A transdermal formulation of ketoprofen was developed to provide effective analgesia in cattle undergoing surgical husbandry procedures. Topical administration would reduce the requirement for veterinary presence and be easy to apply to unhandled animals, improving compliance. The transdermal formulation was at least as effective as a commercially available intramuscular formulation in providing analgesia in cattle following dehorning. Efficacy was assessed using plasma cortisol concentrations, bodyweight (BW) gain and behavioural changes. Plasma ketoprofen concentrations were measured in the plasma approximately 20 min after application and peaked at 2 h, suggesting that transdermal ketoprofen had a rapid onset and would provide suitable analgesia if applied when cattle have been penned prior to the procedure being undertaken.p < 0.05) treatment effect. The IM group exhibited similar responses and both TD and IM cattle had a higher BW gain at 2 and 5 (p < 0.05) weeks post-dehorning, compared to placebo. This study has shown that TD administered ketoprofen was at least as effective as IM to control pain associated with dehorning and facilitates the administration of analgesic drugs prior to the surgical husbandry procedures being performed.There is a critical need to ensure that all cattle undergoing surgical husbandry procedures are provided effective pain relief. Non-steroidal anti-inflammatory drugs (NSAIDs) are most commonly used, and typically are administered by intramuscular (IM) injection. However, administration of NSAIDs via this route to large numbers of cattle which are handled only once or twice a year, typical of many rangeland beef production systems, presents significant occupational health and safety and mis-administration risks. To address this, a novel transdermal (TD) formulation of ketoprofen was developed, and its efficacy assessed in a study of 36 Holstein–Friesian calves which were assigned to a placebo (n = 10), a TD ketoprofen (n = 10), an IM ketoprofen (n = 10) and sham dehorned group (n = 6). TD ketoprofen significantly reduced plasma cortisol concentrations between 1 to 4 h after dehorning compared to placebo treated calves, with concentrations at 2 and 4 h being very similar to those for sham dehorned calves. The expected log count of positively associated pain variables  in the TD group was reduced by 42%, compared to placebo calves, with an overall significant ( Cattle are subjected to surgical procedures  as part of the routine husbandry management on beef and dairy farms . It is nNon-steroidal anti-inflammatory drugs (NSAIDs) can significantly reduce the pain response following surgery by limiting the inflammatory changes induced by tissue damage ,5. For eNSAIDs are currently registered to be administered by intravenous, intramuscular (IM) or subcutaneous injection to ensure rapid onset of analgesia. However, injecting cattle in a handling race prior to performing surgical procedures can be associated with significant occupational health risks for farm staff and handling stress for the cattle. Thus, NSAIDs are currently often administered immediately, before or after surgical husbandry procedure(s) are performed when cattle are individually well restrained. Stafford and Mellor  suggesteTopical NSAIDs offer an interesting alternative to other routes of administration. They can be administered with minimal expertise  and offeCMAX; ~20 µg/mL) by 2 h and an area under the curve (AUC0-Last) of 3940 µg·min/mL. The CMAX and AUC for IM administration were ~11 µg/mL and 2376 µg·min/mL, respectively. The bioavailability of the TD formulation was ~50% [The topical formulation consisted of ketoprofen (20%) dissolved in a combination of 45%:45% ethanol and isopropyl myristate (IPM), mixed with 10% eucalyptus oil . A relatively high concentration of active was used to minimise the volume of formulation that needed to be applied, since transdermal dose rates may be several-fold higher than parenteral dose rates, depending on the active, excipients and species . The plawas ~50% . The IM was ~50% .This study was performed at the Queensland Animal Science Precinct (QASP) at the University of Queensland Gatton Campus and had been approved by The University of Queensland Animal Ethics Committee (SVS/455/14/MLA) by The University of Queensland Animal Ethics Committee (SVS/455/14/MLA). This approval specifically considered and approved the request to dehorn without local anaesthesia as reflecting industry practice, particularly on extensive properties.(i)Dehorning and placebo transdermal treatment (n = 10).(ii)Dehorning and TD ketoprofen .(iii)Dehorning and IM ketoprofen .(iv)Sham dehorning (n = 6).All calves used in this study were sourced from The University of Queensland dairy. On the day before dehorning, a total of 36 weaned male Holstein–Friesian calves weighing on average 151 kg (range 86 to 236 kg) and aged 3 to 6 months were ranked according to BW, and then commencing with the four heaviest to the four lightest were randomly assigned (by blindly selecting ear tag numbers from a bag) to one of four groups:As each calf presented in the crush, they were head bailed, a rail put behind their hindquarters, and then they were restrained using nose pliers to hold the head to the side. The dehorning was performed using a scoop dehorner (Bainbridge) by an experienced large animal veterinarian. Sham cattle were run through the crush but were not dehorned.gluteus medius muscle. Each replicate of calves was subsequently brought back into the cattle handling facility and individual calves were restrained in the crush; they were head-bailed, a rail put behind their hindquarters, and then nose pliers were applied and the head was held to the side. A 10 mL blood sample was collected by jugular venepuncture (0 h) into lithium heparin vacutainer tubes, and then amputation dehorning was performed. The dehorning was performed by an experienced large animal veterinarian using a scoop dehorner (Bainbridge). This method of dehorning was used as the horns had already attached to the frontal sinus. Immediately after severing the horn, wherever possible, severed arteries were grasped with a pair of haemostats and twisted to provide effective haemostasis. Sham calves were restrained as described above and the dehorning device was laid around each horn, but they were not dehorned.To ensure the accurate timing of blood sampling and behaviour observations, the calves were managed as a series of five replicates each containing calves from each treatment group. All ketoprofen treatments were administered 30 min before dehorning, whilst the animals were restrained in the crush. The transdermal ketoprofen or placebo (vehicle only) was applied along the backline, approximately from the shoulder to the mid-point of the backline. The treatments were applied by an operator blinded to the animal ID and wearing gloves. A 10 mL syringe was used to deliver the formulation with the nozzle used to part the hairs and apply the formulation against the skin, approximately 10 cm to the left-hand side of the spinous processes. A standard volume of 10 mL was applied to each calf. The IM ketoprofen was administered into the Blood samples  were subsequently collected at 1, 2, 4, 8, 24, 48 and 96 h for analysis of cortisol concentration. The cattle were weighed  at day -1, at 2 weeks and 5 weeks after dehorning.Plasma total cortisol concentration was determined for each blood sampling time using a commercially available radioimmunoassay (RIA) kit  with assay sensitivity of 2.5 nM, and within assay precision of 2.7% for a quality control of 139.9 nM. Calf live weight was determined at 2 and 5 weeks after dehorning.Calf behaviour was monitored before and after dehorning by the same person, who had been blinded to the treatments. The day before dehorning, the behaviour of all calves was monitored for 2 h to record the baseline variation, which was taken as 0 h observation. After dehorning, behaviour was monitored at 2–4 h, 4–8 h, 8–12 h, at 24 h and at 48 h. A behaviour ethogram adapted from Petherick et al.  was usedp < 0.05) remained in the final SEM model and were aggregated, and compared over time and between groups using a Poisson model. A mixed effect Poisson model with an animal fitted as random intercepts and time as random slope, and unstructured error term for the residuals. Behaviours at baseline (time 0) were centred and added to the fixed effect part of the model . Experimental time was fitted as a categorical variable to derive estimates at each time point; time 2 h was used as the reference category. The analysis only considered the first 24 h, since NSAIDs are only efficacious for this direction. Treatment was also fitted as categorical variable, placebo as reference category. Overdispersion was assessed and it was ruled out as being problematic . Overdispersed models  were refitted using a negative binomial model. Poisson: ear flick, tail wag and negative binomial: headshake, head rub, lying, ruminate, neck extend, grooming.The behavioural changes were analysed using a confirmatory factor analysis (CFA) within the structural equation modelling (SEM) framework . SEM is Bodyweight and plasma cortisol concentrations were analysed using ANOVA.p < 0.05) less than TD and IM cattle at 2 and 5 weeks following dehorning , compared to placebo and sham group calves. The risk of higher mean counts of positively associated pain variables was observed at 2 h and 4 h after dehorning.For the behavioural analysis, seven behaviour variables  had substantial loading and were statistically significant . Animal This study has shown that a topically applied formulation containing ketoprofen could deliver effective analgesia in cattle undergoing dehorning. A range of parameters were used to compare efficacy, including cortisol, BW and behaviour. In addition, similar efficacy was shown between transdermal and IM ketoprofen, which was not surprising, since a pilot study had shown somewhat higher plasma concentration time curves for TD . HoweverThis study was performed on calves at an age when they would normally be dehorned. A cornual nerve block was not used , since this represents the industry standard currently. A sham-handled group was used as a negative control to allow for the stress induced by handling and multiple blood samples collected. The remaining calves were all dehorned and no adverse effects were observed, either from the procedure or the drug administration. There was potentially a higher risk of adverse effects commonly associated with NSAIDs  following TD administration due the higher maximum plasma concentration, compared to IM, although this was considered minimal, since only a single dose was used.There are concerns over the validity of using plasma cortisol concentrations to monitor pain and distress ,23. One Behavioural responses are used to monitor pain and distress in animals ,27. An ep < 0.05) after the procedure. Importantly, BW gains alone would more than justify the cost of administering analgesia, particularly if this was by a non-veterinarian and avoiding call-out fees.Appetite and feed intake also form part of the behavioural response to pain ,29. HoweIt could be expected that the use of a NSAID would ameliorate the physiological and behavioural responses to surgical pain. The initial pain response may be unchanged, but certainly cortisol responses are not as pronounced by 2 h after dehorning if ketoprofen is used . InteresAdministration of a transdermal formulation containing ketoprofen to calves prior to amputation dehorning resulted in a rapid onset of analgesia, very similar in duration and efficacy to that induced by IM administration of ketoprofen. Because of the recognised ease of administration of transdermal formulations to cattle, these findings are likely to facilitate greater routine use of analgesia in cattle undergoing painful husbandry procedures such as dehorning.


