain found in transcripts of Echinacea purpurea, another alkamides producer species, were grouped together with a high bootstrap value of 100%, sharing great similarity. Among the annotated transcripts, we found some coding for the enzymatic domains KS, AT, ACP, DH, OR and TE, which presented higher expression in H. longipes roots than in leaves. The expression level of these genes was further evaluated by qRT-PCR. All unigenes tested showed higher expression in H. longipes roots than in any the other samples. Based on this and considering that the acyl chain of affinin presents unsaturated bonds at even C numbers, we propose a new putative biosynthesis pathway mediated by a four modules polyketide synthase (PKS).Transcriptomics analysis allowed to identify genes highly expressed in alk) previously thought to be absent from the plant kingdom.The global gene expression analysis led to the selection of a set of candidate genes involved in the biosynthesis of the acyl chain of affinin, suggesting that it may be performed by a non-iterative, partially reductive, four module type I PKS complex (PKS  E) or conjugated , either in the α-, intermediate or methyl-ω position, starting at an even C position. Alkamides are plant metabolites comprising a group close to 300 structures produced by a limited number of plant species belonging taxonomically to only eight families. A database of alkamides and other acyl-amides has been recently compiled  where alkamides have been detected in the whole plant. Several species containing high levels of alkamides are found in the Asteraceae, Piperaceae and Rutaceae; being more frequent in the Asteraceae family  bonds, when present, are in a position corresponding to that of the double bonds, most frequently occurring as acetylenic or methyl acetylenic in the ω-position. When more than one acetylenic bond is present, these are conjugated. In those alkamides where the acetylenic bond is at the ω-position, the acyl chain presents an odd number of carbons. In Heliopsis longipes, alkamides accumulate in the roots but are not present in the vegetative aerial tissue. In contrast, alkamides have not been detected in leaves or roots of the related species Heliopsis annua. The most abundant alkamide in H. longipes roots is N-isobutyl-2E,6Z,8E-decatrienamide, named affinin. It represents over 90% of the total alkamide content in well-developed roots. Other C10 acyl chain alkamides in this tissue are structurally related to affinin, such as the N-2-methylbutyl-2E,6Z,8E-decatrienamide and the bornyl ester of C10-2E,6Z,8E  by successive β-oxidation, dehydration, and isomerization (cis configuration (Z) and non-conjugated. The double bonds of fatty acids are incorporated by desaturases at a site different from that on which the acyl chain is synthesized and are strictly positioned in relation to the first desaturation 9Z, such as 12Z, 15Z, or 18Z (trans configuration (E) but may be in cis (Z) when these are conjugated, in contrast to those found in fatty acids. These facts, together, suggest that the biosynthesis of the acyl chain of alkamides could occur through an alternative pathway. Biochemical or genetic information to support the fatty acid or any alternative biosynthetic pathway of alkamides is still scarce, essentially because alkamides are produced in non-model plants for which the genomes have not yet been sequenced. Whole genome sequencing has been successfully used to elucidate biosynthetic pathways in microorganisms, such as bacteria. However, plant genomes are much larger than bacterial genomes, which increases the cost and time associated with sequencing, and plant genomes exhibit particularities such as alternative splicing that can give rise to different transcript versions of a single gene. Therefore, transcript profiling using high-throughput RNA sequencing (RNA-Seq) is a first-rate alternative to investigate the biosynthetic pathways of metabolites in plants without sequenced genomes . Voucher specimens were deposited at Instituto de Ecología, Centro Regional del Bajío. Herbarium IEB (Voucher 263787). Heliopsis annua was collected in October 2014 in the municipality of Vicente Guerrero, state of Durango, México . Voucher specimens were deposited at CIIDIR Durango Herbarium (Voucher id. D. Ramírez 5543). Fresh roots and leaves of both species were collected separately, washed with distilled water, immediately frozen in liquid nitrogen, and stored at −80 °C until processed for RNA extraction.Heliopsis species and processed separately as two replicates, following the protocol established by the PureLinkTM Micro-to-Midi Total RNA Purification System . The quality of the RNA samples was evaluated using the RNA 6000 Nanochip on the BioAnalyzer 2100 . Complementary DNA libraries were constructed with insert sizes from 339 to 697 base-pairs (bp) and sequenced using the Illumina HiSeq 2500 platform  with paired-end reads of 100 bp, at the Unidad de Servicios Genómicos of CINVESTAV, Irapuato, Guanajuato, México.Total RNA was extracted from roots and leaves of eight individual plants of each The resulting FASTQ files were pre-processed by removing adapter sequences and low-quality bases using the software package Trimmomatic-0.32, as previously described . Clean rwww.ncbi.nlm.nih.gov), Protein Family  database, which contains a large collection of protein families, each represented by multiple sequence alignments and hidden Markov models, and Swiss-Prot database (https://www.uniprot.org/), with an e-value cut-off of 1e−5. Gene Ontology (GO) analyses were further used to categorize the function of the unigenes with Blast2GO 5 (https://www.blast2go.com/). Using the basic local alignment search tool (BLAST), the Arabidopsis thaliana transcription factor (TF) database was queried to identify TFs among all unigenes (identity N80%) (The consensus contig sequence files were annotated against the non-redundant protein (NR) database from the National Center for Biotechnology Information  .p-value cut-off ≤0.05 [multiple test correction: false discovery rate (FDR)] and log2 fold change (FC) ≥2 were used to identify significant differences in transcript expression.To estimate the expression level of each transcript in the different tissue samples, high-quality reads from each sample were mapped onto the final transcriptome assembly using the RSEM software . Read coH. longipes and H. annua were obtained from the translated assembled RNA-Seq data. The KS domains were corroborated by BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) and PFAM, a database of large collection with protein families (https://pfam.xfam.org). The domains compared were: the KS domains identified in H. annua roots (A1–A4); the KS domains upregulated in H. longipes roots (B1–B4); the KS domains related to FAS from H. annua (C1–C11); the KS related to FAS from H. longipes (D1–D11); the KS of the type III PKS from H. annua (E1–E4); and the KS of the type III PKS from H. longipes (F1–F4). These KS sequences were compared with KS domains of several microorganisms, plant and mammal species obtained from the NCBI database (www.ncbi.nlm.nih.gov) . Echinacea purpurea also belongs to tribe Heliantheae within the Asteraceae and produces alkamides that are structurally related to affinin (The amino acid sequences of the KS domains of nih.gov) . The KS  affinin . After s affinin . Phyloge affinin .www.primer3.org) and are shown in −ΔΔCT method (H. longipes roots vs. leaves and H. longipes roots vs. H. annua roots.Reverse Transcriptase quantitative polymerase chain reaction assays (RT-qPCR) were performed using the Applied Biosystems 7500 Fast Real-Time PCR system  with the Brilliant II SYBR Green RT-qPCR 1-Step Master Mix Kit . Gene-specific primers for the selected genes in the acyl chain biosynthesis pathway were designed using Primer3 . Roots and leaves of H. longipes generated 45,622,318 and 31,731,475 raw reads, respectively, whereas roots and leaves of H. annua generated 30,326,119 and 30,089,428 raw reads, respectively , followed by the MYB family (223 unigenes), and members of the GRAS family (80 unigenes). In H. annua, among the 938 unigenes related to TFs, the most abundant presented high homology to members of the LSD family (218 unigenes), followed by the WRKY family (162 unigenes), and the members of the HB family (68 unigenes). Finding MYB family members in H. longipes was interesting, since the MYB TFs include a conserved MYB DNA-binding domain that commonly binds to and regulates genes that code for enzymes involved in metabolic pathways , acyl transferase (AT), acyl carrier protein (ACP), oxide reductase (OR), dehydratase (DH) and thioesterase (TE) were selected  and the tissues that do non-produce these compounds .In order to confirm the differential expression of candidate genes in selected . Actin  and H. longipes (leaves), that were annotated as coding for a KS domain or KS domains related to FAS or type III PKS; and B for H. longipes roots upregulated transcripts. Other KS domains of FAS and PKS from mammals, microorganisms and other plants, obtained from NCBI listed in Echinacea purpurea were also included.The list of annotated transcripts differentially and not differentially expressed in H. longipes D1–D11 and H. annua C1–C11, are grouped in a clade together with the FAS from Arabidopsis thaliana KS with a high bootstrap value (98%). This result suggests that these transcripts, obtained from H. longipes and H. annua, encode enzymes with function similar to the KS domain of A. thaliana, which belongs to β-ketoacyl-[acyl-carrier-protein] synthases III (EC 2.3.1.180), enzymes that catalyze the chemical reaction of acetyl-CoA + malonyl-[acyl carrier protein] and produces acetoacetyl-[acyl carrier protein] + CoA + CO2. These enzymes belong to the family of transferases, and particularly to those transferring groups other than aminoacyl groups. They participate in fatty acid biosynthesis and are found in the dissociated or type II FAS biosynthesis system that occurs in plants and bacteria. The KS domains related to type III PKS are represented by the domain found in chalcone synthases (CHS), the enzymatic complexes mediating the