Infants (under 1-year-old) are at most risk of life threatening respiratory syncytial virus (RSV) disease. RSV epidemiological data alone has been insufficient in defining who acquires infection from whom (WAIFW) within households. We investigated RSV genomic variation within and between infected individuals and assessed its potential utility in tracking transmission in households. Over an entire single RSV season in coastal Kenya, nasal swabs were collected from members of 20 households every 3–4 days regardless of symptom status and screened for RSV nucleic acid. Next generation sequencing was used to generate >90% RSV full-length genomes for 51.1% of positive samples (191/374). Single nucleotide polymorphisms (SNPs) observed during household infection outbreaks ranged from 0–21 (median: 3) while SNPs observed during single-host infection episodes ranged from 0–17 (median: 1). Using the viral genomic data alone there was insufficient resolution to fully reconstruct within-household transmission chains. For households with clear index cases, the most likely source of infant infection was via a toddler (aged 1 to <3 years-old) or school-aged (aged 6 to <12 years-old) co-occupant. However, for best resolution of WAIFW within households, we suggest an integrated analysis of RSV genomic and epidemiological data. Global estimates in 2015 indicated that RSV causes ~33.1 million episodes of acute lower respiratory tract illness annually, ~3.2 million of which lead to hospital admissions and ~60,000 deaths in hospitalized children aged under 5 years1. Despite this burden, our understanding of RSV transmission patterns during epidemics, including who infects the vulnerable infant populations remains incomplete2. Defining the patterns of RSV transmission during epidemics, and specifically Who Acquires Infection From Whom (WAIFW) has the potential to inform control strategies4.Respiratory syncytial virus (RSV) is a leading viral cause of bronchiolitis and pneumonia during infancy5. Households are considered an important setting for RSV spread due to likely close person-to-person contacts7. A family study in the United States in the 1970s showed that up to 46% of family members and 62% of infants in the household become infected once the virus is introduced into a household8. Since this study, important advances have been made in diagnostic sensitivity and characterisation of infection sources for household cases, in contact mapping tools and in statistical methods to infer epidemiologically linked case pairs10. Furthermore, household demographic characteristics may differ between developed and developing settings11.RSV transmission occurs during contact with an infectious person or contaminated environmental surfaces13. Impediments to RSV vaccine discovery have been the need to immunize in the first weeks of life when infant immune responses are still sub-optimal and enhanced disease observed during a formalin inactivated vaccine trial in the late 1960s14. Live attenuated vaccines given intranasally, have proved difficult to sufficiently attenuate to limit upper airway congestion during vaccination, while still maintaining immunogenicity16. As a result, alternative approaches are being considered including boosting infant antibody levels through maternal sub-unit vaccine immunization, pre-season delivery of high titre extended half-life immunoglobulin, reducing virus circulation in the community by vaccination of older babies and children or by cocoon vaccination to interrupt chains of transmission leading to infant infection18. To advance the cocoon vaccination strategy, a better understanding of RSV transmission in household settings where most transmissions appear to occur is required18.Currently, there is no licensed RSV vaccine, although there are 19 vaccine, prophylactic or monoclonal antibody candidate products in clinical trials10. It is unclear if the pace of RSV genomic change is sufficient to allow tracking of transmission during epidemics. We have previously shown that partial RSV nucleotide sequences from the highly variable attachment (G) encoding gene (~900 nt) provide insufficient discriminatory power to delineate RSV transmission chains21. However, our initial analysis of RSVA full genome sequences  showed significant promise in providing phylogenetic resolution of viruses circulating in different households10 and similar application of these methods have been shown for norovirus22, foot and mouth disease virus23, influenza A virus24, MERS-CoV25, and Ebola virus27. In this study, we aimed to determine if RSV transmission in households is trackable using viral genomic data and if it is possible to identify who is the likely infector of the under 1-year-old infant.Currently, little is known about the sequence change patterns during individual RSV infection episodes, or during intra-household and inter-household transmission events28. Briefly, 47 households scattered across an area of approximately 21 km2 were followed up over a 6-month period beginning December 2009 and ending June 2010 coinciding with the RSV peak activity months in the area29. Households were defined as a group of people sharing a compound and eating from the same kitchen20. The selected households (abbreviated HHs) were given designated identifiers from 1 to 57 (HH01 to HH57). Twice weekly throughout the study period, a nasopharyngeal-flocked swab (NPS) was obtained from each member regardless of symptom status. The NPS samples were screened for RSV using a multiplex real-time RT-PCR method which subtyped RSV positives into RSVA and RSVB30. For whole genome sequencing (WGS), we targeted 20 select households that documented RSV infection of ≥2 members. A geographical map showing the distribution of the study households is provided in the Additional File: Fig. The study was undertaken within Kilifi County, which is located in coastal Kenya. A detailed description of the study location and study design was provided elsewhere21. Sequencing libraries were prepared using Nextera DNA Library Prep kits and nucleotide sequencing performed using Illumina MiSeq platform multiplexing 15–20 samples per run to generate approximately 1 million paired-end reads (150 bp × 2) per sample21.Viral RNAs from the positive samples of selected households were obtained using the QIAamp viral RNA extraction Kit  following the manufacturer’s instructions. Complementary DNA (cDNA) synthesis and RSV whole genome amplification was achieved using a six-overlapping PCR fragments strategy (each ~2.5 kb) as previously described31. Reads passing quality checks were de novo assembled into longer contigs using the SPAdes v3.5.032. RSV contigs were identified by matching to a database of RSV sequences using USEARCH program33, examined for completeness