The definition of quality controls for cell therapy and engineered product manufacturing processes is critical for safe, effective, and standardized clinical implementation. Using the example context of cartilage grafts engineered from autologous nasal chondrocytes, currently used for articular cartilage repair in a phase II clinical trial, we outlined how gene expression patterns and generalized linear models can be introduced to define molecular signatures of identity, purity, and potency. We first verified that cells from the biopsied nasal cartilage can be contaminated by cells from a neighboring tissue, namely perichondrial cells, and discovered that they cannot deposit cartilaginous matrix. Differential analysis of gene expression enabled the definition of identity markers for the two cell populations, which were predictive of purity in mixed cultures. Specific patterns of expression of the same genes were significantly correlated with cell potency, defined as the capacity to generate tissues with histological and biochemical features of hyaline cartilage. The outlined approach can now be considered for implementation in a good manufacturing practice setting, and offers a paradigm for other regenerative cellular therapies. We prevThe starting material for this approach is a biopsy from the native nasal septum cartilage, which, like articular cartilage, is a hyaline cartilage  composedWhen working with intrinsically variable donor-derived human materials, such as tissues and cells, establishing the quality and consistency of the starting material is key to ensuring reproducibly high quality engineered products, not least to avoid the consequences of cell misidentification . This prEngineered products treat diseases or damage through repairing, replacing, or regenerating tissues or organs . A potenIn this study, we first investigate which cell types are potentially contaminating in human nasal septum cartilage biopsies and their impact on the quality of engineered cartilage. We then investigated whether gene expression analysis could discriminate the contaminant cells found in nasal septal biopsies for the development of a characterization panel for identity and purity quality controls. To assess potency, we compared the gene expression of nasal septum biopsy-derived cells to their ability to produce cartilaginous tissue. Finally, we propose how these purity and potency assays could be implemented in a good manufacturing practice (GMP) compliant process for the translation of our regenerative therapy product.ClinicalTrials.gov, number NCT01605201 and number NCT02673905).Human nasal septum biopsies were collected from 17 donors  undergoing reconstructive surgery after informed consent and in accordance with the local ethical commission . Two samples derived from patients enrolled in the Nose2Knee clinical trials  and a pure perichondrium sample (PC).4 cells/cm2 and cultured in medium consisting of complete medium [Dulbecco’s Modified Eagle’s Medium (DMEM)] containing 4.5 mg/mL D-glucose and 0.1 mM non-essential amino acids, 10% fetal bovine serum (FBS), 1 mM sodium pyruvate, 100 mM HEPES buffer, 100 U/mL penicillin, 100 μg/mL streptomycin, and 0.29 mg/mL L-glutamine . Complete medium was supplemented with 1 ng/mL transforming growth factor beta-1 (TGF-β1) and 5 ng/mL fibroblast growth factor-2 (FGF-2) (both from R&D Systems) at 37°C and 5% CO2 in a humidified incubator (Thermo Scientific Heraeus) as previously described . Cells were isolated from each sample by enzymatic digestion and expanded in complete medium supplemented with TGF-β1 and FGF-2 up to two passages as described above for nasal chondrocytes.2 (N/N0) to T, where N0 and N are the numbers of cells respectively at the beginning and at the end of the expansion phase, log2 (N/N0) is the number of cell doublings, and T is the time required for the expansion.Proliferation rates were calculated as the ratio of log5 cells at 300 × g in 1.5 mL conical tubes (Sarstedt) and cultured for 2 weeks in chondrogenic serum-free medium consisting of DMEM containing 1 mM sodium pyruvate, 100 mM HEPES buffer, 100 U/mL penicillin, 100 μg/mL streptomycin, 0.29 g/mL L-glutamine, 1.25 mg/mL human serum albumin (CSL Behring), and 100 nM dexamethasone , supplemented with 10 ng/mL TGF-β1 (R&D), ITS + 1 , 100 μM ascorbic acid 2-phosphate (Sigma), and 4.7 μg/mL linoleic acid (Sigma). Culture medium was changed twice weekly.Cells expanded until passage two were redifferentiated by culturing as 3D micromass pellets, as previously described . 3D micr2. The resulting constructs were cultured for 2 weeks in chondrogenic medium consisting of complete medium supplemented with 10 μg/mL insulin (Novo Nordisk), and 0.1 mM ascorbic acid 2-phosphate (Sigma) at 37°C and 5% CO2 with media changes twice/week.Passage two cells were seeded on collagen type I/III membranes  at a density of 4.17 million cells per cmThe described protocols match the ones used in the context of the clinical trial, where GMP-grade reagents and autologous serum instead of FBS are used. Grafts for clinical use are produced at the GMP facility at the University Hospital Basel according to standard operating procedures under a quality management system, as described in Samples were fixed overnight in 4% formalin and embedded in paraffin. Sections 5 μm in thickness were stained with safranin O for GAGs and hematoxylin as a nuclear counterstaining as described elsewhere . ImmunohHistological scoring via the modified Bern score (MBS) was performed on safranin O-stained histological images as previously described , as adapWe chose the gene expression markers to investigate based on a literature search. Interested in both purity and potency assays, we focused on matrix associated genes considering the two cell types potentially present in our starting material derive from tissues with structurally different ECM. The gene expression ratios of collagen II to I and aggrecan to versican are well-known chondrogenic markers . HAPLN1 T) value of the reference gene, GAPDH, was subtracted from the CT value of the gene of interest to derive ΔCT values. All displayed gene expression levels are, and statistical analyses were performed on, the ΔCT values. GAPDH was found to be a stable reference gene for both nasal chondrocytes and perichondrial cells with a mean ΔCT value of 18.1 (standard deviation of 0.68) at passage 2 and 22.6 (standard deviation of 0.80) for pelleted cells across both cell types.Total RNA was extracted from expanded cells at both P1 and P2, 3D micromass pellets, and engineered cartilage grafts with the Quick RNA Miniprep Plus Kit (Zymo Research) and quantitative gene expression analysis was performed as previously described . ReverseSamples of engineered cartilage and micromass pellets were digested with proteinase K  for 16 h at 56°C. The GAG content was determined as previously described . BrieflyR2 values were calculated with the pscl R package  function in R was used to build all the models. A logistic regression model was used to predict purity, where the response is a continuous probability between 0 (pure perichondrium) and 1 (pure cartilage) with samples from four donors and 48 independent experiments of known purities. For the logistic regression models, the McFadden pseudo  package  and the  package . For the package  was perft-tests and normality was checked with the Shapiro–Wilk test. To test multiple comparisons, a linear model was fitted, then the glht function of the multcomp R package  were created with the corrplot R package  of the gene expression of cells at passage two that we combined at specific ratios of NC and PC cells revealed statistically significant trends across donors. Due to high donor-to-donor variability, the correlations between cell population purity and gene expression were higher per donor per gene than across donors. The highest correlation was found for the relative expression of aggrecan:versican (ρ = 0.69), where the ratio was higher in purer populations containing more NCs; per donor the correlations were even stronger (ρ = 0.61–0.98) .In general, more significant differences in gene expression in individual genes and cell purity were seen at passage two compared to the pelleted cells’ gene expression . Therefop-value = 2.7e-3, p = 2.8e-3, respectively, and overall, the model was significant (Hosmer–Lemeshow p = 0.95 and McFadden pseudo R2 = 0.53). The coefficient estimates from the model and the ΔCt values for versican and collagen II can be used to estimate the purity of a population of nasal cartilage-derived cells using Eq. 1, where inverse logit is exp (x) / [1 + exp (x)].We performed multiple logistic regression to compare gene expression of collagen type I and II, aggrecan, versican, MFAP5, HAPL1, and nestin to the cell population purity. To gain insight into which genes were most important, stepwise selection  was implR2 value of the observed and predicted values was 0.79 . Due to donor-to-donor variability, potential cross-contamination from the mechanical tissue separation method, and considering the limitations of histological analysis, we show that some donors could still produce cartilage matrix of sufficient quality with up to 40% PC contamination, while the less potent donors could produce cartilage matrix with a PC contamination of up to 30% PC cells .Using the purity and potency assays we developed, the quality was estimated based on the gene expression of passage two cell populations for clinical trial samples and for heterogeneous biopsies collected from patients that underwent plastic surgeries with variable amounts of overlaying perichondrium. The predicted histological score results closely matched the actual values, and the quantified amounts of GAG could be estimated well, predicting if cells would produce high or low amounts of GAG . The cliIn this study, we established novel in-process controls to ensure the quality and standardization of nasal chondrocyte-based engineered cartilage grafts. Histological analysis revealed that nasal septal cartilage may be harvested with some adjacent tissue, and that there may still be fragments of perichondrial tissue overlaying the cartilage even after a trained operator further separates the tissues. Although some researchers claim that perichondrial cells from other cartilage sources have chondrogenic potential , we discIn practice, quantitative reverse-transcription polymerase chain reaction (qPCR) instrumentation is ubiquitous, so a gene expression-based quality control could be easily implemented. The cost of the quality control assay could be reduced by selecting a handful of genes for a standard qPCR analysis compared to transcriptomic analysis or single-cell RNA sequencing, and for a routine test could be enough information to confirm cell identity .To implement such gene expression-based quality controls, a suitable time point during the manufacturing process must be chosen. Biomarkers vary not only spatially within the tissue, but also temporally during monolayer expansion and after tissues are engineered , and it Generalized linear models for the development of gene expression-based quality controls for regenerative medicine is a natural extension of their use in biomarker-based disease diagnosis . Here weWe showed how logistic regression can be used to estimate the histological score, a value bounded between 0 (worst) and 6 (best). Estimating GAG required modeling positive values only, therefore, we demonstrated how a generalized linear model with a gamma distribution and log-link could be implemented, similarly to other biomarker applications . Stepwispure, less than 50% pure, they are labeled as fail, otherwise the estimation is labeled as uncertain. We propose to introduce this uncertain region for the time being until further data can be collected and the estimates can be made more precise. In practice, we