synthesis of flavonoids in plants (H. longipes (F1–F4) are grouped together with a high bootstrap value of 100%. However, they are grouped in a clade far apart from the KS of type III PKS from H. annua E1–E4, which cluster in a clade together with the KS of type III PKS from Arabidopsis thaliana with a high bootstrap value of 98%. Therefore, these transcripts labeled as E1–E4 may share a similar function to the type III from A. thaliana. In addition, the translated transcripts of H. annua A1–A4 are close to the clade of H. annua E1–E4, translated transcripts coding for type III PKS KS domains or Chalcone synthase (CHS). As for the FAS-related sequences, the transcripts coding for these domains are present in both species, which could suggest that they may not be involved in the biosynthesis of the acyl chain of alkamides.The results of the maximum likelihood phylogenetic analysis based on 89 KS sequences are presented in n plants . The nonH. longipes roots, the species and tissue that produces alkamides, grouped with a high bootstrap value of 100% with KS (3-oxoacyl-[acyl-carrier-protein] synthase) of Echinacea purpurea roots. This group is exclusively formed by translated transcripts of E. purpurea and upregulated in H. longipes roots. This association suggest that the KS domains in both species probably have the same function and could be related to alkamide acyl chain biosynthesis, due to their functional nature. Close to this KS clade, KS domains from other plant species include Helianthus annus and Cynara cardunculus. This clade is not far from KS domains from the Chlamydiae phylum microorganism PKS specifically with the two orders: Chlamydiales and Parachlamydiales; and a single class Chlamydiales. These are obligate intracellular Gram-negative bacteria  The first KS domain, associated with plant FAS, includes general H. longipes, H. annua, and other plant transcripts; (2) A second domain type found exclusively in H. longipes root upregulated transcripts and E. purpurea. This KS domain, present in both species, is possibly related to the biosynthesis of the acyl chain of alkamides in H. longipes and E. purpurea. Interestingly, they were grouped in a clade far from FAS and PKS III KS reported in plants, but closer to microorganism PKS  The fourth includes two clades of H. annua KS domains related to type III PKS.In summary, the phylogenetic analysis revealed that the KS domains found in the annotated transcriptomes of the two species of the nism PKS ; (3) TheH. longipes alkamides is proposed. The available information about the different domain activities in published PKS systems, allowed the assembly of a model of a biosynthetic pathway that could produce the C10-2E,6Z,8E acyl chain of affinin. This model incorporates the KS, AT, ACP, OR (including the keto reductase (KR) and ER), DH and TE domains previously found. The model proposes a four-module PKS system where the starting precursor molecule is acyl-CoA, and the extender unit is malonyl-ACP  enzymes in a modular manner. As observed in polyketides biosynthesis from bacteria, fungi and symbiotic marine microorganisms such as in sponges , where uonyl-ACP .GO:0016740) domain accepts and loads the extender unit onto the ACP (GO:0016740) and produces malonyl-ACP. Then, the KS (GO:0016740) domain catalyzes a decarboxylative Claisen condensation with the acyl-CoA and the resulting product is a four carbon (C4) acyl chain with α- and β-keto groups. The KR, including OR (GO:0016491) and DH (GO:0016740) domains, sequentially reduce the β-keto group to a β-hydroxyl group and an α-E double bond, respectively. The α-unsaturated acyl chain is transferred by the AT (GO:0016740) to the second module (Aff-2) and is elongated by the ACP (GO:0016740) with another malonyl-ACP producing a C6-acyl chain. The same enzymatic reactions on the β-keto group as in the first module occur in module 2. The product is a C6-2Z,4E-dien-acyl chain, which is transferred to the module 3 (Aff-3) and condensed with malonyl-ACP to produce a C8-acyl chain with α- and β-keto groups. In this module, which consist of includes the KR including OR (GO:0016491) and DH (GO:0016740) domains, the β-keto group is reduced to a β-hydroxyl group and dehydrated to an α-E double bond. The ER (GO:0016491) enzymatic domain, present only in this module, reduces the double bond. In module 4 (Aff-4), the acyl chain C8-4Z,6E is once more condensed with malonyl-ACP to produce an C10-acyl chain with two keto α- and β- groups. A KR, including OR (GO:0016491) and DH (GO:0016740) domains, in this module reduces the β-keto group to a β-hydroxyl group, and the DH domains to an α-E double bond. The absence of an ER domain in this last module 4 results in the transference of the final C10-2E,6Z,8E-chain to the TE (GO:0016744) domain present at the end of this four module PKS. The proposed sequence of acyl chain biosynthesis, as evidenced by the differential expression supporting a four-modules multi-enzymatic complex, is as follows: in the first module (Aff-1), the AT  that act at the end of the FAS steps, or complexes such as PKS. It is responsible for the condensation of two substrates to generate or elongate an acyl chain, depending on where the enzyme that contains it is present. It can be assumed that such a domain would be participating in the initial steps of the biosynthesis of the acyl chain of alkamides. Indeed, transcripts encoding the KS domain were found among the differentially expressed set. The phylogenetic analysis  revealedn plants .Surprisingly, in the phylogenetic analysis comparing KS domains of FAS, PKS, and the putative alkamide acyl chain biosynthetic genes, KS domains of the latter were found in a clade close to PKS from microorganisms. Thus, PKS-related, specific genes, appear to be conserved within the alkamide producer species, suggesting duplications that led to the evolution of novel functions. A possibility is that these plants acquired these genes by horizontal transfer, as has been suggested for other PKS gene families  or alterH. longipes. However, since RNA-Seq analysis was performed using polyadenylated RNA, it is unlikely that prokaryotic RNA was sequenced, though fungal transcripts would not have been filtered out. Nevertheless, we would expect fungal transcripts to be present at much lower levels than plant transcripts. Moreover, putative endophytes that are exclusively present in H. longipes roots have not been observed in the in vitro root culture in the lab to date.Because of the clustering of KS domains coded by upregulated transcripts with KS domains found in type I PKS systems, found in certain microorganisms, we sought whether other similar domains present in those systems were also found coded by other upregulated transcripts. These results suggest alkamide synthesis is likely carried out by enzymes that are similar to type I PKS. These findings are important, as the differentiation of PKS and FAS will facilitate approaches investigating alkamides biosynthesis pathways in other plant species. They are also interesting as this type of systems were previously thought to be absent from plant species, and mostly present in microorganisms. One possibility is, of course, that these enzymes are expressed by endophytes that are exclusively present in the root tissues of To propose the novel alkamides biosynthetic pathway, we considered the chemical structure of the affinin acyl chain, and the characteristics of the known products of FAS or PKS. Fatty acids derived from FASs comprise completely reduced acyl chains, and the polyketides derived from PKSs include partially unsaturated acyl chains. In the polyunsaturated fatty acids (PUFAs), the double bonds are incorporated by fatty acid desaturases (FADs), iron-dependent metalloenzymes, on the fatty acids released from the FAS enzymatic complex . In contH. longipes roots through a pathway that is similar to the polyketide biosynthesis pathway. This model is further referred as “PKS alk”. The proposed PKS alk  would work in a modular fashion as in the known type I PKSs  module. However, in the Aff-2(γ) module, the DH would generate a cis double bond during the dehydration of an L-β-hydroxy intermediate. This mechanism, by means of which the DH generates cis double bonds when a trans double bound is inserted in the previous module during the synthesis of an acyl chain, has been observed in several PKS type I enzymes in microorganisms  and Ctenium aromaticum (Poaceae).The “PKS H. longipes and H. annua is described. The comparative transcriptome analysis performed for the two Heliopsis species led to the selection of a set of candidate genes putatively involved in the affinin acyl chain biosynthesis, allowing to propose a model for alkamide acyl chain biosynthesis by a PKS complex, here named PKS alk. This complex would be partially reductive and related to type I PKSs, and present in plants that produce alkamides. These findings provide a framework for molecular and biochemical studies to confirm the activity of the proposed pathway. Further studies can also help to better understand the underlying regulatory mechanisms involved and the physiological role of these multifunctional molecules in plants.In the present study, the first transcriptomic high-throughput sequencing of the Mexican endemic plants 10.7717/peerj.10074/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.10074/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.10074/supp-3Supplemental Information 3H. longipes roots and a relevant putative function according to the annotation, and the Locus, % identity, e-value, score and information about the alignments to Arabidopsis (green section) and other plant species (blue section) is presented, for the proposed multi-enzymatic complex PKS alk for the synthesis of Affinin.The table includes the Enzymes name, Unigenes, Sequences and GO that presented increased accumulation in Click here for additional data file.10.7717/peerj.10074/supp-4Supplemental Information 4This data was used to obtain Figure 5: Relative expression of nine candidate genes involved in the putative alkamide acyl-chain biosynthetic pathway.Click here for additional data file.