of the expected open reading frames (ORF) using Geneious v8.1.6 (https://www.geneious.com) and, where necessary, partial contigs were further combined to longer ones using Sequencher v5.0.134. These were subsequently checked presence of intact ORFs, sorted by household, re-aligned and positions of nucleotide variation double-checked if these were supported by majority of the raw reads associated with that sample10. Multiple sequence alignments were prepared in MAFFT v7.22035.The short reads from the MiSeq instrument were de-multiplexed, quality checked (median read Phred score of ≥35) and trimmed using QUASR v6.0836 and RAxML v8.2.1237. The best-fitting models of nucleotide substitution for each alignment were in IQ-TREE v1.4.338. Best tree search was performed by Nearest Neighbor Interchange (NNI). Branch support was evaluated by bootstrapping with 1,000 replicates. Pairwise genetic distances were calculated in pairsnp 0.0.639. The phylogenetic relatedness of the household RSVA and RSVB genomes was assessed at three levels; (i) in combination with global sequences deposited in GenBank , (ii) among the households viruses alone and (iii) among viruses collected from same households only. The potential transmission networks within and between households for each group were inferred in PopART package v1.7.2 using median joining tree (MJT) method with an epsilon of zero40. Evolutionary analyses were determined in maximum-likelihood-based TreeTime program41. Phylogenetic trees were visualized and annotated in FigTree v1.4.4 (http://tree.bio.ed.ac.uk/software/figtree/).Sequence phylogenies were inferred using Maximum Likelihood (ML) methods in MEGA720. We grouped the other participants into 5 age-groups: (i) toddlers (1 to <3 years), (ii) pre-schoolers (3 to <6 years), (iii) school-aged (6 to <12 years), (iv) adolescents (12 to <18 years) and adults (>18 years). We attempted to identify who among these other age-groups were the most likely infectors of the infants by examining the relatedness of the virus genome(s) obtained from the infant to all the viral genomes obtained from the other members in the same household. Information on the dates of the sampling of the sequenced samples was taken into account to position the infant in the transmission network/chain.Infants were defined as the participants aged <1-year-old during the studyThe sequence nomenclature of the household samples has four digits that include the household identifier (first two digits) and subject identifier (the last two digits). All the new 112 full or partial RSVB genome sequences from this study were deposited in GenBank under the accession numbers MH594350 – MH594461. The RSVA genomes are deposited in GenBank under accession numbers KX510136-KX510266.20. All study procedures were performed in accordance with the approved protocol guidelines and in compliance with the relevant regulations.The samples were collected after obtaining informed written consent from each participant if aged ≥18 years or through a guardian or parent if aged <18 years. In addition, children aged above 5 years were asked for assent. The study protocol approved by both the Scientific and Ethics Review Unit (SERU) of the Kenya Medical Research Institute (KEMRI), Nairobi, and Coventry Research Ethics Committee of UK21 with successful amplification and assembly of RSV contigs of >1000 nucleotides length in 246 samples (65.7%). Of these 191 samples (51.1%) yielded contigs >14000 nucleotides (nt) (103 RSVA and 88 RSVB i.e. >90% of RSV full-length genomes) hereafter referred to as genomes. In eight and 14 HHs, two or more RSVA or RSVB genomes were recovered, respectively, allowing our investigation into within-household RSV transmission and variation, Table We targeted 20 households with a total of 226 occupants (range 4–37 persons per household) for WGS. Details of the demographic characteristics of the analysed households, total specimens collected, diagnostic results, genome sequencing success and the observed phylogenetic clades (defined later) are summarized in Table From G gene phylogeny, all RSVA and RSVB viruses sequenced were genotypes GA2 and BA, respectively (results not shown). The genome-based maximum likelihood (ML) phylogenetic trees are shown in Fig. The time-resolved ML trees and temporal signal in nucleotide divergence of the household RSVA and RSVB viruses are shown in Fig. We quantified the genetic diversity observed within the two RSV groups by calculating the number of pairwise single nucleotide polymorphisms (SNPs) (pairwise distance) of viruses within the same group, Fig. . In total, we identified 12 epidemiological strains (five within RSVA and seven within RSVB) that occurred in the study area during the six-month surveillance, eight 66.7%) of which were observed in multiple households while four were found in a single household. For the epidemiological strains that occurred in multiple households, between 5–33  SNPs were observed across their genomes. A comparison of SNP abundance intra-host, inter-host and inter-household is provided in Fig. % of whic10. Following examination of the patterns from all the 20 households, the summary of our deductions on who most likely infected the infant is provided in Table There were 22 infants from the 20 HHs. By our diagnostics, the infant in HH18 did not get RSV infected during our surveillance period. The household-by-household time-resolved infection patterns, genome alignments, phylogenies and minimum spanning sequence networks are provided in the Additional Files 20. This was motivated by unsuccessful vaccines for early infants and that evaluation of other options  requires an improved understanding of WAIFW. Our earlier work, based on temporal case observations, clearly suggested that the older children (siblings or cousins aged <15 years), particularly those attending school, played an important role in introducing the virus into the household leading to infant infection, but was not able to resolve within household transmission chains20. We have subsequently formalised the epidemiological analysis of RSV transmission in the household using an individual-based statistical approach to quantify the risk of infection from a range of host, pathogen and environmental factors7. The present study takes an alternative perspective of the problem, by focusing on the temporal patterns of genomic sequence variation to elucidate who infects whom in the household. This work extends a smaller study based on genomes of RSVA from 9 households10, to the current study of genomes of RSVA and RSVB in 20 households.The origin of this work was a study of who introduces RSV into the household and who infects the infantOur key observation from the present analysis is that RSV consensus genomes incur zero to