would recommend that starting cell populations labeled as pure or uncertain should continue in the production process, however, if the cells fail the purity test, the costly production should be halted. Cartilage engineered from cells of uncertain purity would nevertheless need to pass the release criteria , ensuring the quality of the product.We observed that all pellets that contained at least 70% NC cells pass the clinical trial release criteria, i.e., histological score ≥3, but more contamination could also lead to good results in some cases. To implement the purity assay, we propose a conservative three-category rating scale for the predicted purity (NC%), i.e., if cells are estimated to be more than 70% pure, they are labeled as The proposed models have been generated based on a limited number of genes. In future, it will be valuable to widen the panel of genes analyzed, based on other published studies  or more in silico models. This approach could be relevant for the development of quality controls for other products in the emerging field of regenerative medicine, one of the biggest challenges for advanced therapy medicinal products to overcome for clinical translation.In conclusion, we have put forward gene expression-based assays for identity, purity, and potency to help ensure the safe and effective clinical use of nasal chondrocyte-derived engineered cartilage. More generally, we provide an example of the development and implementation of purity and potency assays based on relatively simple qPCR assays, stepwise selection of the most significant genes, and predictive All datasets generated for this study are included in the article/ClinicalTrials.gov, numbers NCT01605201 and NCT02673905).Human nasal septum biopsies were collected from 17 donors  after informed consent and in accordance with the local ethical commission . Two samples derived from patients enrolled in the Nose2Knee clinical trials (MA conceived and designed the study. MA and LP performed the experiments, analyzed the data, and wrote the manuscript. LP created the models. AB contributed to the study design and revised the manuscript. MH and RK contributed to the sample preparation. DW contributed to manuscript revision. IM contributed to compiling the data and critically revised the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Chlorocebus aethiops. PCA and CAI analyses at the individual ZIKV gene level for both human and Aedes aegypti indicated a clear distinction between the two genotypes. African ZIKV isolates showed higher virulence in mosquito cells than in vertebrate cells. Their higher replication in mosquito cells than African YFV confirmed the role of mosquitoes in the natural maintenance of the African genotype ZIKV. An analysis of individual strain growth characteristics indicated that the widely used reference strain MR766 replicates poorly in comparison to African ZIKV isolates. The recombinant African Zika virus strain ArD128000*E/NS5 may be a good model to include in studies on the mechanism of host tropism, as it cannot replicate in the tested vertebrate cell line.We investigated temporal trends of codon usage changes for different host species to determine their importance in Zika virus (ZIKV) evolution. Viral spillover resulting from the potential of codon adaptation to host genome was also assessed for the African genotype ZIKV in comparison to the Asian genotype. To improve our understanding on its zoonotic maintenance, we evaluated in vitro the biological properties of the African genotype ZIKV in vertebrate and mosquito cell lines. Analyses were performed in comparison to Yellow fever virus (YFV). Despite significantly lower codon adaptation index trends than YFV, ZIKV showed evident codon adaptation to vertebrate hosts, particularly for the green African monkey  Flavivirus belonging to the Flaviviridae family and the Spondweni serocomplex. ZIKV was first isolated in 1947 in Uganda from a febrile sentinel rhesus monkey during a Yellow fever virus (YFV) study , YFV (n = 51) and tobacco mosaic virus (TMV) (n = 68) that had information on year and place of isolation and representing all data available to date on Genbank (www.ncbi.nlm.nih.gov/genbank/) were downloaded and used in this study. The complete coding sequences were aligned and curated using Muscle algorithm (http://www.drive5.com/muscle/) within the Unipro UGENE software (http://ugene.net/download.html) [Nucleotide coding sequences from isolates of ZIKV [ad.html) .Homo sapiens, Macaca mulatta, Erythrocebus patas and Chlorocebus aethiops, and invertebrate vectors including Aedes aegypti, Aedes albopictus and Culex pipiens  was then analyzed in comparison to the Asian genotype ZIKV (n = 356). Sequences of YFV (n = 51) and TMV (n = 68) were used as positive and negative controls, for adaptation and no adaptation to the human genome, respectively. We also did CAI analyses for each viral coding region using both A. aegypti and H. sapiens codon usage tables. Additionally, a principal component analysis to verify clustering patterns according to viral genotypes was performed considering the different genomic regions of the virus.In order to assess the adaptation level of the African ZIKV genotype to invertebrate and vertebrates, codon usage of its sequences  continuous cell lines were inoculated with strains used in this study and incubated at 28 °C without CO2 until a cytopathic effect (CPE) was observable. Infection status was assessed by indirect immunofluorescence assay (IFA) as previously described [escribed , and virescribed , using p5 Ap61 or African green monkey kidney epithelial cells  were seeded into each well in a volume of 400 µL of appropriate medium [3 plaque-forming units (PFU) of virus in 400 µL of medium, resulting in a multiplicity of infection (MOI) of 0.01. Experiments were conducted in 12-well plates using one plate per virus strain with one uninfected well as a negative control. Plates with infected Ap61 and Vero cells were incubated at 28 °C without CO2, and at 37 °C with CO2, respectively. After an incubation time of 4 h, the medium was collected and replaced with 2 mL of new medium to set a start point for the growth kinetics (T0).An amount of 2.4 × 10e medium . In addie medium ,51,52, aAt different time points corresponding to 22, 28, 50, 75, 99, 124, and 146 h post infection (hpi), one well per plate was harvested and the supernatants were collected and frozen at −80 °C in small aliquots until use. Cells were washed once with phosphate-buffered saline (PBS) and then collected in 500 µL PBS. A volume of 20 µL of cell suspension was dried on a glass slide for a subsequent immunofluorescence assay . FinallyData of genome production obtained on Ap61 cells during these experiments with ZIKV were also compared to those we previously obtained for YFV in the same conditions with the same MOI as for ZIKV  to bettePerl and R scripts were used to analyze codon usage data in this study, and are available from the authors upon request. In addition, the percentage of immunofluorescence, the logarithm of RNA copy number and the logarithm of viral titers are supposed to be normally distributed, and a Student’s test was used to analyze differences between virus strains.We explored possible changes in CAI over time for YFV and ZIKV using human, monkey and arthropod vector codon usage tables. Both had large numbers of available, serially stamped, complete polyprotein sequences, allowing us to investigate the temporal trends of ZIKV CAI, and to compare differences in the CAI of YFV strains .p-values < 2.2 × 10−16, rho ranging from 0.988 to 0.999). YFV CAI was significantly higher for mammalian than that for vector codon usage . In addition, YFV CAI was significantly higher for monkeys than for human codon usage , and could be associated with the important role of these monkey species in enzootic transmission of YFV. We also observed significant CAI differences between mosquito vectors with higher CAI for Aedes aegypti than for Aedes albopictus and Culex pipiens codon usage , indirectly confirming Aedes aegypti as the main vector for YFV. No significant differences of YFV CAI were observed for HKgs and AVgs , or between monkey species   A.p-values < 2.2 × 10−16, rho ranging from 0.953 to 0.996). However, ZIKV showed a higher CAI towards HKgs than to AVgs . This effect could have an important impact on the intrinsic biological characteristics of ZIKV during human infection, such as the level of immune response induction.Similar to YFV, a positive correlation was observed in ZIKV codon adaptation to all eight trend lines  codon usage . CAI values were significantly higher for Aedes aegypti, the main vector for ZIKV , and below 1 for Aedes albopictus and Culex pipiens, indicating no evidence for codon adaptation for these mosquito species  indicated a grouping of the two ZIKV genotypes in different groups. This suggested that each genotype may interact with distinct host species . The strain ArD132912 and the human strain HD78788*E showed a production of infectious particles from 75 hpi while the reference monkey strain MR766 released infectious particles very late, i.e., from 99 hpi. Strikingly, we did not detect any infectious particles for the strain ArD128000*E/NS5 isolated from a mosquito over a period of 146 hpi . However, no significant difference was found between other African ZIKV strains from 75 hpi to 146 hpi   G.*E/NS5 and ArD128000*E/NS5 displayed higher infectious particle production, while the reference monkey strain MR766 and the human strain HD78788*E released significantly reduced amounts of infectious particles from 124 hpi to 146 hpi     B. More e 0.0001) D,F. Simi × 10−1) H.10 more infectious viral particles in mosquito cells than in vertebrate cells  increased significantly in mosquito cell supernatants   A,B.p-values ranging from 3.75 × 10−10 to 0.027). In addition, the human strain HD78788*E released fewer RNA copies into supernatants from mosquito cells over 146 hpi, while we found a significantly higher genome copy number in supernatants from vertebrate cells   and the strain ArD128000*E/NS5, which showed a significant increase in viral antigen production in both cell lines from 99 hpi to 146 hpi   C–F. Sign × 10−4) G,H.*E and the monkey strain MR766 were more cell-specific; both replicated more abundantly in vertebrate cells   A–H. Thes*E/NS5, presenting the N-glycosylation motif NDI at Asparagine 154 of the E protein, showed more efficient growth replication than strains ArD128000*E/NS5 and HD78788*E with NDT motif or the strain MR766, which showed complete deletion of that motif . This effect was more marked in the vertebrate cell line, and showed that N-glycosylation on the E protein can play a major role in changes during growth of African ZIKV strains   from each time point for each strain in each cell line. We found significant differences in replication efficiency with much higher and increasing ratios over 146 hpi observed in mosquito cells ( 0.0013) A,B. Afri 146 hpi A,B. RefeAedes aegyti, their growth behaviors were compared in mosquito AP61 cells.Since YFV and ZIKV share the same main vector, i.e., The growth dynamics of African YFV showed that strains 307, 357, 345 and 314  exhibited high growth efficiency regarding the number of RNA copies and infectious particles produced in mosquito cells. The reference wild-type strain Asibi and the attenuated vaccine strain 17D replicated less efficiently, while strain 333 (lineage 3) displayed the lowest replication efficiency A–C.