Conductive and degradable nanofibrous scaffolds have great potential in promoting cell growth, proliferation, and differentiation under an external electric field. Although the issue of inferior electrical conductivity in body fluids still exists, polyaniline (PANI)-based degradable nanofibers can promote cell adhesion, growth, and proliferation.To investigate whether the effect is caused by the PANI morphology, we selected three inorganic acids as dopants in the process of PANI in situ oxidative polymerization: hydrochloric acid, sulfuric acid, and perchloric acid. The obtained polyaniline/polylactic acid (PANI/PLA) composite nanofibers were characterized via SEM, FTIR, and XPS analysis, and we confirmed that the PLA nanofibers were successfully coated by PANI without any change to the porous structure of the PLA nanofibers. The in vitro mechanical properties and degradability indicated that the oxidation of acid dopants should be considered and that it was likely to have a higher oxidation degradation effect on PLA nanofibers. The contact angle test demonstrated that PANI/PLA composite nanofibers with different surface morphologies have good wettability, implying that they meet the requirements of bone tissue engineering scaffolds. The surface roughness and cell viability demonstrated that different PANI morphologies on the surface can promote cell proliferation. The higher the surface roughness of the PANI, the better the biocompatibility. Consequently, the regulated surface morphology of PANI/PLA composite nanofibers via different acids doping has positive effect on biocompatibility in tissue engineering. Extracellular matrix (ECM) is a type of macromolecular network secreted by cells into the extracellular stroma. It presents the foundation of cells, tissues, and organ, accompanied with organs, and is characterized by a complex grid structure , 2. MoreConductive polymers  have good in vitro and in vivo biocompatibility, which can significantly affect cell adhesion, proliferation, and differentiation as well as tissue regeneration –13. Amon2SO4, SA), and perchloric acid , were selected as the dopants in a PANI in situ oxidative polymerization. Then, the mechanical properties, wettability, surface morphology, biocompatibility, and cell adhesion of PANI/polylactic acid (PLA) nanofibers were investigated under different acid dopants. The results indicated that the higher the surface roughness of the PANI, the better the cell proliferation, thus showing better biocompatibility.Polyaniline doped with inorganic acids generally has good electrical conductivity. However, the anions introduced by various inorganic acid dopants will affect the conductivity and structure of polyaniline –27. In tMw = 60,000) was purchased from Solarbio, dichloromethane (DCM) was purchased from Tianjin Fuyu Fine Chemical Co., Ltd., and N, N-dimethylformamide (DMF) was purchased from Macklin. Meanwhile, ammonium persulfate (APS) was purchased from Aladdin, HCl and H2SO4 were purchased from Guangzhou Chemical Co., Ltd., and HClO4 was purchased from Macklin.Aniline (AN) was purchased from Sigma, PLA (PLA particles with a specific mass were added into a mixed solution of DCM and DMF (volume ratio of 7:3) before being stirred until they dissolved, and a mixed solution of 10% PLA was obtained. The PLA solution was then dispensed into a syringe and connected with a high-voltage power supply. The electrospinning machine  was set with a voltage of 15 kV and at distance of 15 cm. The obtained PLA nanofibers were vacuum-dried overnight at 40 °C.2SO4, and HClO4, were used as dopants for in situ oxidative polymerization in the preparation of PANI/PLA nanofibers [The PLA nanofibers were placed in the plasma cleaning machine chamber  and discharged for 2 min at 30 W RF power. In this paper, three common inorganic acids, namely HCl, Hnofibers , and thePANI/PLA nanocomposite nanofibers were prepared under ice-bath conditions , 28. TheThe uniaxial tensile tests for the PLA nanofibers and PANI/PLA composite nanofibers were conducted via a strain–stress test . Here, the specimen was cut into a dumbbell-like shape, with the tensile speed maintained at a constant 3 mm/min. Young’s modulus was calculated from the 0–15% strain linear region in the stress–strain curve, and the tensile strength and fracture tensile rate of the curve were determined from the fracture of the nanofiber membrane.−1. X-ray photoelectron spectroscopy  and Al-Kα were used as the X-ray emission sources to further determine the surface composition of the PANI/PLA nanofibers, while their wettability was measured in terms of the contact angle of the water droplets at ambient temperature via contact angle analysis . The degradation of the nanofibers was evaluated using a mass loss method [The morphology of the nanofiber scaffolds was characterized via field emission scanning electron microscopy (FE-SEM)  to observe the different morphologies of the PANI doped with different inorganic acids. Prior to the SEM observation, the nanofiber samples were sprayed with gold for 60 s to allow for clearer observation of the morphology. Meanwhile, the surface roughness of the PANI/PLA composite nanofibers was measured using atomic force microscopy . To confirm that PANI was completely loaded on the PLA nanofibers, Fourier transform infrared spectroscopy (FTIR) (Thermo Nicolet iS50) was used to measure the wavelength change of 2000 ~ 500 cms method , 30. The2. When the cell growth reached a fusion degree of 90%, the cells were passaged at a ratio of 1:3.In this paper, the biocompatibility of PANI/PLA composite nanofibrous scaffolds was characterized by human osteosarcoma (HOS) cell activity experiment. HOS cells were purchased from the cell bank of the Chinese Academy of Sciences, Shanghai. The HOS cells were cultured in low-glucose Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal bovine serum, 100 U/mL penicillin, and 100 U/mL streptomycin before being incubated at 37 °C and 5% CO4 well density, while a blank group and a control group were simultaneously set up. The cells were then incubated in a cell incubator at 37 °C for one, three, and five days, with the medium refreshed every two days.The HOS cells had to be seeded on the PANI/PLA nanofibers prior to the cell proliferation test. Here, the nanofibers were placed into a 96-well plate such that they completely covered the bottom of the plate before being sterilized via UV for 30 min and via a 75% ethanol solution for 30 min. They were then washed with PBS. The nanofibers were then inoculated with a 1 × 10Cell viability of the PANI/PLA nanofibers was evaluated using a 3--2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay. Following incubation at one, three, and five days, the medium was removed from the 96-well plate and washed with PBS three times before a 1-mL DMEM containing 10% 5 mg/mL MTT was added. The medium was then incubated at 37 °C for 4 h and then removed before DMSO was added to dissolve the methylprednisolone. The medium was vibrated for 10 min, and then, the absorbance was determined .The HOS cells were incubated in the PANI/PLA nanofiber incubator for 24 h and were washed with PBS three times. Then, the cells were fixed with 4% paraformaldehyde for 10 min at room temperature. The fixed cells were washed with PBS three times (10 min each time) and 10 μL of 100 nM FITC-labeled peptide was added before the cells were incubated for 30 min at room temperature and then washed with PBS three times (5 min each time). The extracellular actin of the HOS cells was stained, with confocal microscopy  used to observe the cell staining at a 20 × magnification.The adhesion of the HOS cells on the PANI/PLA composite nanofibrous scaffolds was observed via SEM. Here, the culture medium was removed after the 24-h PANI/PLA nanofiber HOS cell culture and was then washed with PBS three times before the addition of 4% PFA. The medium was fixed overnight at 4 °C, washed with PBS three times, dehydrated with gradient ethanol solution , and then freeze-dried for 24 h. Prior to the SEM observation, the nanofibers were sprayed with platinum for 120 s to allow for better observation.. The HOS cells were cultured on different PANI/PLA composite scaffolds for the designated 7d. The cells were lysed using 50 μL of Tris–HCl  with 0.1% (v/v) triton X-100. The ALP activity is analyzed by quantifying the concentration of p-nitrophenol from p-nitrophenyl phosphate (PNPP), which is estimated by recording the absorbance at 405 nm. The percentage ALP activity of the cells cultured along the PANI/PLA nanofibers is calculated by comparing the ALP activity of the cells cultured on pristine PLA nanofibers.ALP is one of the commonly used early osteoblast differentiation markers that depend on alkaline phosphatase enzyme expression. Herein, ALP activity was performed using the ALP Assay Kit p < 0.05 (∗) and very significant when p < 0.005 (∗∗).The statistical significance of the results was assessed via one-way analysis of variance (ANOVA) using GraphPad Prism (version 8.02). Here, the differences in mechanical properties, in vitro biodegradability, and cell viability among the different PANI/PLA composite nanofibrous scaffolds were analyzed. The results were considered to be significant when The mechanical properties of tissue-engineered scaffolds are important indicators in the evaluation of whether the scaffolds can withstand fluid dynamics. The presence of inorganic acids may affect the physical and chemical properties of the PLA matrix of PANI/PLA composite nanofibers in the in situ chemical oxidation polymerization process of PANI. Therefore, exploring the mechanical properties of PANI/PLA composite nanofibers doped with inorganic acids is necessary. Here, the mechanical properties of PANI/PLA composite nanofibers were evaluated via a tensile test, which is shown in Fig. 4, which resulted in the cleavage of the ester bond in the PLA molecular chain and the oxidative decomposition of the carboxyl group, subsequently leading to inferior mechanical properties [2SO4, while the introduction of the APS in the reaction process can also have had a slight impact on the PLA nanofibers, with the comprehensive effect of these factors exhibiting different mechanical properties [As shown in Fig. operties .Meanwhioperties .Cell adhesion, proliferation, and differentiation are affected by the morphology, with a rough surface generally believed to be conducive to cell adhesion . The hydThe PANI morphology on the surface of the different PANI/PLA fibers was observed via FE-SEM Fig. . The figCell adhesion, migration, and proliferation are significantly affected by the wettability of the scaffolds , 36. In −1 correspond to a C=C stretching of the quinoid rings and a C = C stretching, C–N stretching, and = C–H stretching of the benzenoid rings, respectively. In the pure doped PANI spectrum . These results indicate that PANI was successfully loaded on the surface of the PANI/PLA nanofibers doped with inorganic acids. To further investigate the chemical composition of the PANI/PLA nanofibers, XPS was used to analyze their surface composition. In addition, in the XPS spectra , and protonated imine (=N+), respectively [The FTIR spectra of pure PANI and PANI/PLA composite nanofibers doped by different inorganic acids are shown in Fig. rum Fig. b, in addectively , 41. Wit4 aggravated the degradation of PLA and accelerated the mass loss of the PANI/PLA-PA nanofibers, which is consistent with the mechanical properties presented in Fig. As a template for tissue repair and regeneration, bioactive scaffolds are degraded and excreted out of the body following induced cell and tissue repair . In thisThe biocompatibility of bioactive scaffolds is the basis for promoting cell adhesion, growth, and proliferation . Herein,To investigate the biocompatibility of PANI/PLA composite nanofibers, their cell viability was evaluated using a MTT method. Figure PLA is biodegradable, but hydrophobic, meaning that it is not conducive to cell adhesion, growth, and proliferation. Following the plasma treatment, the surface of the PANI/PLA composite nanofibers was loaded with oxygen-containing groups, and the functional surface demonstrated good hydrophilicity. The above morphology and AFM results indicate that the PANI doped with different inorganic acids exhibited different morphologies and roughness levels on the surface of the PLA nanofibers. Meanwhile, the PANI/PLA composite nanofibers exhibited excellent wettability. Therefore, we considered that the different morphologies of PANI doped with inorganic acids led to the enhancement of the surface energy and polarity of the PANI/PLA composite nanofibers, which consequently affected the cell growth, migration, and proliferation, resulting in improved performance in terms of cell activity .To further study the cell behavior of the PANI/PLA composite nanofibers, the growth and adhesion on the nanofibers were observed via fluorescence immunostaining Fig.  and SEM Meanwhile, the cell immunofluorescence staining and cell adhesion results indicated that the different PANI morphologies on the surface of the PANI/PLA composite nanofibers affected the growth, adhesion, and proliferation of the HOS cells, which was consistent with the above results.As an early osteogenic marker, the ALP test was conducted on PLA and PANI/PLA composite nanofibers scaffolds for 7 days. Compared to the pure PLA nanofibers, the result Fig.  showed tIn this paper, PANI/PLA composite nanofibers of different surface morphologies were prepared by three types of inorganic acid as dopant in in situ polymerization. We confirmed that PANI could be successfully loaded on the surface of PLA without changing the porous structure of the nanofibers. The mechanical properties and in vitro degradation experiments demonstrated that oxidizing acids can significantly weaken the mechanical properties and accelerate the degradation of polyester nanofibers. Meanwhile, the rougher surface resulted in a better wettability and promoted the cells adhesion, growth, and proliferation, which indicated a better biocompatibility. In conclusion, the regulated PANI morphology via different acids doping has positive effect on biocompatibility in tissue engineering.