just a few nucleotide substitutions within infected individuals (median: one SNP per episode) or between infected individuals of the same household (median: three SNPs). Combined with the rapid spread of RSV within households and incomplete sequencing (~50%) of the positive samples challenges the reconstruction of the transmission using genomic data alone. For six households (32%) where the infant was infected (n = 8), we could identify the 1–2 most likely individuals who infected them. The infant suspected infectors were mostly household co-occupants <12 year of age  and school-aged (50%) age-groups). Only in a single instance was an adult co-occupant (mother) suspected to be the infant infector. In the remaining households , the infant was identified as either the household index case, a co-index case or the sequencing of key samples failed, making it difficult to infer their infection source.43. The conclusion of the work was that shared minor variants provide little additional resolving power to discern chains of transmission beyond that possible through consensus sequences.Elsewhere we attempted to utilise shared minor variants identified from deep sequencing data for RSV in these same households to draw out patterns of transmission44. In these studies, notably, it was assumed that a single infection source was responsible for the cases occurring in the same household, whereas temporally it can be difficult to fully establish this. Furthermore, without virus genotyping and, ideally, full-genome sequence data, the composition of outbreaks cannot be definitively established; as we have seen multiple concurrent virus introductions into households are not uncommon. Furthermore, for study of Heikkenen et al.44, the investigators followed up the household only after the index infant had been admitted to hospital, which limits the possibility of observing preceding transmission events including who infected them.Previously, only two other studies focused on transmission of RSV infections within households7. Similar to previous studies based solely on epidemiological (not sequence) data44, we highlight the importance of the infant’s elder siblings especially those under 12 years of age as a source of the infant infection. Adults in the households played only a minor role when considered either as household RSV infection introducers or as infant infectors. Furthermore, by analysis of RSVA viruses from nine households, we had previously shown that most  RSV infections in a household outbreak result from a single introduction of the virus10. Here we have extended the analysis to RSVB, confirming a closely similar pattern to RSVA.Our study involved sampling irrespective of symptom status, coupled to sensitive molecular diagnostics and genomic sequencing, which has given a clear indication that households are indeed a common space for RSV transmission45. Despite the close genetic relatedness of RSVA viruses detected in the study, our analysis showed that the 9 infected households were invaded by up to 5 distinct RSVA “epidemiological strains” that diverged at least 6 months before their collection date. For RSVB we determined that the 14 infected households were invaded by up to 7 distinct RSVB epidemiological strains. Highly similar intra-household and intra-host genomic variation patterns were observed between the two groups.The unique household study design here allowed us to compare the phylodynamics of RSVA and RSVB viruses. Overall, the sequenced RSVB viruses showed ~7 times greater genomic diversity compared to RSVA. It is likely that the observed difference reflects annual stochasticity in the number of introduced strains rather than an inherent biological difference although a few previous reports indicated existence of subtle differences between the two groups in transmissibility and local persistence20. The genome sequencing work targeted 20 households where ≥2 members were found to be RSV infected. Despite these households occurring in a small geographical area (~20 km2) it was surprising to see up to 12 epidemiological strains in circulation. Most of the sampled viruses clustered by household. Some households shared the infecting strain with other households, suggesting a shared infection source although direct transmission between these households was unlikely given the large fraction of non-sampled households. Four out of the 12 identified epidemiological strains occurred only in one household each. Notably, households in close physical proximity did not necessarily end up being infected with similar virus clades or subclades implying other unobserved epidemiological factors rather than physical proximity may be more important in determining WAIFW in this community46.Due to the intense logistics involved in undertaking such a study, only 50 households from one administrative unit  within Kilifi County were recruited20. Perplexingly, the study infants were the second most frequent index cases (32%) and were co-index in a further 14% of the household episodes20. It is possible that some of the infant co-index cases were the infectors of infants in the household, but our diagnostic method  failed to detect the virus in the preceding samples. This may occur perhaps due to limited virus replication in older individuals or our 3–4 days sampling interval may have been too wide to capture index cases before onward transmission. By our diagnostic method, a parent was the index case only in one household.Our earlier epidemiological analysis suggested school-going house-members are the sub-population (39%) most likely to introduce the infection into the household48. Alternatively, if these were indeed reinfections, then this observation calls for an interrogation of protective RSV immune responses and this has implications to the development of effective RSV vaccines50.It was surprising to find few to no SNPs in RSV genomes from individuals appearing to shed RSV for up to 2 months. These individuals may have been true prolonged shedders of the virus or were virus re-infected. If prolonged shedders, then it is perplexing that in some individuals, there was one or more negative sample(s) separating the positive samples. Alternately, these could be false negative assay results which may have arisen due to the sensitivity of our sampling or diagnostic method or that the virus was temporarily absent from the upper respiratory tract airway but was still present elsewhere in the individual’s respiratory tract. Prolonged shedding of RSV of up to 2 months has been previously reported especially in immune-compromised populations43. Since in close to half of the study households the infant participant was the infection index or co-index case, for future studies we recommend sampling protocols that also