*E, all African ZIKV strains replicated more abundantly (from 2 to 6Log10) than YFV strains and produced significantly more infectious particles in cell supernatants   A–C. ThesAs seen for YFV and West Nile virus (WNV), first isolated in Africa, ZIKV is another flavivirus which spread from Africa to other continents ,17. The The evolutionary adjustment of viruses to hosts through codon usage adaptation may reveal the role of host in natural maintenance of particular viruses.Chlorocebus aethiops (green African monkey) is not surprising, because several monkey species have been identified as primary reservoirs of ZIKV [Sapajus libidinosus) highlights the possibility that other host species could be involved in a sustained sylvatic cycle in the Americas [Temporal analyses of CAI are informative in better identifying time points, which were marked by advantageous nonsynonymous changes in different hosts. Despite low CAI changes observed in its evolution, ZIKV shows slow evolutionary dynamics just like those of YFV ,36,57,58 of ZIKV , and theAmericas , but perThe abrupt variation observed in ZIKV population sizes over time could be correlated to the rapid increase of available sequencing data  and to tAedes aegypti mosquito vector could explain the low competence for ZIKV for Aedes aegypti in the Americas [Aedes aegypti in Africa, where it is transmitted by several other Aedes spp. mosquitoes, such as Aedes vittatus and Aedes luteocephalus [Aedes vector for ZIKV in Africa.The lower CAI values obtained for the Americas  or the icephalus ; this raDespite multiple isolations from mosquito populations and serological evidence of ZIKV infection in Africa, a major outbreak in humans attributed to the African genotype ZIKV has not yet been documented. Codon bias is a common mechanism across the genomes of several organisms and plays a major role in their evolution. The significant ZIKV adaptation to the genome of monkeys could lead to possible spillover of the African genotype ZIKV to other vertebrates such as humans, as exhibited by the high CAI for the human genome, which is actually already higher than the CAI of Asian genotype ZIKV. This clearly shows that the African genotype ZIKV also has the potential to efficiently replicate in humans. This is particularly true for the CAI for the E and NS5 proteins , which hAfrican genotype ZIKV presents two major clades, which have previously experienced probable recombination and glycosylation mechanisms in their natural cycle ,28. To bWhile some differences were previously reported between African and Asian ZIKV strains ,68,69,70The replication levels of African ZIKV strains isolated from mosquitoes in the vertebrate cell line confirms that these isolates are able to efficiently infect and replicate in vertebrate cells, as, for example, reported for neuronal cells ,71. HoweIndeed, the African genotype ZIKV has been shown to induce less pathogenicity in human cells than the epidemic Asian genotype ZIKV, which shows long-term persistence, more consistent with clinical manifestations ,73,74. TAfrican ZIKV strains are more virulent in mosquito cells . This ob*E/NS5, ArD165522, ArD132912 and ArD128000*E/NS5 replicated more efficiently in the mosquito cell line, while the human strain HD78788*E and the monkey strain MR766 showed higher replication in the vertebrate cell line. It is of note that the prototype ancestral ZIKV strain MR766 replicated significantly more slowly. At least three MR766 strains exist with genetic differences [However, cell-specific phenotypic differences observed between African ZIKV strains used in our study might be related to the intrinsic properties of the African ZIKV strains, their species origin or differential susceptibility to ZIKV infection for the cell lines used in our study ,78,79. I*E/NS5 and HD78788*E, particularly in mosquito cells, points to the detrimental impact of recombination in the NS5 protein on virulence and viral infectivity. The effect of recombination on replication efficiency and virulence of ZIKV has long been suspected based on phylogenetic studies [*E/NS5, which replicated efficiently in mosquito cells but exhibited no production of infectious particles in the vertebrate cell line over 146 hpi, indicated that recombination has an effect on host tropism.Like other RNA viruses cycling between arthropod vector and mammal hosts, it was previously found that the majority of codon sites in E and NS5 proteins of ZIKV undergo purifying selection ,51,81. N studies ,84,85. TIn contrast to a previous in vivo study demonstrating that complete deletion—as observed in most of the African ZIKV strains—or single amino acid substitution in the NDT motif cause less infectivity , our stuThe recombination and N-glycosylation may lead to potential future adaptation and pathogenicity of the African genotype ZIKV, and the biological and genetic characteristics of new isolates need to be continuously monitored.Aedes aegypti mosquito vector has been correlated with the signature A188V mutation in NS1, which potentially facilitates transmission, and therefore, contributed to the spread of the virus from Asia to the Americas [However, considering functional differences between in vitro and in vivo systems in terms of physical barriers and antiviral responses, the fitness of African ZIKV strains with a NDI motif on the E protein should be studied in vivo, since many other amino acid substitutions contributing to ZIKV pathogenicity have been previously identified ,88,89. EIn contrast to faster in vitro replication of African YFV in mosquito cells, the African ZIKV strains exhibited more efficient replication, yielding more infectious particles. In contrast to YFV, which has a long history of human infection in Africa ,90, the *E/NS5 may be a good model for use studies on the mechanism of host tropism.In summary African genotype ZIKV isolates replicate more efficiently in mosquito cells than in vertebrate cells. Their CAI indices, however, indicate a higher adaptation to vertebrates than observed for Asian genotype ZIKV, which may reflect efficient sylvatic transmission cycles but possibly also asymptomatic transmission in a peri-urban cycle. An analysis of the growth characteristics of individual African genotype ZIKV strains revealed that strain MR766 is not suitable as a reference strain, while recombinant strain ArD128000


In the past decade, researchers have carried out a massive amount of research on the application of biochar for contaminants removal from aqueous solutions. As an emerging sorbent with great potential, biochar has shown significant advantages such as the broad sources of feedstocks, easy preparation process, and favorable surface and structural properties. This review provides an overview of recent advances in biochar application in water and wastewater treatment, including a brief discussion of the involved sorption mechanisms of contaminants removal, as well as the biochar modification methods. Furthermore, environmental concerns of biochar that need to be paid attention to and future research directions are put forward to promote the further application of biochar in practical water and wastewater treatment. Biochar with rich carbon content is a thermal decomposition product derived from biomass under a condition that lacks oxygen . These iWater and wastewater treatment is one of the emerging subsets of biochar application. Due to the properties of large surface area and pore volume, rich organic carbon content and mineral components, abundant and diverse functional groups, biochar displays prominent sorption ability for both inorganic and organic contaminants in aqueous solutions . Traditi3−), ammonium ion (NH4+), phosphate (PO43−), fluoride (F−)) from wastewater (detailed discussion is given in the section “Biochar application in water and wastewater treatment”).Biochar is a by-product of thermochemical transformation such as pyrolysis, hydrothermal carbonization, gasification, and torrefaction . ReportsWith increased application of biochar being carried out in the water and wastewater treatment, this paper reviews recent advances in the biochar application, including a brief discussion of the involved mechanisms in the removal of specific organic and inorganic contaminants. Moreover, this review briefly covers the modification methods of biochar based on different emphases and explains how the modification alters the properties of biochar, as well as the removal efficiency. Furthermore, remained environmental concerns and future research directions are highlighted, with possible solutions put forward.The literature reviewed in this paper was obtained on databases of ScienceDirect, Web of Science, Google Scholar, and the Chinese journal database CNKI. The keywords used to search for literature on the databases are as follows: biochar, cellulose, lignin, pyrolysis, and carbonization associated with the feedstocks and biochar production methods; industrial, agricultural, pharmaceutical, heavy metals, dyes, pesticides, antibiotics, and persistent contaminants reflecting biochar application; electrostatic interaction, precipitation, complexation, hydrophobic effect, and chemical bonds referred to sorption mechanisms; porosity, surface area, functional groups, magnetization, and biochar-based composites related to modification methods. Besides, literature research was specially conducted within the papers on “Special Issue on Biochar: Production, Characterization and Applications - Beyond Soil Applications” published on “Bioresource Technology,” and papers published on “Journal of Chemical Technology and Biotechnology,” which were presented in the 2017 European Geosciences Union session “Novel Sorbents for Environmental Remediation” .The sorption ability of biochar for contaminant removal has been well documented. However, there are lacking studies on corresponding sorption mechanisms for target contaminants, which have fundamental meanings for improving the removal efficiency. Sorption mechanisms vary according to the properties of both contaminants and biochar. Here, the dominant mechanisms in the removal of heavy metals and organic contaminants are illustrated in π electron-rich domain or surface functional groups; (iv) co-precipitation to form insoluble compounds. Here, specific examples are used to explain each mechanism.Heavy metals in the water environment mostly come from anthropogenic activities such as smelting, mining, and electronic manufacturing effluents . BiocharPZC is the solution pH at which the net charge of the biochar surface is zero. Biochar is positively charged at solution pH < pHPZC and binds metal anions such as HAsO42− and HCrO4−. On the contrary, biochar is negatively charged at solution pH > pHPZC and binds metal cations such as Hg2+, Pb2+, and Cd2+ (PZC >7) to sorb As(V) from water at pH 7, with a maximum sorption capacity of 0.3 mg g−1. As(V) mainly exists in the form of HAsO42− at pH 7. The biochar surface is positively charged since the solution pH < pHPZC. In that case, HAsO42− interacts with the protonated functional groups on biochar surface by electrostatic attraction.Solution pH could strongly influence the surface charge of biochar. pHand Cd2+ . These p+, K+, Mg2+, and Ca2+ released from the biochar, probably as a result of metal exchanges with Pb2+. Biochar pyrolyzed from biomass has plenty of exchangeable cations on the surface, such as some alkali or alkaline earth metals  that can be replaced by heavy metal ions during the sorption. 