Ischemia reperfusion injury (IRI) is related to different surgical interventions such as organ resection and transplantation, and therefore its prevention is of great interest. However, several decades of investigations have not, unfortunately, lead to a definitive solution for the treatment of IRI due to its complex and multifactorial pathophysiology with a plethora of underlying mechanisms that are shared among different organs  ,2,3. TheIRI is the most important cause of graft failure in transplantation besides immunological rejection. This is the result of cumulative damage that depends on the strength, duration and intensity of the different episodes inherent to transplantation, and leads to the appearance of organ primary/secondary failure, which may happen as a consequence of a re-transplantation ,5.Along this Special Issue “Molecular Mechanisms and Pathophysiology of Ischemia-Reperfusion Injury 2.0” we provide a broad overview of the recent insights on several strategies adopted influencing different molecular and pathophysiological mechanisms involved in different organs such as heart, brain, liver and kidney. These strategies in clinical surgery and transplantation embrace the current understanding of some of the underlying pathophysiological molecular pathways and in turn better knowledge of them is determinant to counteract the IRI deleterious effects. Here, we report new advances in IRI molecular mechanisms associated with different strategies, such as ischemic preconditioning (IP) and post-conditioning (Post C) ,7,8, whiIt is well-known that the activation of molecular sensors, such as nitric oxide and AMPK, are tied to IP and PostC ,14,15. OMoreover, there is growing evidence in the brain that factors such as CELSR1  laminin G (LAG) seven-pass G-type receptor) are responsible for neuroprotective effects on cerebral ischemia injury . This isRecent efforts have been conducted to avoid the detrimental injury effects that occur during liver resection interventions by inhibiting iNOS with L-NAME , as wellSeveral MP investigations in kidney have shown the benefits of incorporating antioxidants, such as Vectisol, which decreases the early levels of the detrimental radical oxygen species (ROS)  or mesenIn conclusion, the challenge for the upcoming years is to explore in depth the complex molecular pathophysiological mechanisms inherent to IRI in order to establish new frontiers for future interventions in clinical transplantation.


The composite nanofibers maintained their cylindrical fiber morphology, structure and composition after multiple heating–cooling cycles and retained their thermal management functionality. The contact angle measurements showed that the addition of PCMs imparted hydrophobicity to the nanofibers.Nanofibers with thermal management ability are attracting great attention in both academia and industry due to the increasing interest in energy storage applications, thermal insulation, and thermal comfort. While electrospinning is basically a fiber formation technique, which uses electrostatic forces to draw ultrafine fibers from a wide variety of polymers, with the addition of phase change materials (PCMs) to the electrospinning solution it enables the production of shape stabilized phase change materials with thermal management functionality. In this study, polyacrylonitrile (PAN) nanofibers containing paraffinic PCMs were produced by electrospinning method and the composite nanofibers obtained were characterized in terms of their morphology, chemical structure, thermal properties, stability, thermal degradation behaviour and hydrophobicity. Besides, PCMs with different phase transition temperatures were added simultaneously into the nanofiber structure in order to investigate the tunability of the thermoregulation properties of the nanofibers. Uniform nanofibers with thermal management functionality were obtained. It could be possible to obtain composite nanofibers showing thermoregulation ability over a wider temperature range by simultaneous addition of PCMs with different melting points into the nanofiber structure. 50 wt% PCM could be added to PAN nanofiber structure wherein the resulting nanofiber exhibited 58.74 J g PCMs are materials, which change phase at certain temperatures and accordingly have the ability to absorb, store and release large amounts of energy in the form of latent heat depending on the surrounding temperature5. PCMs can be grouped into three major classes such as organic compounds, inorganic compounds, and eutectic compounds of the first two. While organic PCMs are polyethylene glycols (PEGs), paraffin waxes, and fatty acids, inorganic PCM compounds consist of metals, metal alloys, salts, and salt hydrates. Melting temperature, temperature range of the phase change and heat storage capacity are important factors for a PCM and the selection depends on the end application5.Being one of the basic elements of clothing comfort, the thermo-physiological aspect includes the heat and moisture transmission characteristics of clothing. Thermo-physiologically comfortable clothing supports the thermoregulation of the body and helps the wearer to keep a comfortable temperature. In this regards, new textile concepts have been introduced, one of which is the addition of PCMs to the structure of textile materials6. Although majority of the regulation is done by skin, textile materials with thermoregulation properties can be used to help the skin preserve its temperature within the comfort zone, especially during abnormal fluctuations of temperature1. Thermoregulation properties can be achieved in textile materials by different ways: Textile fibers can be functionalized by mixing/blending PCMs into the fiber spinning solution/melt7, loading PCMs into the core of hollow fibers9 or incorporation of PCMs to the polymer chain during synthesis10. PCMs can also be applied to fabrics by coating12 or finishing processes15. Electrospinning is another preferred technique especially in research studies showing the feasibility of producing textile materials with thermal management properties25.When added to textile materials, PCMs improve the thermal comfort of textile materials by providing proactive temperature management which is especially preferred in some applications such as everyday clothing , professional clothing , interior decoration and essentials , shoe linings, automotive textiles (seat covers) and is of vital importance in some other applications such as surgical gloves, bedding materials, bandages and products which are used in hospitals and especially in intensive care units to regulate patients’ temperatures and keep within the comfort range28. Electrospun nanofibers have attracted great attention due to their ease of fabrication, unique properties such as high surface-area-to-volume ratio, low density, and high pore volume and possibilities of functionalization31 and used in a wide range of applications such as filtration32, energy harvesting and storage33, tissue engineering34, drug delivery35, wound healing36, sensors37, and polymer reinforcement38, etc. Composite nanofibers produced with the addition of different types of additives displayed additional functionalities. It has been possible to produce antistatic nanofibers with polyaniline39, antibacterial nanofibers containing silver nanoparticles40, and antistatic nanofibers showing enhanced tensile properties with the addition of carbon nanotubes41.Electrospinning is a simple and cost-effective technique to produce fibers from a wide variety of polymers with diameters ranging from nanometres to submicrons. It is a unique approach that uses electrostatic forces to produce fibers from polymer solutions (or melts). The polymer solution filled into a syringe is introduced to the tip of a needle to form a pendant drop by a syringe pump, charged by the application of high voltage and then subjected to electric field. When the electric field overcomes the surface tension of the polymer solution, a charged jet ejects from the tip of needle, undergoes unstable whipping and bending motions between the tip of the needle and the collector, where the solvent evaporates and polymer in the nanofiber form is collected on the collector25. Chen et al. conducted a series of studies on the incorporation of fatty-acids and fatty-acid derivatives as PCMs into nanofiber structure using conventional electrospinning18. They successfully electrospun nanofibers of polyethylene terephthalate (PET)/lauric acid, which had smooth surfaces and sufficient tensile strength using conventional electrospinning. With their highest melting enthalpy of about 70.76 J g−1, the nanofibers were suggested for applications such as thermo-regulating textiles and solar energy storage16. They also produced composite PET nanofibers using different types of fatty acids such as lauric acid, myristic acid, palmitic acid, and stearic acid as additives. The morphology and the average diameter of the nanofibers were affected by the amount of the fatty acid. The thermal storage ability and the thermal transition temperatures observed were dependent on the type of the fatty acids. While the melting temperature obtained varied between 45 and 69 °C, heat of fusion varied between 54.91 and 67.88 J g−118. Producing composite nanofibers of polyamide/lauric acid, Cai et al. reported about the formation of ribbon shaped nanofibers with melting enthalpies changing between 63.66 and 74.12 J g−1 as the fatty acid content increased from 80 to 150%19. Producing PET nanofibers with a series of different fatty acid eutectics in another study, they reported that the latent heat and phase transition temperature of composite fibers were dependent on the type of the fatty acids. Thermal analysis suggested that the melting and crystallization enthalpies of composite fibers increased gradually with increasing fatty acid eutectic amounts, with melting enthalpies ranging from 33.91 to 112.7 J g−120. Ke et al. electrospun composite PET nanofibers using a series of fatty acid eutectics for thermal energy storage. PET/PCM ratios were selected as 100/50, 100/100, and 100/150. The composite nanofibers’ diameter ranged from 100 to 300 nm and they showed different thermal behaviours depending on the type and content of PCMs. The melting enthalpies observed were between 34.18 and 95.24 J g−121. Using PEG as