consider, in addition to households, other potential RSV transmission settings in the community e.g. child-care centres, post-natal clinics, schools, school transportation, sporting events etc. Contact data should be collected to reinforce the viral sequence data and epidemiological data to support robust inferences of transmission pairs46. The protocols for genomic sequencing also need to be optimised to obtain virus sequences even from samples with diminishing virus titres. Given the imperfections of analyses of epidemiological data or genomic data in isolation, there is a clear need to undertake the joint analysis of both sources of information using a probabilistic framework7, that will allow inference of events not directly observable with inevitably imperfect data.Our study illustrates both the value and the limitations of RSV genomic data in tracking transmission of this rapidly spreading infection in a household setting. The pace of RSV substitutions was demonstrated to be insufficiently fast to enable the full inference of within household RSV transmission trees. Additionally, we have previously shown that patterns of sharing of minor variants does not add insight beyond the consensus sequence approachAdditional FileDataset 1


Blast-induced traumatic brain injury (bTBI) has been recognized as the common mode of neurotrauma amongst military and civilian personnel due to an increased insurgent activity domestically and abroad. Previous studies from our laboratory have identified enhanced blood-brain barrier (BBB) permeability as a significant, sub-acute (four hours post-blast) pathological change in bTBI. We also found that NADPH oxidase (NOX)-mediated oxidative stress occurs at the same time post-blast when the BBB permeability changes. We therefore hypothesized that oxidative stress is a major causative factor in the BBB breakdown in the sub-acute stages. This work therefore examined the role of NOX1 and its downstream effects on BBB permeability in the frontal cortex (a region previously shown to be the most vulnerable) immediately and four hours post-blast exposure. Rats were injured by primary blast waves in a compressed gas-driven shock tube at 180 kPa and the BBB integrity was assessed by extravasation of Evans blue and changes in tight junction proteins (TJPs) as well as translocation of macromolecules from blood to brain and vice versa. NOX1 abundance was also assessed in neurovascular endothelial cells. Blast injury resulted in increased extravasation and reduced levels of TJPs in tissues consistent with our previous observations. NOX1 levels were significantly increased in endothelial cells followed by increased superoxide production within 4 hours of blast. Blast injury also increased the levels/activation of matrix metalloproteinase 3 and 9. To test the role of oxidative stress, rats were administered apocynin, which is known to inhibit the assembly of NOX subunits and arrests its function. We found apocynin completely inhibited dye extravasation as well as restored TJP levels to that of controls and reduced matrix metalloproteinase activation in the sub-acute stages following blast. Together these data strongly suggest that NOX-mediated oxidative stress contributes to enhanced BBB permeability in bTBI through a pathway involving increased matrix metalloproteinase activation. In spite of the increase in blast-induced neurotrauma (BINT) studies, there are still significant gaps in the understanding of how the acute shockwave induced biomechanical injury plays a role in the subsequent secondary biochemical injuries in the chronic stages. A recent survey reported that more than 30 phase III clinical trials aimed at targeting TBI have failed and, to this effect, our group has attempted to elucidate the temporal and spatial neuropathology of bTBI, in relation to blast-overpressure, as it pertains to two commonly identified pathological factors: oxidative stress increase and blood-brain barrier (BBB) breakdown10.Blast-induced traumatic brain injuries (bTBIs) are the most prevalent form of neurotrauma amongst soldiers and some in civilian populations due to asymmetric warfare with increased acts of terrorism domestically and abroad12 and is mainly induced by reactive oxidative species (ROS). These include, but are not limited to, superoxide (O2⋅−), hydroxyl radical (HO⋅), and hydrogen peroxide (H2O2)14. While a basal level of ROS is to be expected to occur during normal reactions in redox-reactions during and the electron transport chain operation, an excess amount, as seen after injury, can be harmful. NADPH oxidase (NOX) is a multi-subunit enzyme that catalyzes the formation of superoxide radicals from available molecular oxygen. As previously reported by our group and other investigators17, NOX is upregulated in multiple brain regions following blast injury and neurons maximally contribute to the highest increase in hippocampus compared to other neural cells. It is also worth noting that a single blast substantially increases superoxide levels in multiple brain regions from the pre-frontal cortex to hippocampus to cerebellum, all along the direction of shock wave propagation17.Oxidative stress has been implicated in multiple modes of TBI18. The BBB is a selectively-permeable membrane separating the brain from the circulatory system, consisting of tight junction complexes, which attach adjacent endothelial cells together, as well as a host of modulating neural cells including pericytes and astrocytes, which wrap around the endothelium. In a recent work by our group, increased permeability of the BBB was most significantly seen four hours post-blast across the cerebral hemisphere, with highest permeability seen in the frontal cortex18. Damage to the BBB was established through extravasation of tracers Evans blue and sodium fluorescein, the dislodging of tight junction proteins, and leakage of blood-borne cells into the brain parenchyma and brain parenchymal molecules into the blood18. Increased permeability of the BBB has been observed in several other animal models of TBI including closed cortical injuries21, weight drop models23, as well as blast models29.We also recently identified that direct blast biomechanical loading causes disruption of brain vascular integrity (BBB) in multiple brain regions within minutes of loading and that this BBB disruption further increased in four hours following blast before returning to normal at 24 hours post-injury32. However, no studies have been performed on the interaction between oxidative stress and BBB permeability changes in blast TBI. Since our recent studies indicate blast injury results in disruption of BBB and activation of factors conducive to oxidative stress18, we sought to examine the interaction of these two well-known