2Hg and (-O)2Hg attributed mostly to Hg sorption on bagasse biochar. The sorption capacity decreased by 18% and 38% when using methanol to block -COOH and -OH functional groups. Nevertheless, the blocking did not affect Hg sorption on hickory chips biochar since the formation of Hg- π bindings between Hg and π electrons of C=O and C=C dominated the sorption. −1 as a result of the increased mineral content in biochar, especially the soluble carbonate (from 2.5% to 2.9%). X-ray diffraction following Cd sorption evidenced that Cd-carbonate and phosphate formed in the biochar (3(CO3)2(OH)2 and Pb-phosphate Pb9(PO4)6 contributed most to the high removal rate of Pb  interaction existed between the graphitic surface (π electron donors) and SAs (π electron acceptors), accounting for the strong sorption.Different results also showed electrostatic interaction to be an essential mechanism of polar organic contaminant sorption . Xu et a−1 reflecting C=O stretching vibration shifted little, and the peak at 3447 cm−1 corresponding to -OH stretching vibration had a bit change. There was a good chance that the intermolecular hydrogen bonds (O-H- - -O bonds) existed between the H atom in -OH of BB molecules and the O atom in C=O on biochar surface, vice versa. The negatively charged properties for both biochar and BB also supported this weak interaction.The co-existence of carbonized and non-carbonized proportions makes the biochar surface heterogeneous; meanwhile, the two types represent different sorption mechanisms. In addition to the sorption of organic compounds onto the carbonized proportion, the partition into the non-carbonized organic matrix is also essential when biochars are produced at lower temperatures .Among the increased number of published reports, biochar can be directly used in water and wastewater treatment as a sorbent for contaminants removal, or be used in constructed wetlands (CWs) and in the soil to improve the water quality. As a dominant source of water contamination, the quantity of industrial wastewater and types of water contaminants are booming due to the rapid development of the industry. Biochar is becoming a new approach to remove various contaminants from industrial wastewater, both for heavy metals and organic compounds.2+, Pb2+, Cu2+, Hg2+, Cr6+, and Ni2+ have received much attention due to the adverse effects they could bring if released to the environment. Batch sorption experiments by 2+, Pb2+, and Cu2+) from solutions. Further research of Pb2+ sorption implied that the biochar had a comparatively high Langmuir sorption capacity of 14.3 mg g−1, despite the slow sorption kinetics. −1, with the maximum Hg(II) sorption capacity of 103 mg g−1. Their later study showed that the Hg sorption capacity by MSR biochars increased by a maximum factor of 6 for high-temperature (750−900 °C) biochars compared to the raw material (2-modified glue residue biochar for Cr(VI) sorption. The maximum sorption capacity reached 325.5 mg g−1, higher than the previously reported sorbents. Removal of Cdmaterial . Hg(II) material . Shi et 3 mg g−1, assuming pore-filling to be the primary sorption mechanism, and the high sorption capacity was attributed to the high specific surface area  and pore volume (2.7 cm3 g−1) of biochar. Researchers produced pecan nutshell biochar to remove Reactive Red 141 from water. The biochar was claimed to be low-cost and environmentally friendly, which could be a substitute for other conventional sorbents  and SAs are two of the most commonly used antibiotics and are also used in intensive agriculture as feed additives, bringing potential hazards to the environment and human health when extensively used . The reme cycles . Peiris Biochar application in the removal of indicator organisms and pathogens mainly aims at the treatment of urban stormwater runoff, which contains a wide range of contaminants and eventually runs into surface water . Irrigation with these contaminated waters can lead to microbial contamination of vegetables. Biochar filters for microbe removal from water have received considerable attention.10 CFU Saccharomyces cerevisiae was successfully removed from diluted wastewater under the condition of on-farm irrigation. The particle size of biochar is the main influencing factor accounting for the microbial removal efficiency. The minimum particle size (d10 = 1.4 mm) could consistently remove at least 1 log10 CFU of most target microbes. More micropores and smaller pore size of biochar could increase straining effect and contact time between bacteria and sorption sites. Escherichia coli and prevented their mobility during continuous, intermittent flows. The improved removal capacity of pathogens was attributed to higher retention on the biochar filter, which increased the attachment of E. coli.− in drinking water.In virtue of the convenience and little generation of secondary contamination , biochar4+ sorption by hydrous bamboo biochar. Results found that the biochar had a sufficient sorption capacity for NH4+, with a maximum of 6.4 mM g−1. The sorption was enhanced at higher ionic strength conditions, indicating that physical reactions possibly made contributions to the sorption process such as electrostatic interactions. Potential mechanisms for NH4+ sorption was further studied by 4+ sorption capacity by changing the surface charge of biochar. Negatively charged biochar in higher pH (pH > pHPZC) solutions easily sorbed NH4+ due to electrostatic attraction. FT-IR patterns showed that the -OH and -C=O groups weakened after the sorption, indicating that the NH4+ acted with these functional groups through surface complexation. In addition, ion exchange between NH4+ and the negatively charged functional groups such as -OH and -COOH also led to the NH4+ sorption. For NO3−, the sorption mechanisms are governed by multiple interactions, primarily electrostatic attraction, and ionic bonds with exchangeable cations from the biochar, based on the sorption study of NO3− by bamboo biochar ( biochar .43− sorption from eutrophic water (−1 in a wide pH range and was the best option for PO43− sorption among the biochars. 43− from wastewater by biochars before and after magnetic modification. Results showed that the sorption ability of magnetic biochar was twice (25–28 mg g−1) than that of the unmodified biochar (12–15 mg g−1). The PO43− sorption on magnetic biochar is dominated by simultaneous mechanisms including electrostatic attraction, surface precipitation, and complexation, while for the original biochar, the sorption mainly depends on electrostatic attraction.Walnut shell and sewage sludge were co-pyrolyzed to prepare biochar for POic water . The bio− is characterized by high electronegativity and small ionic size, resulting in a strong affinity towards metal ions such as Al(III), La(III), and Fe(III)  . Thus, asorption .In recent years, CWs have been widely used in wastewater treatment, including removal of N, P , and som4-N (39%), TN (39%), and organic contaminants (85%) were better than those of conventional VFCWs, especially for the high-strength wastewater. A seven-month study by 4-P removal. The waste biochar has the potential for regeneration, which can be applied as soil fertilizer to improve soil quality, while this application still needs more investigations. 4-N and TN by SFCWs with biochar were higher than those by pure gravel-filled SFCWs. The additive of biochar promotes N removal by changing the structure of microbial communities and increasing the abundance of dominant species. Besides, biochar improves the metabolism of high molecular compounds and convert them into low molecular compounds. These results provide new insights into strengthening N removal through microbial metabolism with the effect of biochar.2+, Mg2+) on biochar surface can replace the monovalent cations  on exchange sites of clay particles, which enhances clay flocculation and thereby improves macropores size and network in the soil . This system promises highly efficient removal of phosphorus and mineral contaminants from wastewater; meanwhile, it makes use of the minerals stripped from water to produce fertilizer, which is also cost-effective. The system was then licensed and promoted in real wastewater treatment systems in the USA, England, and South Korea. This case demonstrates the scalability of biochar engineered application and provides guidance as well.To date, biochar-based filters have been an attempt to advance the engineered application of biochar. Sand filters and biofilters amended with biochar , and filAlthough biochar has been extensively applied in the removal of diversiform contaminants in water solutions, its applicability is limited because of the lower removal efficiency for some selected contaminants or in some specific water conditions. The unmodified biochars have much lower removal ability than the modified ones, especially in high-strength wastewater . ResearcIn general, biochar with larger surface area has more sorption sites, facilitating the sorption capacity. Plenty of modification methods of biochar have been proposed to achieve this favorable property.2 3) are most frequently used to realize the attachment of metals. After heating under atmospheric conditions at 50−300 °C, the chlorides or nitrates were driven off as Cl2 and NO2 gases, and the metal ions were converted into metal oxides . The sorption capacity at pH 6.5 was an order of magnitude larger than that at pH 3, indicating the pH-dependent and chemical sorption on oxygen-containing functional groups. 2SO4 and HNO3, the O/C ratio of rice straw biochar was higher in the final product, implying that oxygen-containing functional groups were enriched in the structure of biochar.The acidic treatment provides additional oxygen-containing functional groups on the biochar surface and increases the potential of chemically binding with positively charged contaminants via specific sorption. The biochar forms carboxylic groups on its surface when exposed to acidic solutions . Hadjitt4 solution. A MnOx-biochar was prepared after another pyrolysis. Compared with the original biochar, the O/C ratio increased from about 0 to 0.5. XPS analyses showed that the increased oxygen existed mainly in the Mn-OH and Mn-O structure, which primarily accounted for the enhanced sorption ability for Cu2+ (from 19.6 to 160.3 mg g−1). 2O2. The cation exchangeability of the biochar was almost doubled than that of the untreated one, as a result of cation exchange on the more abundant oxygen-containing functional groups on the modified biochar surface.Since biochar modification by strong acids is costly in a large-scale application and causes environmental concerns when disposing of the modification agents, researchers have made efforts to come up with cheaper and cleaner oxidants as alternatives to modify biochar. Alkaline solutions play a similar role to acids and oxides in increasing the number of oxygen-containing functional groups on the biochar surface. 2+ raised with the increase of the proportion of graphene oxide in the composite. When the maximum percentage of graphene oxide is 1%, the removal rate of the composite was 8.7% more than that of the unmodified biochar. FT-IR showed that the abundant oxygen-containing functional groups dominate the sorption behavior of Hg2+ on the biochar-graphene oxide composite.Among various biochar-based composites, the biochar-graphene oxide composite material, which is obtained by impregnating the raw material in a graphene oxide suspension and then pyrolyzing, also displays more oxygen-containing functional groups after incorporating the graphene structure . The remIncorporating amino functional groups onto the biochar surface improves the sorption ability through inducing strong complexation between contaminants and the amino sites. The modification is obtained either by chemical reactions or blending biochar with amino-rich polymers such as polyethyleneimine (PEI) and chitosan .3, H2SO4, and Na2S2O4 to modify biochar via nitration and reduction reactions as a selective and efficient sorbent for Cu2+. Although there was little significant difference in the physical structure before and after the modification, attenuated total reflectance FT-IR and XPS results showed that the amino groups chemically bound with the functional groups on the biochar surface. The amino modification made the sorption capacity for Cu2+ increased by five times. −1) than that of the unmodified biochar (23.1 mg g−1).2+, Cu2+, and Pb2+ in aqueous solutions by almost all chitosan-modified biochars were enhanced, compared with the unmodified biochars. Further studies of Pb sorption on chitosan-modified bamboo biochar found that, even though the sorption kinetics were slow, the modified biochar had a relatively high Langmuir Pb sorption capacity of 14.3 mg g−1, significantly reducing the toxicity of Pb. Characterization of the Pb-loaded biochar after sorption exhibited that the sorption of Pb is primarily caused by the interaction with amino functional groups on the biochar surface.The magnetization of biochar is a new modification frontier. It develops in situations where the separation of biochar from aqueous solutions face great difficulties. The application of a magnet for magnetic biochar enables such difficulty to be solved.3+/Fe2+ solution. It was found that the iron content increased from 1.4% to 80.6%, indicating that the biochar was effectively magnetized. In the application of Pb2+ and Cd2+ removal from solutions, the biochar showed significantly higher sorption capacity. Except for conventional pyrolysis, microwave heating is extensively applied in the synthesis of magnetic biochar. 