additive, Chen et al. produced cellulose acetate/PEG nanofibers. According to thermal analysis, the heat capacities of the composite fibers with 10–70 wt% PEG varied between 3.8 and 120.2 J g−122. Additionally, they studied the effects of molecular weight of PEG on morphology, thermal properties, and mechanical properties of the composite fibers. They used PEG with five different molecular weights, which varied between 2000 and 20,000 g mol−1. According to the thermal analysis results, the composite fibers displayed thermal storage and release properties in different temperature ranges, which were dependent on the molecular weight of PEG. The melting enthalpy of the composite fibers varied between 53.23 and 86.03 J g−1 depending on the molecular weight of PEG23. Hu et al. produced core–shell nanofibers using natural soy wax (bio-based PCM) as the core and polyurethane as the shell by coaxial electrospinning. Uniform fiber morphology with a core–shell structure, and a homogeneous wax distribution throughout the core of the fibers, were obtained. Thermal analysis results showed that the enthalpy increased with wax content. The melting enthalpy increased from 9.24 to 36.47 J g−1 as the wax content in the fiber structure increased from 10 to 60%24.It has also been possible to produce composite nanofibers with thermoregulating properties by adding PCMs into the nanofiber structure. The studies on the production of composite nanofibers containing PCMs mainly focused on the use of individual fatty acids, their eutectics, and polyethylene glycolsAlthough there are many studies in literature investigating the properties of PCMs, composite PCM materials and composite nanofibers with fatty acids and polyethylene glycols, there is very limited number of studies showing the use of pure paraffins in the production of functional nanofibers with thermal management functionality. In this study, two different PCMs, both of which were 100% paraffin with different thermal transition temperatures, were directly added into PAN nanofiber mats to impart thermal regulation properties to nanofibers. They were also added simultaneously to the nanofiber structure to investigate the possibility of tuning the thermoregulation properties (impart higher heat storage/release capacity over a wider temperature range). The composite nanofibers were analysed comprehensively regarding their morphology, chemical structure, thermal properties, stability, thermal degradation behaviour and hydrophobicity. This study provides substantial contribution to the literature by showing the development of chemically, morphologically and thermally stable composite PAN nanofibers with thermoregulation properties, pointing out the required process parameters for the production of uniform-structured PAN/PCM nanofibers, showing the possibility of finetuning the properties of these nanofibers by incorporating different types of PCMs simultaneously into the nanofiber structure and changing their ratios in order to obtain higher storage/release capability over a wider temperature range, and providing comprehensive discussion regarding morphology, chemical structure, thermal properties, stability, thermal degradation behaviour and hydrophobicity of the composite PAN nanofibers containing PCMs.−1), provided by Sigma Aldrich, was used as the fiber forming polymer. Dimethylformamide (DMF) was supplied from Sigma Aldrich and used as the solvent. PCMs with two different melting points of 28 °C (PCM28) and 32 °C (PCM32) were provided by Microtek Laboratories, Inc. and added to the nanofiber structure in order to obtain nanofibers with thermal management functionality. The properties of the PCMs provided by the manufacturer are presented in Table PAN . Ultrasonic tip was utilized for the homogenization of PCM/DMF dispersions. After homogenization of the dispersions with ultrasonic tip for 10 min, PAN was added to the dispersion and dissolved by stirring magnetically for 3 h at 40 ℃. The concentration of PAN was 10 wt%. Reference nanofibers were also produced from 10 wt% PAN/DMF solution.Composite nanofibers containing PCMs were produced on horizontal electrospinning setup. Illustrated in Fig. −1 and the applied voltage as 15 kV. Electrospinning was performed under standard atmospheric conditions (Temperature: (20 ± 2) ℃, Relative humidity: (65 ± 5) %). The nanofiber mats were collected for 4 h. The compositions of nanofiber mats produced are presented in Table Composite electrospinning solutions of PAN/DMF with PCMs were fed through a blunt needle with a diameter of 1.25 mm, using a syringe of 10 mL. The nanofibers were collected on a rotating metal collector which was covered by nonwoven, respectively. The distance between the tip of the needle and the collector was set as 10 cm, the flow rate as 1 mL h−1 with a resolution of 4 cm−1. 32 scans were taken for each experiment and averaged to obtain the FTIR spectra of composite PAN/PCM nanofiber mats. FTIR was also utilized to investigate the chemical structure of composite nanofiber mat after 50 consecutive thermal cycles. Differential Scanning Calorimetry (DSC) (Mettler Toledo) was used to investigate the thermal properties of PCMs, neat PAN and composite PAN/PCM nanofiber mats. PCMs were tested in a temperature range from 0 to 50 °C, with a heating/cooling rate of 5 ºC min−1, under nitrogen (N2) with a flow rate of 50 ml min−1 in order to understand their phase transition properties such as thermal transition temperatures and enthalpies. The composite nanofibers were also tested in a temperature range from 0 to 50 ºC, with a heating/cooling rate of 5 ºC min−1 in nitrogen environment in order to evaluate the latent heat storage and release ability of the nanofibers. The thermal characteristics were evaluated from the second heating of the samples. Besides, 50 consecutive heating–cooling cycles between 0 and 50 °C with a heating rate of 5 °C min−1 were applied to both PCMs and composite PAN nanofiber mat with 50% PCM32 monitor the variations in the latent heat storage and release ability of the nanofibers by repeated thermal cycles and evaluate the thermal stability of the composite nanofibers. The phase transition temperature was taken as the peak point of the DSC curves. The enthalpy of melting (ΔHm) and the enthalpy of crystallization (ΔHc) were calculated based upon the areas under the solid–liquid and liquid–solid phase change peaks of paraffins using the thermal analysis software affiliated with the equipment. Thermogravimetric analysis (TGA) (Mettler Toledo) was used to determine the thermal decomposition behaviours of PCMs, neat PAN and composite PAN/PCM nanofiber mats. The heating rate was set as 10 °C min−1 and the TGA curves were recorded from room temperature to 1000 °C under N2 flow with a rate of 50 ml/min. Derivative TGA (DTGA) curves were obtained from the TGA curves. Water contact angle measurements were also conducted to characterize the nanofiber mats in terms of hydrophobicity.The viscosity and conductivity of PAN/DMF and PAN/%50PCM/DMF electrospinning solutions were measured. The viscosity was measured with Brookfield DV2TR using SC4-21 spindle at 50 RPM. The conductivity was measured using Hanna HI 8633 conductivity meter. Scanning electron microscope (SEM) (Jeol Quanta 200 FESEM) was used to analyse the morphology of the nanofibers. Samples were coated with gold using a sputter coater before SEM analysis. SEM images were taken at an accelerating voltage of 20 kV. The fiber diameters were measured from the SEM images using Image J. The average nanofiber diameters and the standard deviations were based on 50 measurements per sample. Average nanofiber diameters were expressed as the mean ± standard deviation (S.D.). In addition, SEM was also utilized to show the form stability of composite nanofibers after 50 consecutive heating–cooling cycles. Fourier Transform Infrared Spectrometer (FTIR) (Perkin Elmer-Spectrum Two) was used to record absorption spectra of composite nanofibers in a range from 4000 to 650 cmThe results obtained are discussed below with the help of the relevant tables and figures.SEM images of neat PAN and composite PAN/PCM nanofiber mats, taken with 25 kX magnification, are presented in Fig. The white colour of the nanofiber mats Fig.  and also17. Since polymer concentration and the electrospinning parameters were fixed in this study, the morphology of the electrospun fibers considered to be affected mainly by the PCM content. The effects of the incorporation of additives into nanofiber structure are widely discussed in literature. Jo et al. reported an increase in fiber diameter with titanium dioxide addition42. In many studies, decrease in nanofiber diameter was reported with silver nitrate addition45. The addition of fatty acids and PEG and increase in their contents were reported to result in larger nanofiber diameters46.SEM images showed that the diameters of the nanofibers were affected by the incorporation of additives. From the previous studies, morphology of the electrospun fibers are primarily influenced by the type of polymer, the solution properties and the process parameters47, increase in conductivity leads to the formation of thinner nanofibers to some extent and then coarser nanofibers after a critical value45. Depending on the compromise between viscosity and conductivity, the average nanofiber diameter is determined. The viscosity and conductivity of PAN/DMF solution were measured as 174 mPa s and 158 µS/cm, respectively. When 50% PCM was added into the PAN solution, the viscosity increased to 332 mPa s and the conductivity remained almost constant (was measured as 150 µS/cm). Thus, the viscosity effect was dominant when PCM was added and as a result of this, increase was observed in average nanofiber diameter. While the neat PAN nanofibers had an average diameter of 257.15 nm, the average nanofiber diameter increased to about 350.15 nm with 50% PCM addition (PCM-50PCM28-50PCM32).The viscosity and the conductivity of the electrospinning solutions are two important parameters that have effects on nanofiber diameter and are affected by addition of additives to a great extent. While increase in viscosity of the electrospinning solution leads to the formation of thicker nanofibers18, the maximum with PEG was 70 wt%22. In this study, it could be possible to add up to 50% paraffinic PCMs to the