pathogenic factors in BINT. We hypothesize that while BBB is acutely disrupted immediately following blast injury through biomechanical means, and enhanced BBB disruption in later stages (zero to four hours) is due, at least in part, to the activation of NADPH oxidase (NOX) and associated increase in oxidative stress. This study therefore employed apocynin, which is known to block the assembly of NOX subunits and prevent activation of NOX, and reduce superoxide production to ascertain if NOX plays a role in the manifestation of BBB breakdown. Apocynin was used to examine the role of NOX on BBB disruption and not as a therapeutic agent though such a treatment possibility exists; however, that is not the purpose of the present study.Oxidative stress is a well-known factor for BBB disruption in blunt TBI as well as other neurological disorders including Alzheimer’s and Parkinson’s diseasesAdult (10-week old), male Sprague-Dawley rats (Charles River Laboratories) weighing between 350 ± 50 g were used in accordance with protocols approved by Rutgers University Institutional Animal Care and Use Committee (IACUC). Animals were housed at 22 °C with free access to food and water in a 12 hour light-dark cycle. Animals were divided among sham, blast, and treatment groups, all to be sacrificed 15 min or four hours post-blast . All methods used throughout the study were performed in accordance with protocols, guidelines, and regulations approved by Rutgers University IACUC.35. A total of 54 rats were used in the study for different experimental methods with at least 4–5 animals in each experimental group  based on a priori power analysis. Prior to blast exposure, all animals  were anesthetized with 5% isoflurane, released in a chamber containing 95% air and 5% CO2, until rats were unresponsive to noxious stimulation. Apocynin  was administered intraperitoneally 30 min prior to blast exposure. Sham animals were kept near the shock tube to expose them to noise but not any shock wave. A separate group of animals with sham + apocynin treatment was not included in the present study, since several investigators have reported apocynin (5 mg/kg wt) administration to control/sham animals did not exert any toxicity nor affected physiological and behavioral parameters assessed in parallel to animals subjected to traumatic brain injury36, splanchnic artery occlusion37, liver injury38 and experimental epilepsy39. Following blast injury, animals were closely monitored for any signs of apnea, loss of motor coordination and neurological severity score (NSS) was evaluated five minutes post exposure. None of the animals included in this study displayed NSS scores that differed from sham animals. Animals were exposed to a single blast of 180 kPa peak overpressure. Prior to transcardial perfusion, animals were anesthetized with a mixture of ketamine and xylazine (1:10 ratio), perfused either 15 min or four hours post-blast. For immunofluorescence and extravasation studies, animals were first perfused with PBS followed by 4% paraformaldehyde, whereas for ELISA studies, animals were perfused with only PBS and brain tissue was collected for preparation of homogenates. Prior to initiation of perfusion, CSF was extracted from the cisterna magna using a 25 gauge butterfly needle from scalp vein set  and was freely allowed to collect into catheter. Blood (about 4 ml) was collected by cardiac puncture (left ventricle) and allowed to settle in vacutainer tubes (BD Bioscience) containing 3.2% sodium citrate for 10 minutes. Plasma was separated from blood by centrifuging at 2000 g. Extravasation of Evans blue was assessed by method recently published by us18. All the blast exposures were continuously monitored with high-speed video recording (5000 fps) as described previously18 as a quality control measure to ensure that potential secondary/tertiary injuries are absent.Rats were exposed to a single shock wave at the New Jersey Institute of Technology  in the shock tube described in previous publications17. Fixed tissues were incubated overnight at 4 °C with respective primary antibodies to NOX1  and RECA-1 . Double immunofluorescence was performed using Alexa Fluor 488 for NOX1 and Alexa Fluor 594 for RECA-1. Quantification of the fluorescence intensities was performed following the method recently published by us18. Briefly, slides containing mounted coronal sections of frontal cortex were imaged at 20x magnification using a Leica Aperio Versa 200 digital pathology scanner. Control sections were used as reference for adjusting the exposure times and gray scale balance for optimal image quality; once set, these parameters were fixed and used for image acquisition of the remainder of both control and experimental groups. To remove variability that might arise, all sections to be compared were scanned on the same day using the same established scanning protocol. For the immunostained sections, three channels were collected for each coronal section. Blue: 405 nm (DAPI), red: 594 nm (NOX1), and green: 488 nm (RECA-1). For the extravasation studies, two channels were collected. Red: 594 nm (Evans blue) and green: 488 nm (NaFl). Values were expressed as average fluorescence intensity/unit stained area. For the EB extravasation and dihydroethidium (DHE) study we expressed results as percentage change from control due to an absence of a negative control that can be used to remove autofluorescence from the analysis. A maximum intensity threshold was also set to remove any oversaturation from excess fluorescent dye. The algorithm outputs the area of positive staining for each brain region, the average intensity of each channel, and intensity profile of each antigen.In order to evaluate the increase in NOX1 expression in vascular endothelial cells following blast injury, double immunofluorescence studies were conducted for NOX1 and RECA-1  in the frontal cortex. Briefly, four hours post-injury, both sham and injured animals were transcardially perfused with PBS and brains were fixed with 4% paraformaldehyde (PFA). Tissue section and staining procedure was done in similar manner described in our previous publication2.