2+/Fe3+ solution into an ammonia solution with biochar particles dispersed, followed by ultrasound irradiation at 60 °C. The magnetic biochar exhibited an increased number of carboxyl functional groups on the surface, resulting in a more negatively charged property, which improved the sorption rate and capacity for heavy metal ions.Impregnation-pyrolysis and co-precipitation are the most commonly used preparation method for magnetic biochar, accounting for about 69.6% of all preparation methods . Impregn2O3, Fe3O4, FeO, and Fe0, also play an important role in improving the sorption ability  removal by directly reduction , attributed to the Fe(II) and Fe(III) in octahedral coordination in Fe3O4, which act as active chemical sorption or reduction sites  was larger than that of the lignin biochar (200 m2 g−1) when carbonized at the same temperature because of the resistance of lignin, showing the cellulose biomass to be preferable than lignin biomass for biochar production; the surface area and total pore volume of pinewood biochar pyrolyzed at higher temperature were much higher due of the more complete carbonization of lignin, implying that higher temperatures induce well-developed pore structure  and then pyrolyzed for the available biochar. Modification steps are also required for an ideal sorption effect. Compared with conventional activated carbon, these treatments for biochar will inevitably increase the production cost. Therefore, future researches should attempt to find a compromise between optimizing the production process and maximizing the applicability of biochar to minimize the cost . Meanwhitructure .The stability of biochar and biochar-based composites should be considered in the practical application of biochar. So far, most researches have focused on the sorption of single contamination in aqueous solutions. However, the prevailing situation in real water application is the coexistence of a variety of contaminants, where synergistic and antagonistic sorption effects can be observed. The presence of multiple contaminants potentially results in ionic interference and competition of sorption sites, eventually reducing the removal efficiency. At present, empirical data based on sorption of co-contaminant is limited, appealing for the establishment of simultaneous sorption models, which could reveal the involved synergistic or antagonistic sorption mechanisms. To facilitate such studies, reports on biochar sorption should contain sufficient information about the sorbent properties and sorption conditions as detailed as possible in case of providing future directions. Several efforts have been reported, such as the simulated molecular equations for studying competitive sorption of co-contaminant ; new anaAlthough it is consensus that biochar is low-cost, renewable, and sustainable compared with activated carbon , to achiThis review gives a systematical overview of the broad application of biochar in water and wastewater treatment to remove common and emerging organic and inorganic contaminants. The involved sorption mechanisms are demonstrated as a foundation of studies on biochar sorption behavior. Based on the mechanisms, attention has been paid on biochar modification to improve its performance, which aims to increase the surface area, porosity, or surface sorption sites of the biochar. Exciting frontiers of magnetic biochar and biochar-biofilm combination are also presented. Meanwhile, existing environmental concerns of biochar application are discussed in the aspects of cost, performance, stability, co-contaminant, and sustainability. Finally, future research directions are put forward to facilitate the practical application of biochar.


Skeletal muscle dysfunction, articular cartilage degeneration, and bone loss occur essentially in parallel during aging. Mechanisms contributing to this systemic musculoskeletal decline remain incompletely understood, limiting progress toward developing effective therapeutics. Because the progression of human musculoskeletal aging is slow, researchers rely on rodent models to identify mechanisms and test interventions. The Dunkin Hartley guinea pig is an outbred strain that begins developing primary osteoarthritis by 4 months of age with a progression and pathology similar to aging humans. The purpose of this study was to determine if skeletal muscle remodeling during the progression of osteoarthritis in these guinea pigs resembles musculoskeletal aging in humans. We compared Dunkin Hartley guinea pigs to Strain 13 guinea pigs, which develop osteoarthritis much later in the lifespan. We measured myofiber type and size, muscle density, and long-term fractional protein synthesis rates of the gastrocnemius and soleus muscles in 5, 9, and 15-month-old guinea pigs. There was an age-related decline in skeletal muscle density, a greater proportion of smaller myofibers, and a decline in type II concomitant with a rise in type I myofibers in the gastrocnemius muscles from Dunkin Hartley guinea pigs only. These changes were accompanied by age-related declines in myofibrillar and mitochondrial protein synthesis in the gastrocnemius and soleus. Collectively, these findings suggest Dunkin Hartley guinea pigs experience myofiber remodeling alongside the progression of osteoarthritis, consistent with human musculoskeletal aging. Thus, Dunkin Hartley guinea pigs may be a model to advance discovery and therapeutic development for human musculoskeletal aging. Musculoskeletal aging broadly describes the progressive decline in skeletal muscle, bone, tendon, and articular cartilage that contributes to disability, chronic disease, and impaired quality of life in older adults . One facMusculoskeletal decline is heterogenous and multifactorial in nature. The decline in skeletal muscle is slow and heterogeneous with the average human over the age of 65 losing 1% muscle mass per year . Among tCritical to both discovery and translational research to improve human health is availability of laboratory and pre-clinical models that bear close resemblance to the human condition. Musculoskeletal system components interact mechanically, communicate biochemically, and often decline in parallel collectively contributing to falls, fracture risk, and loss of mobility . Additioad libitum, this strain of guinea pig becomes overweight/obese  OA that closely resembles the onset and disease progression in humans . When feht/obese  and begiht/obese . While tht/obese . Dunkin ht/obese . Importaht/obese . Howeverht/obese . If the n = 6 at each age per strain). Animals were maintained at Colorado State University’s Laboratory Animal Resources housing facilities and were monitored daily by a veterinarian. All guinea pigs were singly-housed in solid bottom cages, maintained on a 12–12 h light-dark cycle, and provided ad libitum access to food and water. At the time of euthanasia, the guinea pigs were 5, 9, or 15 mo. Two 15 mo Dunkin Hartley guinea pigs had to be urgently euthanized 1 week prior to harvest due to lung and gastrointestinal complications according to the necropsy conducted by a veterinarian. In accordance with the standards of the American Veterinary Medical Association, animals were anesthetized with a mixture of isoflurane and oxygen; immediately afterward, the anesthetized animals were transferred to a chamber filled with carbon dioxide for euthanasia. To confirm euthanasia, bilateral thoracotomy was performed. After euthanasia, a portion of the belly of gastrocnemius and soleus muscles of one leg was excised, mounted, fixed, and frozen in isopentane in OCT for fluorescent immunohistochemistry for myofiber type and size analysis, while the remaining muscle was snap frozen in liquid nitrogen for isotope analysis. We could not collect these measurements for the two premature deaths of the 15 mo guinea pigs. The other leg was formalin fixed with the knee and ankle joints at 90o prior to being individually dissected at their attachments to the bone. All limbs were fixed and treated in an identical manner to control for the possibility of fixation-related artifacts. The muscles for this leg were used for volumetric analysis, fiber angle, and immunohistochemistry.All procedures were approved by the Colorado State University Institutional Animal Care and Use Committee (Protocols 16-6755AA and 19-9129A) and were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals. 18 male Dunkin Hartley were obtained from Charles River Laboratories  and 18 male Strain 13 guinea pigs were obtained from the United States Army Medical Research Institute of Infectious Diseases  at approximately 3.5, 7.5, and 13.5 months of age  was used to obtain volume in both heads of the gastrocnemius and soleus, which had been formalin fixed with the knee and ankle joints at 90° prior to being individually dissected at their attachments to the bone. MRI scans were performed at Rocky Mountain Magnetic Resonance of Colorado State University, with a 2.3 T Bruker BioSpec, equipped with a 20.5 cm, 100 mT/m gradient system, using a custom built 3.4 cm internal diameter, single channel RF Litz coil  tuned to detect 1 H at 100.3 MHz. The excised muscles were weighed and then imaged in groups of 8. In the T1- weighted gradient echo images, a fast low-angle shot sequence was used to acquire volumetric images resolved with 0.5 mm isotropic resolution in three-dimensions: echo time = 4.73 ms; repetition time = 15 ms; field of view = 96.0 mm × 33.5 mm × 29.5 mm; matrix size = 192 × 67 × 59.