nanofiber structure without any deteriorations in the nanofiber and nanofiber mat structure.A good shape stabilizer should be able to host a significant amount of PCM to have better thermal management properties since the thermal regulation is performed by those compounds. According to literature, morphology analysis showed that the maximum PCM that could be incorporated into fiber structure changed depending on the PCM type used. While it could be possible to add as much as 100% fatty acids without disturbing fiber structureFTIR spectroscopy was used to confirm the incorporation of PCM into PAN nanofiber structure. Besides, the spectra obtained was used to compare the spectral differences between the composite nanofiber mats and the neat PAN nanofiber mat.3 stretchings observed at 2922 cm−1 and the spectra of neat PAN and composite nanofibers were normalized to the characteristic C≡N stretching band of PAN which appears around 2242 cm−1 to be able to compare them and obtain comparable absorbance values. The normalized FTIR spectra of PCMs, neat PAN and composite PAN/PCM nanofiber mats are presented in Fig. The spectra of PCMs were normalized to the largest peak observed, namely the –CH−1 are the characteristic of the aliphatic C–H stretching vibration. Peaks at 1470 and 1465 cm−1 are the bending vibration of the –CH2 group; 1375 cm−1 is the in-plane bending vibration of C–H and C–C; 728 and 720 cm−1 are the rocking vibration of C–H in the paraffin49. The following peaks are observed in the spectra of PAN nanofibers , 2922 and 2870 cm−1 , 2242 cm−1 (C≡N stretching), 1622 cm−1 (C=C stretching), 1452 cm−1 (CH3 bending and CH2 scissor vibration), 1356 cm−1 (CH3 symmetric bending vibration in C–CH3), and 1252 cm−1 (C–N stretching), 1070–1040 cm−1 (C = N bending), and 778 cm−1 (–C–CN– stretching)51. Displaying the characteristic peaks of PCMs in addition to the characteristic peaks of PAN, the FTIR spectra of composite nanofibers, shown in Fig. −1) increased with the increase in the PCM content.In Fig. m), crystallization temperature (Tc), enthalpy of melting (ΔHm), and enthalpy of crystallization (ΔHc) of the samples) via DSC and also compared to the PCMs. DSC is an effective method for testing the functionality of PCM materials. It provides information about the amount of energy that can be stored and released by a material. The energy that can be stored is associated with the enthalpy of melting, whereas the energy that can be released is related to the enthalpy of crystallization52.For the textile material with thermal management functionality, appropriate phase transition temperatures which are close to skin temperature along with high latent heat storage capability are the most desirable characteristics. In this study PCMs, which had melting points close to the skin temperature were especially chosen. The composite nanofiber mats containing PCMs were analysed with regards to their thermal properties  of composite fibers were dominated by the type of PCMs and had no obvious variations compared with those of PCMs. The slight decrease, which was less than 2 ℃, was likely caused by weak molecular interactions between PCMs and PAN nanofiber mat, which affected the required energies for phase transitions to take place and led to the depression of the phase change temperature53. On DSC thermograms of the composite nanofibers produced with the simultaneous addition of PCM28 and PCM32, two endothermic peaks and three exothermic peaks resembling the thermal transition behaviour of PCMs were obvious. It could be possible to obtain composite nanofibers showing thermoregulation ability over a wider temperature range by simultaneously adding PCMs with different melting points (PCM28 and PCM32) into PAN nanofiber structure. The composite nanofibers of PAN containing PCM28 and PCM32, displayed thermoregulatory properties between 22.75 and 31.65 °C during heating and between 29.68 and 12.62 °C during cooling which were much wider compared to nanofibers containing only PCM28 and PCM32. Enthalpy storage values and enthalpy release values increased with the increase in the concentration of PCMs. The melting enthalpy increased from 11.42 to 24.71 J g−1 and the crystallization enthalpy increased from 11.53 to 26.45 J g−1 as the PCM28 content increased from 20 to 33%. For the PCM32 containing composite PAN nanofibers, the melting enthalpy increased from 14.88 to 31.89 J g−1 and the crystallization enthalpy increased from 17.16 to 37.64 J g−1 as the PCM 32 content increased from 20 to 33%. The highest storing and releasing enthalpy values, which were 58.74 and 57.42 J g−1 were observed for composite PAN nanofibers containing the highest amount of PCMs. For the composite nanofibers produced with the simultaneous addition of PCM28 and PCM32, melting enthalpy increased from 13.57 to 58.74 J g−1 and the crystallization enthalpy increased from 13.75 to 57.42 J g−1 as the total PCM content increased from 20 to 50%.DSC thermograms of neat PAN and composite PAN/PCM nanofiber mats are presented in Fig. 16. From Table 56. During the electrospinning process, the evaporation of solvent occurs in milliseconds, and the molecules of PCMs may not have enough time to form well-defined structures in the composite fibers57. Besides, paraffins have a disadvantage that they have low thermal conductivity which usually falls short of providing the required heat exchange rate between the PCM and substrate4. The variations between the theoretical and actual values became less significant with the increase in the PCM content potentially due to the diluent effect of PAN surpassed by the larger amount of PCM.The enthalpy values of composite PAN/PCM nanofibers were lower than that of PCMs powder because PAN in the composite fibers did not have any contributions to enthalpy values at this temperature range. Additionally, the enthalpy values measured showed some variations from the theoretical enthalpy values. The theoretical enthalpy values calculated, and experimental enthalpy values obtained by DSC are presented in Table Electrospinning enables it to produce novel thermoregulating materials which imparts form stability to PCMs in their molten form with the polymeric component of nanofibers acting as a scaffold for the PCMs. Composite PAN nanofiber mat containing 33% PCM32 (PAN-50PCM32) was tested regarding its thermal stability against 50 consecutive thermal cycles. The thermograms of 1st and 50th cycles are presented in Fig. 17.The PCM particles were observed to be infused to each other Fig. a,b after−1, which were the vibrations of the C–H groups of the paraffinic PCMs.FTIR was used to investigate the chemical structure of composite nanofiber mat before and after 50 consecutive DSC thermal cycles. The spectra Fig.  demonstr5%) and the residue at 800 ℃ for PCMs, neat PAN and composite PAN/PCM nanofiber mats are presented in Table The thermal decomposition behaviour of PCMs, neat PAN and composite PAN/PCM nanofiber mats were evaluated using TGA in nitrogen atmosphere. While the TGA and DTGA curves of PCMs and neat PAN nanofiber mat are shown in Fig. max) values at 365.4 and 409 ℃ while PCM32 showed one-step degradation, with Tmax at 420 ℃, the difference of which was likely due to the molecular weight differences of the PCM materials  and the residue at 800 ℃ for PCMs, neat PAN and composite PAN/PCM nanofiber mats  could be incorporated into the PAN nanofiber structure wherein the resulting composite nanofiber exhibited 58.74 J g


Trichoderma reesei were prepared for the biodegradation of crystalline cellulose nanofibers, which carrier system could be incorporated in tissue engineering biomaterials to degrade the crystalline cellulose nanoreinforcement in situ and on-demand during tissue regeneration. Both alginate beads and microparticles were processed by extrusion-dropping and inkjet-based methods, respectively. Processing parameters like the alginate concentration, concentration of ionic crosslinker Ca2+, hardening time, and ionic strength of the medium were varied. The hydrolytic activity of the free and encapsulated enzyme was evaluated for unmodified (CNFs) and TEMPO-oxidized cellulose nanofibers (TOCNFs) in suspension (heterogeneous conditions); in comparison to solubilized cellulose derivatives (homogeneous conditions). The enzymatic activity was evaluated for temperatures between 25–75 °C, pH range from 3.5 to 8.0 and incubation times until 21 d. Encapsulated cellulase in general displayed higher activity compared to the free enzyme over wider temperature and pH ranges and for longer incubation times. A statistical design allowed optimizing the processing parameters for the preparation of enzyme-encapsulated alginate particles presenting the highest enzymatic activity and sphericity. The statistical analysis yielded the optimum particles characteristics and properties by using a formulation of 2% (w/v) alginate, a coagulation bath of 0.2 M CaCl2 and a hardening time of 1 h. In homogeneous conditions the highest catalytic activity was obtained at 55 °C and pH 4.8. These temperature and pH values were considered to study the biodegradation of the crystalline cellulose nanofibers in suspension. The encapsulated cellulase preserved its activity for several weeks over that of the free enzyme, which latter considerably decreased and practically showed deactivation after just 10 d. The alginate microparticles with their high surface area-to-volume ratio effectively allowed the controlled release of the encapsulated enzyme and thereby the sustained hydrolysis of the cellulose nanofibers. The relative activity of cellulase encapsulated in the microparticles leveled-off at around 60% after one day and practically remained at that value for three weeks.Recent advances in nanocellulose technology have revealed the potential of crystalline cellulose nanofibers to reinforce materials which are useful for tissue engineering, among other functions. However, the low biodegradability of nanocellulose can possess some problems in biomedical applications. In this work, alginate particles with encapsulated enzyme cellulase extracted from  The outstanding mechanical properties of crystalline cellulose nanofibers, the high availability of cellulose in nature, and the renewability and proved cytocompatibility of the nanofibers make them an excellent candidate for the reinforcement of