−) levels in different brain regions were measured using DHE following the method of Kim et al.39, and was previously described17. Briefly, animals were injected with 5 mg/kg DHE  intraperitoneally 30 minutes prior to blast and 4 hours after exposure; animals were transcardially perfused first with PBS followed by 4% PFA . Brains were extracted and 50 µm sections were cut using Leica VT 1000S vibratome and mounted. DHE immunofluorescence in each region was visualized and quantified using similar protocol as described above.Superoxide  using sonicator . Samples were then centrifuged at 14,000 g at 4 °C. The protein concentration in the samples was estimated by bicinochoninic acid (BCA) method . Subsequently, samples were diluted in PBS and loaded onto specific ELISA plates for MMP3, MMP9, occludin, and claudin-5 and GFAP . Plates were read in microplate reader  at wavelength of 450 nm. Steps of ELISA were conducted in accordance with manufacturer instructions and samples plotted against a standard curve made up of eight samples using SoftMax Pro 6.5 software.Slides containing EB extravasated tissue sections were digitized (10x magnification) using Leica Aperio Versa 200 digital pathology grade slide scanner. Fluroescent intensities were quantified after excitation at 594 nm, 125 ms exposure, using AreaQuant, software specifically designed for this imaging application (Leica Biosystems) using similar protocol described in the previous section.Data are presented as mean ± standard error of the mean. Statistical significance was determined using one-way analysis of variance (ANOVA) to compare mean fluorescence intensities between control, blast, and blast + treatment groups with a post-hoc analysis using Tukey pairwise test to determine differences between individual groups. Normalcy and population variance homogeneity were assessed with Shapiro-Wilk and Levene’s tests respectively. Differences between means were assessed and probability levels of p < 0.05 were considered statistically significant. Minitab 17 Statistical Software was used for all analyses and Origin 2017 was used for generation of bar plots. Bar plots presented in semi-log scale are done so as to capture the intensities when the differences between groups are several orders of magnitude. Fluorescent images were taken using Aperio Versa software and analysis and export done via ImageScope software (LEICA Corp.).17. In the present study, we examined the levels of NOX1 in the vascular endothelial cells in the frontal cortex. The double immunofluorescence for NOX1 and RECA-1  showed a significant increase in amount of co-localization following blast  across the cerebral hemisphere and cerebellumast Fig. , whereas41. After demonstrating the increase of NOX1 concentration in neurovasculature in the frontal cortex, we sought to determine if this increase leads to an increase of superoxide production. In-vivo levels of superoxide were measured using DHE and we observed a clear increase in superoxide produced in the frontal cortex, which not only correlated well with our earlier observation17 but is also consistent with the upregulation of NOX1 in the present study  of EB in the brain parenchyma compared to the control in the frontal cortex  and such extravasation was completely attenuated by treatment with apocynin (p < 0.01).The extent of extravasation was evaluated in the frontal cortex at both fifteen minutes and four hours post-blast injury Fig. . Our groConcentration of GFAP in the blood plasma was determined by ELISA Fig. . Statist42. Four hours following blast, a statistically significant increase over control was observed in the injured group . Comparison between blast and treatment group at this point also revealed statistically significant difference .In order to determine the ratio of albumin in the CSF and blood plasma, ELISA was conducted on both samples Fig. . While aper se are responsible for MMP activation.In order to examine the role of matrix metalloproteinases in the breakdown of the BBB, we conducted an ELISA for MMPs 3 & 9 for control, blast, and blast + apocynin groups Fig. . MMP3 inThis work aims to investigate the role of NOX-mediated oxidative stress as a mechanism for increased BBB permeability in blast-induced traumatic brain injury, immediately following blast and further increase between blast and 4 hours post-blast. Using apocynin, which inhibits the assembly of various NOX subunits rendering the enzyme inactive, we found that apocynin significantly attenuated the extravasation, preventing the reduction in tight junction proteins as well as reduced the levels of MMPs, between the time points of blast and 4 hours post-exposure. These data together strongly suggest that NOX-mediated oxidative stress significantly contributes to BBB permeability.It should however be noted that in the current study, we employed apocynin as a proof of concept to examine whether oxidative stress contributes to BBB disruption after blast exposure, rather than a therapeutic paradigm. Additionally, we sought to examine whether apocynin has any impact on BBB permeability changes that we earlier observed immediately (15 min) following blast exposure. Accordingly, apocynin was administered 30 min prior to blast exposure. Noteworthy that apocynin did not show any beneficial effects during the acute phase (15 min) of the injury  suggesting that oxidative stress and associated effects on BBB permeability are predominantly secondary biochemical factors. As shown in Fig. 44. A dose of 5 mg/kg employed in the current study also has been used by several investigators to examine the role of NOX-mediated responses in various experimental conditions including stroke45, and blunt model of TBI51. A complete restoration of blast-induced BBB permeability at 4-hours’ time point by apocynin strengthens our hypothesis that though initial BBB increase is minimal, sustained BBB disruption is primarily contributed by oxidative stress. However, few studies have reported anti-inflammatory properties of apocynin. Thus, Anter et al., 52 reported that topical application of apocynin showed anti-inflammatory properties as indicated by reduction in paw swelling in rats. Likewise, another study showed that apocynin significantly reduced the levels of TNF-alpha in animals subjected lateral fluid percussion model of TBI54. Additionally, diapocynin (a dimer of apocynin) significantly reduced microglial activation in MPTP model of Parkinson disease55. Further, apocynin derivative dihydrocoumarin also exerted anti-inflammatory properties in vitro in erythrocytes subjected to oxidative injury56. Similarly, apocynin has shown anti-inflammatory effects in mouse model of stroke57 by reducing the NLRP3 inflammasome. Taken together, these reports also attribute anti-inflammatory actions of apocynin. Noteworthy that we identified robust activation of microglia as early as 4 h after moderate blast injury (180 kPa) (unpublished observation) and that such activation was more pronounced near the site of vascular rupture (increased blood brain barrier permeability) we observed earlier18 as well as in the current study. Based on these reports, it is also likely that protective effect of apocynin in the present study may in part mediated by suppressing the neuroinflammation following blast injury.The pharmacokinetics and the bioavailability of apocynin have been well established. Apocynin appears to be a stable