§ was used for segmentation and ROI analysis of muscle volume. Muscle mass and volume were utilized to calculate density – mass divided by volume (mg/mm3).The volumetric images were exported as DICOM and Analyze 11Tibial length was determined using calibrated digital calipers. Measurements were collected on the posterior/caudal aspect of the bone from the intercondylar eminence to the articular surface of the medial malleolus. Measurements were taken in triplicate with the mean recorded.r2 = 0.5953).From the formalin fixed limb, both the medial and lateral gastrocnemius heads were stained in India ink, imaged, and analyzed for fiber angle (θ), defined as the angle of the fiber from the muscle’s line of action . MeasureFrom the fixed limb and after fiber angle analysis, the gastrocnemius and soleus were paraffin embedded, cross sectioned, and stained with Masson’s Trichrome following an established protocol at the Colorado State University Diagnostic Medicine Center. Masson’s Trichrome staining results in collagen fibers stained blue, nuclei tissue stained black, and background tissue stained red. We imaged the stained cross sections using an upright microscope at 10x magnification and used ImageJ to determine the percentage of area stained blue. To do this, two reviewers set a threshold to quantify the amount of blue present in the cross section and results were subsequently averaged.Additionally, we used a spectrophotometric method to quantify relative collagen content in tissue using Sirius Red and Fast Green . Portions of the gastrocnemius and soleus were frozen at the time of tissue harvest and 5 μm skeletal muscle cryosections were mounted on microscope slides. Sections were rinsed with 1X phosphate buffered saline (PBS), immersed in the Dye Solution for 30 min at room temperature, and covered to prevent evaporation of the solution. After incubation, we aspirated the solution and then rinsed the tissue section with distilled water. We then applied the Dye Extraction Buffer and then transferred the Buffer to a 96 well plate for spectrophotometric analysis. We measured the OD values at 540 nm and 605 nm and calculated collagenous and non-collagenous using manufacturer instructions.From the fixed limb, knees were decalcified, sectioned coronally at the level of the medial tibial plateau, and paraffin embedded such that a 5 μm intact central section could be stained with toluidine blue. Following recommended published guidelines, the four anatomic compartments of the joint  were scored using a semiquantitative histopathologic grading scheme (OARSI) such that a total joint score was assigned .We employed fluorescent immunohistochemistry to measure fiber type and size distribution in the gastrocnemius and soleus muscles. We cryosectioned portions of both the soleus and gastrocnemius that were embedded in OCT and frozen in isopentane cooled by liquid nitrogen during harvest. We then mounted 5 μm skeletal muscle cryosections on microscope slides, allowed them to air dry for 10 min, fixed them in −20°C acetone for 10 min, and rehydrated them in 1X PBS. We then blocked the samples in 10% normal goat serum (NGS) for 1 h and rinsed them in 1X PBS for 30 s. Samples were incubated in the following primary antibodies diluted in 10% NGS for 2 h at room temperature protected from the light: Laminin: Abcam 11576, 1:500; MyHC I: DHSB BA-F8, 1:50; MyHC IIB: DHSB 10F5, 1:50; MyHC IIA: DHSB 2F7 1:50. Following 3, 5 min rinses in 1X PBS, we incubated the cross sections with secondary antibodies also in 10% NGS for 1 h , applied an anti-fade reagent , and adhered cover slips to the microscope slides.2 bins. The number of fibers in each bin was then divided by the total number of fibers analyzed to determine the percent distribution of each bin.The slides were imaged by the Center of Muscle Biology at the University of Kentucky as described . Briefly2H2O) to measure long-term skeletal muscle protein synthesis rates. 30 days prior to euthanasia, all guinea pigs were given a subcutaneous injection of 0.9% saline enriched with 99% 2H2O equivalent to 3% of their body weight. Drinking water was then enriched with 8% 2H2O to maintain deuterium enrichment of the body water pool during the 30-day labeling period. Body water enrichment was determined from plasma as previously described  was used for statistical analysis and to create figures. While statistical significance was set a priori at p < 0.05, because of the relatively small sample size, we also chose to report values of p < 0.10 as noteworthy observations. Data are normally distributed and presented as mean ± standard error when data from individual animals cannot be shown .A two-way ANOVA was performed when outcome measures  were assessed at all three ages of 5, 9, and 15 mo, to analyze the main effects of both age and strain. If there was significant main effect of age, we conducted a Tukey’s p < 0.001) on body mass. Body mass increased with age in both strains, with 9 mo guinea pigs weighing significantly more than 5 mo, and 15 mo guinea pigs tending (p = 0.06) to weigh more than 5 mo (p < 0.05), although this was observed predominantly in Strain 13 . Dunkin Hartley guinea pigs had more severe OA than Strain 13 counterparts  in both strains of guinea pigs. Dunkin Hartley guinea pigs had larger gastrocnemii than Strain 13 guinea pigs  on soleus mass, with 15 mo guinea pigs having greater (p < 0.05) mass than their 5 mo counterparts (p < 0.05).To determine whether or not there were muscle-specific declines concomitant with the progression of OA, we measured skeletal muscle mass, volume, and density in the hindlimb of the guinea pigs. There was a non-significant positive effect of age on gastrocnemius mass (terparts . Howeverp = 0.06) accounted for by Strain 13 guinea pigs (p = 0.06) on soleus mass relative to body mass , but again, this was not observed in Strain 13 guinea pigs  difference in soleus myofiber CSA between 5 mo and 15 mo Strain 13 guinea pigs  in gastrocnemius fiber angle  were all lower in 15 mo guinea pigs compared to 5 mo in Dunkin Hartley guinea pigs only  in collagen FSR in both Strain 13 and Dunkin Hartley guinea pigs  contributes to the age-related decline in skeletal muscle mass. Accordingly, we measured to skeletal muscle protein synthesis rates using the stable isotopic tracer nea pigs .p < 0.05; p = 0.07; Similar to the gastrocnemius, soleus synthesis rates were slower in the older Dunkin Hartley guinea pigs in each fraction except for collagen . MyofibrMusculoskeletal decline is a key contributor to disability  and cardOver the time course of the current study, we observed that Dunkin Hartley guinea pigs have a change in muscle fiber size distribution favoring smaller fibers, fiber type composition shifting toward type I and IIA fibers, lower muscle density, and slower rates of muscle protein synthesis. Collectively, these changes suggest skeletal muscle remodeling that is consistent with the aged skeletal muscle phenotype in humans. Interestingly, these observations occurred by an age (15 mo) that is young relative to the maximal predicted lifespan of other strains of guinea pigs [∼10% of the predicted 12 years ]. The obA potential limitation worth noting is that there is growth between the 5 and 9 mo time points. While this is not entirely reflective of human musculoskeletal aging, humans do experience an increase in bodyweight, primarily in adiposity, with age . Guinea In humans, muscle with mixed fiber types  are more susceptible to loss of mass compared to muscles comprised primarily type I muscle fibers, such as the soleus, which are generally unaffected by age . This isAs humans age, there is generally an increase in fat deposition within  or around  skeletal muscle. Only in Dunkin Hartley guinea pigs did we observe a lower gastrocnemius density in 15 mo guinea pigs compared to 5 mo counterparts . The onlFew published studies include data focused on the influence of age on collagen content in human skeletal muscle. Most suggest there is no change in collagen content with age in humans , a decreIn humans, fiber angle has been shown to decrease in the gastrocnemius and soleus with age, which is likely due to decreases of sarcomeres both in series and in parallel . We obse2H2O), we measured the cumulative protein synthesis over 30 days, capturing both resting and anabolic episodes of protein synthesis in these guinea pigs. 15 mo Dunkin Hartley guinea pigs had lower rates of protein synthesis in all the subfractions of both gastrocnemius and soleus muscles compared to 5 mo counterparts (Humans experience a decline in skeletal muscle mass at a relatively slow rate (∼1% decline per year; terparts , 10. In terparts , 10. Thuterparts . This diterparts , the ratterparts .2H2O methods to facilitate long-term assessment of protein synthesis rates found no difference in myofibrillar FSR between young and older adults prior to exercise training, but an age-related impairment in the response to training (A recent study leveraging training . In our At the moment, there is no clear, established mechanism for the early onset of the skeletal muscle aging phenotype we observed in the Dunkin Hartley guinea pigs. This strain of guinea pig develops primary OA that histologically resembles human OA . Strain ad libitum fed controls and delay the onset of OA in this model. In humans, both knee OA and elevated levels of inflammatory mediators are associated with sarcopenia (Another mechanism that could explain the early onset muscle aging phenotype is increased systemic inflammation. Compared to a strain of guinea pig that is not prone to primary OA at a young age, Dunkin Hartley guinea pigs have higher levels of the pro-inflammatory mediator interleukin-1β IL-1β; . Intervercopenia . Additiorcopenia . Inflammrcopenia  and implrcopenia . TherefoLaboratory models, by definition, do not provide perfect replications of human clinical conditions. Much of what is currently known about skeletal muscle aging comes from research using models designed to study muscle disuse in either rodents or humans , which hWhile we assessed a number of well-recognized aspects of skeletal muscle remodeling associated with human aging, there are other important components of the aging muscle phenotype we have yet to measure in this unique guinea pig model, most notably muscle function. We predict that the changes in muscle phenotype we observed here would be paralleled by declines in muscle function. The observed loss of type IIB fibers is known to be a key determinant of muscle strength . FurtherThe original contributions presented in the study are included in the article/The animal study was reviewed and approved by Colorado State University Institutional Animal Care and Use Committee.This study was conducted at Colorado State University. KH, KS, and RR contributed to the conception and design. All authors contributed to data acquisition and/or interpretation of data. RM and MW wrote the first manuscript draft. All authors critically revised the manuscript and provided intellectual and approved the final version of the manuscript submitted for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


PLGA scaffold without Medpor® was created to serve as the control. Scaffolds were vacuum-seeded with rabbit chondrocytes, freshly isolated from the ear by enzymatic digestion. Then, cell-seeded scaffolds were implanted subcutaneously in the dorsal pockets of nude mice. After 12 weeks, explants were analyzed by histological, biochemical, and mechanical evaluations. Although the PLGA group resulted in neocartilage formation, the PLGA–Medpor® group demonstrated improved outcome with the formation of well-surrounded cartilage around the implants with higher mechanical strength than the PLGA group, indicating that Medpor® has an influence on the structural strength of engineered cartilage. The presence of collagen and elastin fibers was evident in the histological section in both groups. These results demonstrated a novel method of coating implant material with engineered cartilage to overcome the limitations of using biodegradable scaffold in cartilage tissue regeneration. By utilizing the patient’s own chondrocytes, our proposed method may broaden the choice of implant materials while minimizing side effects and immune reaction for the future medical application.Inert biomaterials used for auricular reconstruction, which is one of the most challenging and diverse tasks in craniofacial or head and neck surgery, often cause problems such as capsule formation, infection, and skin extrusion. To solve these problems, scaffold consisting of inert biomaterial, high-density polyethylene (Medpor The loss of normal ear morphology due to congenital defect (microtia), trauma, or cancer requires a reconstructive surgery. Specifically, auricle is an outer most part of auditory system that can be easily damaged by external force because of its thin structure. To repair damaged ear, the framework for architectural support has to precede . The earSeveral materials, such as cartilaginous, osseous, and alloplastic have been reported as potential alternatives for the auricle reconstruction since 1891 ,5. AmongAdvancements of materials in medical application