biomaterials in tissue engineering applications ,4,5,6,7.Aspergillus, Humicola, Penicillium, and Trichoderma. They are composed of three enzymes that act synergistically in the hydrolysis of cellulose into soluble sugars [2+, enables biomolecules and cells to be incorporated into the biocompatible and biodegradable alginate matrix with the retention of the three-dimensional arrangement of the encapsulated molecule [Cellulase enzymes can be isolated from cellulolytic fungi such as e sugars ,49,50. Te sugars ,51. Polye sugars ,53,54. Se sugars . Among te sugars . The mone sugars . A relatmolecule . In a numolecule ,60.2+, addition of NaCl to change the medium ionic strength, and hardening time. A statistical design enabled us to report the optimum conditions for the processing of spherical particles presenting the highest enzymatic activity. The activity and stability of the immobilized cellulase were compared to those of the free enzyme. The comparative kinetics behavior of the enzymatic degradation of the different cellulose nanofibers both by using alginate beads and micro particles could be discussed.The aim of this work is the preparation of cellulase-immobilized polymer particles enabling the enzyme controlled release for the hydrolysis of crystalline cellulose nanofibers of the type nanofibrillated cellulose. Furthermore, the cellulase-immobilized particle system should find application in the in situ degradation of the nanocellulose incorporated in biomaterials, while this latter serves as a matrix for tissue regeneration. In this work, cellulase was encapsulated in alginate particles and the enzymatic activity was evaluated on three different cellulose substrates: solubilized carboxymethylcellulose (CMC), non-modified (CNFs), and 2,2,6,6-tetramethylpiperidinyloxy (TEMPO)-oxidized cellulose nanofibers (TOCNFs) in suspension. Comparative studies of cellulose nanofiber biodegradation were performed with cellulase-entrapped in alginate beads and microparticles. Microsized particles should possess advantages over larger particles in drug-controlled release and other biomedical applications. These latter were manufactured to have a more uniform size and shape with high surface area-to-volume ratio. Different parameters were varied in the processing of the cellulase-encapsulated alginate particles like the concentration of alginate and ionic crosslinker Ca3 adjusted to pH 7, which was also used as eluent. Then, they were filtered through 0.45 µm pore size membrane . The chromatographic equipment was composed of an IsoChrom LC pump  connected to PL aquagel-OH MIXED-M and PL aquagel-OH MIXED-H columns . A multiangle laser light scattering (MALLS) detector DAWN DSP  operating at 664.0 nm was coupled on line to a WATERS 410 differential refractometer. The refractive index increment dn/dc was 0.134 mL/g. The size exclusion chromatography (SEC) coupled to MALLS allowed determining a weight (Mw) and a number-average molecular weight (Mn) of alginate of 9.62 × 104 (±0.66%) and 4.55 × 104 (±2.21%) g/mol, respectively; with a polydispersity index (Mw/Mn) of 2.11 (±2.30%), showing a relatively narrow molecular weight distribution  was purchased from Sigma Aldrich . Weight- and number-average molecular weights of ALG were determined by size exclusion chromatography (SEC). Alginate aqueous solutions at 0.1% (w/v) were prepared in 0.1 M NaNOribution . The gulspectrum , a compoand Kohn .Carboxymethylcellulose (CMC) was supplied by Sigma Aldrich (Germany). It is a derivative of cellulose, which is soluble in aqueous buffers and others solvents.Cellulose nanofibers of the nanofibrillated cellulose type were used:® (Novozyme) at pH 5.0. The digested samples were further refined to obtain a pulp suspension of SR (Schopper-Riegler) number higher than 80 and mean fiber length lower than 300 µm. 2% (w/w) fiber suspensions were processed with an Ariete homogenizer, involving one pass at 1000 bar followed by three passes at 1500 bars.(i) Non-modified cellulose nanofibers (CNFs): Their suspensions were obtained from bleached pine sulfite dissolving pulp at the Centre Technique du Papier , by a mechano-enzymatic method adapted from Pääkkö et al. (2007) . The pul(ii) TEMPO-oxidized cellulose nanofibers (TOCNFs): They were prepared according to the protocol described by Benitez et al., 2016 . Kraft pTrichoderma reesei was purchased from Novozyme . Celluclast 1.5 L had a protein content of 124 mg/mL and activity of 700 EGU/g.The enzyme cellulase Celluclast 1.5 L from the fungus 2 coagulation bath was adjusted to 10 cm. Different CaCl2 concentrations (0.2 M or 0.4 mol/L) were considered. In some experiments, 0.8 mol/L NaCl was added to increase the medium ionic strength and thereby promote the formation of homogeneous spherical beads. These later were allowed to harden for 1 or 3 h in the CaCl2 crosslinking bath. Finally, the beads were collected, washed several times with Milli-Q water and directly stored as hydrogel beads in pH 4.8 acetate buffer at 4 °C, or freeze-dried to obtain dry particles. To prepare cellulase-encapsulated alginate beads, Celluclast 1.5 L from Trichoderma reesei was mixed with the alginate solution and left gently stirring for 1 h at 4 °C. A relation of 1 mg of enzyme protein to 290 mg of alginate was used. Then, same dispensing procedure as above was followed to obtain cellulase-entrapped alginate beads.(i) Beads: Alginate was dissolved at a given concentration ) in Milli-Q water, by mechanical stirring for 24 h at 60 °C. The obtained viscous solution was filtered through 0.8 µm pore size membrane . Then, alginate beads were prepared by extruding the alginate solution by means of a dispenser PerformusTM I . It allowed the pumping, under controlled dry air pressure (0.3 bar) of the solution contained in a syringe cartridge, through a needle of inner diameter 200 µm. The dropping distance between the needle tip and the surface of the CaCl2 and 0.2 M NaCl, by applying a pressure of 0.7 MPa, a dosing distance 0.03 mm and a valve opening and closing time of 250 and 300 µs, respectively. They were allowed to harden for 1 h while gently magnetically stirring. Finally, the microparticles were washed several times with Milli-Q water and freeze-dried. To achieve cellulase-encapsulation in the microparticles, in the inkjet ‘printing’ procedure, alginate solutions containing the same cellulase/alginate ratio as above (1/290 mg/mg) were considered.(ii) Microparticles: An inkjet-based method was used to prepare alginate microparticles. A 4% (w/v) alginate viscous solution (bioink) was loaded into a cartridge, which was set to a temperature-controlled nozzle system, constituting a printhead of a 3D bioprinter . Preliminary works were performed with lowest alginate concentrations ), but distinct spherical particles of homogeneous size distribution were not achieved. The alginate microparticles were ‘printed’ into a gelling bath containing 0.2 M CaClThe cellulose degradation was carried out by enzymatic hydrolysis with cellulase immobilized in the alginate particles or as free enzyme. Measures of 20 mg of cellulase-encapsulated alginate particles or the corresponding amount of free enzyme dissolved in few µL of acetate buffer at a given pH were incubated with the solubilized carboxymethylcellulose (CMC) or with the cellulose nanofibers in suspension. The cellulose hydrolyses were performed at varied temperatures between 25 and 75 °C, pH values between 3.5 to 8.0, and incubation times of 21 days. On a practical level of understanding the effect of environmental parameters on enzymatic activity, previous studies have called for the caution of the common practices of using soluble substrates , like th−1, respectively. Height and phase images were analyzed with NanoScope Analysis 1.5 software.(i) Atomic force microscopy (AFM): The morphology of the cellulose nanofibers was characterized by atomic force microscopy (AFM). A droplet of a 0.001% nanofiber suspension was placed on a freshly cleaved mica surface. After sample drying, the observation was performed in tapping mode with an AFM  equipped with a tube scanner from Veeco Digital Instruments , using silicon tips  with resonance frequency and spring constant of 360 kHz and 50 N·mdmax is the maximum diameter passing through a bead centroid; and dper is the diameter perpendicular to dmax.(ii) Optical microscopy: The dimensions and morphology of the alginate particles, both as hydrogel and dry state, were analyzed by light optical microscopy. To this end, a digital camera  coupled to a binocular optical microscope  was used. The particle micrographs were analyzed with the software ImageJ 1.52u. Concerning bead shape analysis, the sphericity factor SF was determined by using the Equation (1). This factor varies from 0 for a perfect sphere up to unity for an elongated object ,65,66.S(iii) Fluorescence microscopy: Fluorescence microscopy allowed observing the local distribution of the enzyme within the alginate particles. The cellulase was stained with fluorescein isothiocyante (FITC). In dark conditions, 700 µg of FITC reactive dye were dissolved in dimethyl sulfoxide (DMSO) to achieve a concentration of 10 mg/mL FITC. 2 mg of cellulase were added to 1 mL of sodium bicarbonate buffer (pH 9.0) and the obtained solution was stored at 4 °C for 1 d. The FTIC solution from above was added into the flask containing the enzyme solution and the mixture was incubated for 8 h at 4 °C in darkness. Dialysis was used to remove the unconjugated FITC. The inner content of the dialysis tubing containing the FITC-labeled cellulase was collected. The stained enzyme was used to prepare alginate particles with encapsulated fluorescent-labeled cellulase, following the above described procedure to prepare the cellulase-encapsulated alginate particles. The appearance of the FITC-labelled cellulase alginate beads after immersed in PBS solution (pH 7.4) for different times  was followed by fluorescence microscopy. The fluorescent beads were imaged by using a confocal laser scanning microscope  equipped with detectors and filter sets for monitoring fluorescence.