compound once administered into the system with a bioavailability of >8%; half-life of the compound has been determined using LC-MS to be 48 h in the blood circulation58.In the present study, we observed a robust (10-fold) increase in NOX1 expression four hours following blast injury in endothelial cells in the frontal cortex as demonstrated by double immunofluorescence staining of NOX1 with RECA-1, an endothelial cell marker. Our recent observations along with other investigations also report increased activation of NOX1 between four hours and a few days in animal models of TBI17 may exert a paracrine effect on vascular endothelial cells and cause BBB disruption. Alternatively, vascular endothelial cells per se could produce superoxide since our current study display a robust increase in NOX1 levels following blast in these cells, and such superoxide production within the endothelial cells would have caused BBB disruption in an autocrine mechanism. We further established that the superoxide production regardless of its cellular origin, is indeed mediated by NOX1 expression as apocynin inhibits assembly of NOX subunits and renders NOX functionally inactive, completely reducing the production of superoxide consistent with our previous work17.In order to confirm that increased NOX1 expression activates downstream pathways, we examined the superoxide production by prior administration of dihydroethidium (DHE). There was a robust increase in superoxide production in animals subjected to blast injury, which parallels the increased NOX1 expression on vascular endothelial cells. While precise cellular origin of superoxide in the present study is not identified, it is highly likely that the superoxide produced by neural cells including neurons, astrocytes and microglia as a result of increased activation of NOX18. The BBB permeability changes were represented not only by the extravasation of EB but also by the reduced levels of tight junction proteins as well as cross translocation of substances between brain and blood and vice versa. A significant reduction in both occludin and claudin-5, four hours after blast injury is consistent with extravasation of tracers since loss of tight junction protein integrity is well known to allow leakage of tracers and other blood borne macromolecules into brain parenchyma. Accordingly, increased presence of astrocytic protein, GFAP, in plasma as well as increased CSF-blood ratio of albumin observed in the present study confirm the reciprocal translocation of brain and blood-borne macromolecules as a result of BBB disruption. Noteworthy that a pretreatment with apocynin significantly attenuated the extravasation of tracers, prevented the loss of TJPs and brought back the CSF-blood albumin ratio to that of controls, as well as attenuated increased presence of GFAP in plasma. Together these data again strongly suggest that NOX-mediated oxidative stress contributes to heightened BBB disruption four hours following blast injury.It is interesting to note that our present study observed a close correlation between increased NOX1 expression, its associated increase in superoxide production, and the heightened disruption of the BBB 4 hours after blast injury63. MMPs are endopeptidases that degrade the extracellular matrix (ECM) in various cell types. While the activity of MMPs are tightly controlled in normal, “uninjured” conditions by endogenous MMP inhibitors, uncontrolled activation has been observed due to oxidative stress67. This in-turn degrades the brain endothelium and substantially increases the permeability of the BBB. The role of MMPs in BBB degradation has been well-investigated63. The present study showed that the levels of both MMP3 and MMP9 significantly increased over controls four hours after blast exposure. Rats pretreated with apocynin displayed significant reduction in the levels of MMPs as compared to animals subjected to blast injury again indicates that NOX-mediated oxidative stress contributes to increase in MMPs, likely leading to degradation of tight junction proteins ultimately causing BBB breakdown.Several groups have attempted to establish a connection between oxidative stress and BBB breakdown by implicating matrix metalloproteinasesWhile it is strongly suggested that oxidative stress is a secondary event that contributes to heightened BBB disruption in the later stages after blast injury, we further extended studies to address whether BBB disruption immediately after blast injury is caused by a direct mechanical loading or in part mediated by any presence of oxidative stress. Accordingly, the ability of apocynin to reduce BBB breakdown only at 4 h post injury but not in the acute phase (prior to 4 h post-injury) in conjunction with our observation of robust increase in NOX1 four hours after blast strongly suggest that oxidative stress appears to be a secondary event occurring following blast injury. Further, our study also strongly suggests that the disruption of BBB immediately after blast is attributable to a direct mechanical loading created by shockwave. Figure It is notable that although we did not observe any changes in the neurological severity scores (NSS) following blast, our recent studies display that either single blast injury at moderate BOP (180 kPa) or repeated (5 successive times) low-level blast (70 kPa) resulted in significant neurobehavioral deficits as identified by anxiety/depression like symptoms, loss of short-term memory as well as sleep disturbances (unpublished observations). Together these observations strongly suggest that (1). Blast induces long-term neurobehavioral deficits and (2). There will be a higher propensity that repeated blast exposures at lower BOP will cumulatively impact on the neurobehavioral changes, in both conditions without any overt NNS NSS change.In summary, our studies demonstrate that moderate blast injury causes disruption of BBB by loss of TJPs and results in leakage of macromolecules across brain-to-blood as well as increase in NOX expression on vascular endothelial cells and associated increased production of superoxide 4 h after blast injury. BBB breakdown occurs immediately due to direct stress-cell interactions, and further increase in BBB between injury and 4 hours can be attributed to oxidative stress. These events corroborate with the heightened activation of MMP-3 and MMP-9. Pretreatment with NOX inhibitor apocynin, BBB permeability is significantly reduced and the increase in MMPs levels were prevented. This study therefore indicates a strong relationship between NOX-mediated oxidative stress and blood brain barrier breakdown, which may lead to further investigation delineating relationships between other injury mechanisms, a prerequisite for better therapeutic interventions.