enabled variety of implantable substitutes to replace damaged human tissues. However, many artificially manufactured biomaterials cause inflammatory reaction, fibrosis by fibroblasts, infection, skin necrosis, extrusion, and more after implantation. To date, silicone and polyethylene have been most widely used as permanent tissue substitutes because of their reasonable biocompatibilities. Silicone and polyethylene are also the most popular implants for cartilage reconstruction. However, silicones do not become integrated into the surrounding tissues and cause a high rate of extrusion through the skin and infection . PreviouRecently, the technique to cover polyethylene implant with cartilage tissue using gel type of polymer such as Pluronic and fibrin was proposed ,16. Howe® scaffold seeded with isolated chondrocytes for auricular cartilage tissue regeneration  (PLGA)-coated Medporneration . Medpor®truction . By maniChondrocytes were successfully isolated from rabbit ear cartilage for monolayer culture. Cells were expanded by subculture A and tes® structure  engineered cartilage can possess various physical properties based on the choice of inert materials; and (3) possible capsule formation or intrusion, or tissue necrosis can be minimized by the inert material.Although regenerating cartilage tissue from its specific chondrocyte without foreign material would be ideal as a non-immunogenic implant, this approach may lack the mechanical strength. Thus, multilayer approach may be considered as a practical solution due to certain unique advantages: (1) autologous chondrocytes can provide a non-immunogenic coating, preventing the foreign body reaction elicited by Medpor® implant was attempted to be coated with gel-type materials such as fibrin gel and Pluronic F-127 mixed with cells, and the outcome was somewhat optimistic [® implant was covered with cartilage tissues and the other side was disturbed during in vivo implantation. In most tissue engineering applications, fibrin is introduced through injection. However, if these polymers can be successfully converted to stiff solid polymers in multiple ways, including crosslinking and bonding reinforcement, in the near future, gel-type of materials in cartilage tissue engineering application will be a strong candidate for overcoming their weak structural properties.In the previous study, Medportimistic ,22. EvenConversely, PLGA can provide adequate structural integrity without early deterioration.This study focused on the proof of concept by using a small scale of samples to test the feasibility of regenerating the whole structure of the human ear or nose cartilage on a larger scale of regeneration in the future. However, we utilized autologous primary chondrocytes as the cell source to regenerate neocartilage, which is a therapeutic model in the clinical setting. As an alternative option for autologous cell source, current advances in stem cell technology may offer various choices of stem cell sources that can differentiate into chondrogenic lineage cells, including mesenchymal stem cells (MSCs), induced pluripotent stem cells (iPSCs), embryonic stem cells (ESCs), and amniotic stem cells (ASCs), demonstrating comparable differential potential to chondrogenic lineage similar to primary chondrocytes ,24,25,26The monolayer expansion of autologous primary chondrocytes usually dedifferentiate, and this limits its applicability in large-scale cartilage tissue engineering. As an alternative use for stem cells, co-culturing of both autologous chondrocytes and MSCs together would be an option in the pursuit of the shortage of an autologous cell source. Cohen et al. demonstrated the successful outcome of patient-specific human ear cartilage engineered exclusively with human cell sources without extensive in vitro cell expansion prior to implantation. This study was a critical step towards the clinical application of tissue engineering for ear cartilage reconstruction .® did not form much cartilage-like tissues, but instead formed mostly fibrous tissues. This might have been caused by the poor seeding method, which led to a lower cell seeding efficiency and less homogeneous distribution of the cells throughout the scaffold when compared with dynamic seeding techniques [® with tissue-engineered cartilage.A key to successfully engineering cartilage is homogeneously distributing chondrocytes throughout the PLGA scaffold. Static seeding techniques are the most frequently used method, as they are easy to use and have less chance of damaging cell by external force. However, additional studies on seeding will be useful because the optimal seeding method can increase seeding efficiency, allowing a higher number of cells in the seeding suspension to adhere to the scaffold. It would also be reproducible and easy to perform, leading to consistent outcome. In fact, our early outcome for the engineered tissue with PLGA and PLGA–Medporchniques ,29. To oRecent 3D printing technology can provide an opportunity to combine different types of materials to engineer a complex structure of scaffold. Therefore, human ear shape scaffold, for example, can be printed using 3D printing technology in a more precise and controlled manner . Further® implant. Although we demonstrated engineered cartilage using PLGA–Medpor® scaffold in a small scale in this study, we will move on to a larger, whole ear or nose-size scale of implant material in the near future.It is concluded that cartilaginous tissue can successfully regenerate around a Medpor2 at 37 °C for further expansion. The medium was changed every 3 days.New Zealand white rabbit  was euthanized, and elastic cartilage was obtained from its ear. Animal studies were carried out according to the protocol approved by the Institutional Animal Care and Use Committee at the University of North Carolina at Chapel Hill (protocol no. 17-151). All procedures were performed under sterile conditions. The perichondrium was removed, and the cartilage was finely minced, and then enzymatically digested in 0.2% collagenase type II  solution at 37 °C with constant agitation. After 2 h, chondrocytes were separated by centrifugation and washed with 1× PBS. Isolated chondrocytes were gently mixed with chondrogenic media , plated in a 10 cm culture dish, and stored in incubator maintaining 5% CO5 cells) using an RNeasy kit , and the total RNA was reverse-transcribed into cDNA by RT-PCR. Oligonucleotide primers for the PCR were designed according to the published sequence for the type II collagen . The PCR conditions used for type II collagen production were 22 cycles of denaturation (95 °C/l min), annealation (60 °C/1 min), and extension (72 °C/1 min). The oligonucleotide primers for beta-2M were used as a control. Then, the sample was electrophoresed through a 2% agarose gel containing ethidium bromide.RNA was isolated from the cultured chondrocytes , and visualized using an enhanced chemiluminescent substrate . Anti-collagen type II antibodies  were used to detect secreted collagen type II.For immunostaining of type II collagen, chondrocyte culture was fixed in 4% paraformaldehyde solution  for 30 min. The cells were incubated with bovine anti-collagen II polyclonal primary antibody (ThermoFisher Scientific) diluted to 1:100 in 1% Bovine serum albumin (BSA) solution at 4 °C overnight followed by incubation with 3% BSA in 1×PBS for 30 min to block unspecific background staining. After washing with 1xPBS three times, Alexa Fluor Plus 488 (ThermoFisher Scientific) as secondary antibody was then applied for 30 min and then subjected to three more washes in 1xPBS, which was followed by treatment with propedium idodie  for nuclei staining. Images were acquired using Nikon Eclipse Ti-U inverted fluorescent microscope .® scaffolds were fabricated as disk shapes. A cylindrical Teflon mold with a hole (100 mm in diameter and 10 mm in depth) in the middle was created. A total of 5 g PLGA  with the ratio of PLA to PGA as 65:35 was dissolved in 10 mL CHCl3 and mixed with 40 g of salt (sized between 200 and 250 µm). To make the three-dimensional porous structure of the polymer scaffold, salt leaching methods were applied. Medpor® (0.8 mm in thickness) was cut to be 8 mm in diameter. Dissolved PLGA with salt was poured into the mold, up to half of the mold volume; Medpor® was placed in the mold; and then the remaining mixture was poured in to acquire the desired thickness. After complete drying, the mold was placed in distilled water to leach out salt, and was dehydrated and then sterilized with ethylene oxide gas. PLGA scaffold was made the same way, without Medpor®.Both PLGA and PLGA–Medpor6 cell suspension in 25 µL media for each scaffold. Statically seeded scaffold was used to study the effect of vacuum. A sterile bottle top filter (ThermoFisher Scientific) was placed on a glass bottle and connected to a vacuum hose. After the vacuum ran on the surface of the filter, the level of vacuum was adjusted to slowly let 25 µL of media drop on the surface in order to get absorbed into the scaffold. After the scaffold was placed on the filter, vigorously mixed 25 µL of cell suspension was seeded on top of the scaffold surface using a pipette. The scaffold was quickly removed from the filter once the cell suspension was absorbed into the scaffold and was placed in growth media for least 2 h. The same procedure was repeated on the other side of the scaffold  for 2 days at room temperature. After thorough washing with PBS, the explants underwent critical point drying process by dehydrating through an ethanol gradient  and drying in hexamethyldisilazane . Subsequently, the cross-sectional surfaces of the samples were sputter-coated with platinum at thickness of 4 nm in a vacuum and were examined using scanning electron microscopy .Each sample was harvested 12 weeks after implantation, and fixed in 10% neutral buffered formalin solution for 2 days. After dehydrating in 95% and 100% ethanol sequentially, the samples were transferred to the tissue processor for paraffin embedding. Then, the blocks were sectioned at 5 μm, deparaffinized with xylene, and stained with Weigert’s hematoxylin  and counter-stained with eosin (Sigma-Aldrich) to assess cell morphology. Masson’s trichrome and Verhoeff staining were conducted using ready-to-use kits following the company’s instruction. Briefly, for the Masson’s trichrome staining , slides were immersed in Bouin’s solution for 15 min. Subsequently, the slides were stained with Weigert’s hematoxylin for 5 min and with Biebrich scarlet acid fuchin for 5 min. The stained slides were differentiated in phosphotungstic/phosphomolybdic acid solution for 5 min to enhance the staining of the collagen. Finally, the samples were dyed with aniline blue solution for 5 min and differentiated in 1% acetic acid solution for 2 min to avoid color removal. Verhoeff staining was carried out using Elastic Stain Kit . Slides were stained in Verhoeff’s solution for 60 min and then differentiated in 2% ferric chloride for 1 to 2 min after rinsing in tap water. The differentiation was stopped by immersing the slides in tap water after checking microscopically for black elastic fiber. The counterstaining in hematoxylin solution was carried out for 5 min. Then, slides were mounted and neocartilage formation was analyzed using a Nikon Eclipse Ti-U inverted microscope (Nikon Instruments Inc.). Collagen and elastin were quantified in as percentages using ImageJ software .® were tested using Instron . The explants were stored in PBS until the compression test was performed. Specimens were compressed by 0.5 mm/min uniaxial force. Five specimens were tested per each group. The maximum strength of each specimen was determined from the stress–strain curve.The compressive strengths of the explants made with PLGA and PLGA–Medport-test was used to compare the means between the groups.One-tailed Student’s