(iv) Scanning electron microscopy (SEM): The dimensions and surface morphology of the beads and alginate microparticles was also investigated by scanning electron microscope  at an accelerating voltage of 15 kV. For SEM measurements, the freeze-dried particles were deposited on conductive carbon tabs and coated with gold under vacuum using a sputter-coater.X-ray diffraction (XRD) patterns of the cellulase-encapsulated alginate particles, the sodium alginate, and the cellulose nanofibers both CNF and TOCNFs after different hydrolysis times were determined using a diffractometer AXS D8 , operating at 40 kV and 40 mA with the Cu Kα radiation (λ = 1.542 Å). The diffraction angle 2θ varied between 5 and 120° by steps of 0.04° for the starting materials: alginate, CNFs, TOCNFs, and for the obtained cellulase-encapsulated alginate particles; and between 5 and 70° by steps of 0.02° for the CNFs and TOCNFs obtained after different times of enzymatic degradation.Thermogravimetric analysis TGA of the cellulase-encapsulated alginate particles and starting alginate was performed with a STA 409 C equipment . Approximatively 10 mg of the dried samples were weighed in a platinum pan and heated from room temperature up to 700 °C, with a heating rate of 10 °C/min under nitrogen atmosphere.2 coagulation bath, addition or not of NaCl, and hardening time (HT). In general, the size of the beads ranged from (2.0 ± 0.4) to (2.5 ± 0.2) mm for the hydrated and (1.1 ± 0.2) to (1.7 ± 0.2) mm for the dried beads. The size and shape of the beads should be influenced by internal and external factors including surface tension of alginate solution and gelation bath, collecting distance, CaCl2 concentration, needle diameter, M/G ratio, distribution in alginate chains, and stirring rate of gelation bath [ion bath ,69. Whenion bath . The sphTrichoderma reesei present carbohydrate binding moieties which might favor interaction of the enzyme with the alginate matrix [e matrix . It shoue matrix .co–glycidylmethacrylate), gelatin and polyacrylamide provides high stability due to the protective microenvironment of the gel matrix [l matrix ,73,74,75The stability of entrapped cellulase was initially investigated along 6 h incubation at pH 4.8 and at 60 °C towards degradation of solubilized cellulose substrate. The relative activity of the cellulase encapsulated in the alginate particles was 44.9%, 42.1%, and 38.4% after 6 h for alginate capsules prepared with alginate 2% , 3%, and2, NaCl, and hardening time (HT) were considered in a statistical analysis. As mentioned above, the c(ALG) was varied at three levels, while that of CaCl2 and NaCl, and the HT were varied at two levels. A 23∙3 full factorial design was used. To determine the most significant parameters affecting particle sphericity and relative activity of immobilized cellulase after 1 h incubation at the obtained pH and temperature of maximum activity , the four independent variables: concentrations of alginate, CaClp < 0.05) influencing on both particle sphericity factor (SF) and relative activity of immobilized cellulase. The interaction of these two factors and in a less extension that between the c(CaCl2) and the hardening time (HT) have significant influence on the sphericity factor (SF). This is described in more detail in the following section.ANOVA analyses of the four factors showed that c(ALG) and c(NaCl) were the most significant variables ) including quadratic (only for the three-level factor) and linear interactions was used to calculate the predicted response.The software STATISTICA 10.0  was used in all calculations and a significance level of 0.05. Two regression models depicting the relative activity and the SF were obtained. Equations (3) and (4) represent the final empirical models for relative activity and SF, respectively.2. In both equations the R2 was close to one and to the adjusted R2 (R2adj). The significance of linear interaction and quadratic model terms was determined using Fisher’s statistical test and p-value, as displayed in ANOVA analyses in The quality of the models could be verified by observing the determination coefficient R2), the c(NaCl) and the interaction between c(ALG) and c(NaCl) are the significant model terms for the enzyme relative activity (2)*HT are significant model terms (The c(ALG) and their quadratic term, the c might compete with the Ca2+  to link to alginate chains, which could contribute to destabilization of the so called egg-box structure of Ca2+-crosslinked alginate. Thus, the Na+ could contribute to a partial disruption of the Ca-alginate network constituting the particles. This effect diminishes the protective role of these latter for the enzyme, as the alginate particles serve as support for enzyme sustained delivery and activity preservation.According to Equation (3), the c(ALG) has a negative effect on the relative activity, indicating that lower alginate concentration leads to higher enzyme activity. For SF, the c(ALG) has a positive effect (Equation (4)) but it means that better particle sphericity was achieved for lower alginate concentration ,65,66. F2): 0.3 mol/L; hardening time: 2 h). The enzyme activity significantly increased when the concentration of alginate and NaCl were the lowest. 2) and hardening time (HT) on the SF when the other variables were at center points (c(ALG): 3.33 % (w/v) and c(NaCl): 0.4 mol/L). Better sphericity (low SF) was obtained with the decrease of c(ALG), c(NaCl), and c(CaCl2), and by shortening the hardening time.The response surface and contour plots of significant combined effects obtained for each response are shown in 2) = 0.2 mol/L, c(NaCl) = 0 and 1 h hardening time. These optimum conditions corresponded to those used in the formulation F1  = 2% (w/v), c for the free enzyme, while the encapsulated cellulose retained a high activity of 60% until at least 504 h (21 d) d.S was estimated for the different hydrolysis times. S is related to the number-average degree of polymerization DP accordingly to S = 1/DP − 1/DP0, where DP0 is the initial number-average degree of polymerization. The pseudo zero-order kinetics Equation (5) is traditionally used for the hydrolysis of carbohydrates as cellulose [n0 is the fraction of available glycosidic bonds (accessibility) [In order to quantitatively study the kinetics of the cellulose nanofibers enzymatic hydrolysis in heterogeneous conditions, the fraction of hydrolyzed glycosidic bonds ellulose ,77(5)1Dequation (6)S=n0·ibility) .S correspond to a few units, i.e., S << n0 or kt <<1. Thus, Equations (5) and (6) have been successfully used for the carbohydrate degradation in solution, where all glycosidic bonds are available for the degradation [Nevertheless, Equation (6) could not be used to model the experimental data of the hydrolysis in heterogeneous conditions. In the literature, Equation (5) appears largely satisfied when radation ,81. In hradation . In cellradation , the sumradation (7)S=nw0S (Equation (5)) to the concentration of sugar reducing ends (RE) produced during the degradation of the cellulose nanofibers  are clearly observed in the hydrolysis kinetics plots of cellulose nanofibers, corresponding to heterogeneous conditions. As expected for semi-crystalline cellulose with high DP0 [kt << 1, the so-called Ekenstam’s approximation could be applied to the ‘weak links’ degradation kinetics. Thus Equation (7) could be re-written asIn heterogeneous degradation of cellulose, a global deviation was explained invoking the presence of the so-called weak links (w) or “high defects” ,85,86. Tbers see  for diffhigh DP0 , as presk′1) of the weak links from the slopes of the degradation kinetics curves in the first regime (1) below 6 h , in which the slowest depolymerization occurs (k′3) due to topological constraints constituted by crystallites [k′4 was similar to the k′2 estimated in regime 2 (assigned to the degradation of amorphous regions). In k′1, k′2, k′3, and k′4 are summarized, corresponding to the different regimes observed during the enzymatic degradation of cellulose nanofibers. These values were deduced for the free enzyme and the cellulase immobilized in alginate beads or micro-sized particles and for the degradation of the cellulose nanofibers CNFs and TOCNFs.These interpretations allowed us to calculate the hydrolysis pseudo rate constants  was obtained from the subtraction of this amorphous contribution from the total diffractogram area . This allowed calculating CrI by integration of the crystalline peaks and the total diffraction pattern, according to the Equation (11)The crystallinity index CrI values of the starting cellulose nanofibers and those obtained after different times of enzymatic degradation with the cellulase encapsulated in the alginate microparticles are reported in CrI of the CNFs decreased from 23% to 7.5%. This CrI decrease confirms the hydrolysis of crystallinity regions of the cellulose nanofibers during the degradation of these latter by the immobilized cellulase.The obtained 2 gelation bath and leaving hardening the particles for 1 h. These optimum conditions were considered in the processing of enzyme-entrapped alginate particles for the successful biodegradation of crystalline cellulose nanofibers in suspension. The effect of temperature, pH, and incubation time on the enzymatic activity was investigated. The encapsulated cellulase displayed higher activity compared to the free enzyme over large ranges of temperature and pH and for longer times. The storage stability of immobilized enzymes in comparison to free enzymes demonstrated that the cellulase encapsulation effectively prevented the deactivation of the enzyme. The presented results allowed us to develop a new technological approach to achieve the biodegradation of crystalline nanofibers of both non-modified and TEMPO-oxidized cellulose, looking for an effective application of cellulose nanofibers as reinforcement of biomaterials for tissue engineering.Extrusion-dropping and inkjet-based technologies were successfully used to produce alginate beads and microparticles, respectively, in which the enzyme cellulase could be immobilized to degrade cellulose substrates including crystalline cellulose nanofibers in heterogeneous conditions. The maximum catalytic activity of the enzyme was achieved at pH 4.8 and 55 °C for studies performed with cellulase encapsulated in alginate particles. The optimization of the factors affecting the activity of immobilized cellulose in the hydrolysis of cellulose substrate in solution revealed that the highest enzyme activity with spherical particles could be achieved by dispensing 2% (w/v) alginate into a 0.2 M CaCl