Although electroanatomic mapping techniques have been previously applied to openchest epicardial ablation procedures, such efforts have often been limited bysignificant geometric distortions introduced by the need to use nonstandardmapping patch placements and by intrathoracic conductance changes introduced byhaving the pericardial space exposed. In this article, we present a case of apatient with recurrent hemodynamically unstable ventricular tachycardia whounderwent a successful open chest epicardial ablation procedure withelectroanatomic mapping in which geometric distortions were minimized byjudicious placement of mapping patches and the use of a saline bath within thepericardial space. Modern EAM systems allow clinicians to visualize complexanatomical relationships and catheter positions, and to track delivered ablationlesions while limiting a patient’s exposure to fluoroscopy and harmfulradiation.We herein present a case report of a patient with recurrent ventricular tachycardia(VT) following coronary artery bypass grafting and a mitral valve replacement (MVR).The patient underwent an open chest epicardial catheter ablation in which an EAMsystem was successfully used using a novel irrigated saline bath configuration tofacilitate mapping and lesion tracking in a situation where direct visualization ofthe myocardial VT substrate was not possible due to its posterobasal location andthe patient’s hemodynamic instability during attempts at retracting the heart.2)-branchstenosis, a 90% proximal-first obtuse marginal (OM1) stenosis, an 80%mid-right coronary artery (RCA) stenosis, and a 95% distal-RCA stenosis; his leftventricular ejection fraction was 35% with posterobasal akinesis seen onventriculography. A radionuclide uptake study showed viability of the inferolateralwall and anteroapical septum; the base of the posterior wall was not viable. Atransesophageal echocardiogram confirmed severe eccentric mitral regurgitation. Heelectively underwent 4-vessel coronary artery bypass grafting consisting of a leftinternal mammary artery (LIMA) → LAD, saphenous vein graft (SVG) → D2,SVG → OM1, and SVG → RCA; he also underwent a MVR with a #29 bovinepericardial valve.A 72-year-old male with new-onset heart failure and severe mitral regurgitationunderwent coronary angiography, which demonstrated a 60% proximal-left anteriordescending (LAD) stenosis, an 80% ostial-second diagonal  inserted at the bedside. Over the next several days, hecontinued having multiple episodes of sustained and hemodynamically unstable VT (upto 5 per 24 hours) despite ongoing antiarrhythmic drug therapy ; he also had a flail chest from multiple rib and sternal fracturessustained during his many episodes of cardiopulmonary resuscitation.On the 12th postoperative day, a decision was made to proceed with a combined sternalrepair and open chest epicardial catheter ablation in order to stabilize thepatient’s condition.1-V6 were placed posteriorly to allowfor surgical access to the anterior chest wall. In order to accommodate themedian sternotomy, the 4 torso mapping patches were rotated 45° along thepatient’s craniocaudal axis from their usual positions. This allowed formaintenance of the relative orthogonal relationship of each patch pair  with respect to oneanother; the neck and thigh patches remained in their usual locations (The patient was brought to the electrophysiology laboratory from thecardiothoracic intensive care unit (CT-ICU), intubated, and on mechanicalventilatory support. Electrocardiography limb leads were placed in the usualposition, but leads Vocations . The patA deflectable decapolar catheter  andquadpolar catheter  were positioned within thecoronary sinus and at the right ventricular apex, respectively. EAM wasperformed utilizing EnSite Velocity , whichemploys a low-level 8.128 kHz current through the orthogonally located skinpatches. The recorded voltage and impedance at each catheter’s electrodesgenerated from this current allows their distance from each skin patch, andultimately, their location in space, to be triangulated with the help of areference electrode.In order to provide a stable conducting medium around the exposed myocardium, thepericardial space was continuously irrigated with normal saline prewarmed to37°C and delivered via sterile vinyl tubing. A low level of continuous suctionwas applied via an outflow line to maintain a stable fluid level within the openpericardial cavity. Validation was performed only after the chest had beenopened to its fullest excursion and the pericardial space had been filled withsaline.A Safire TX 8 mm tip catheter  was placedwithin the open pericardial space, below the fluid level; a 3D-electroanatomicshell of the left and right ventricles were created using EnSite Velocity onlyusing geometry collected from the distal bi-pole of the catheter. The geometriesof the right and left ventricles were divided based on the observed courses ofthe LAD and right posterior descending coronary arteries as determined by directvisual inspection. Voltage mapping was performed using a scare range of 1.5 mVto 0.3 mV.All ablations were carried out utilizing power settings of 70 to 90 Watts and atemperature cutoff of 65°C to 80°C. The efficacy of delivered lesions wasmonitored by the following: (1) loss of local electrograms and (2) loss ofpacing capture. Lesion locations were tracked utilizing EnSite Velocity.After reopening of the sternum, inspection demonstrated obvious dehiscence andfractures between the manubrium, sternal body, and xiphoid process, as well asthe presence of a large hematoma. All wires were removed and the clotevacuated.Manual inspection of the heart demonstrated a large well-circumscribed area ofinfarct along the posterobasal wall; however, attempts to maintain exposure ofthe posterior wall via manual retraction of the heart apex were precluded bysevere hypotension necessitating intravenous vasopressor support, presumably dueto significantly impaired left ventricular filling dynamics with apical tractionin the setting of the patient’s recent MVR.Sustained VT at a cycle length of 380 ms was readily induced via overdriveventricular pacing at 280 ms from the right ventricular apex . The cycGiven the patient’s poor hemodynamic tolerance of his VT, a decision was made topursue pace mapping along the edge of the identified posterior wall scar forwhat was presumed to be re-entrant VT. As the patient did not tolerateretraction of the heart apex for visualization of the posterior wall, EAM wasperformed with the heart exposed but in situ utilizing EnSite Velocity.Three-dimensional geometries of the left and right ventricles were created asdescrTotal radiofrequency application time:approximately 15 minutes.Pace mapping along the edge of the posterobasal infarct zone identified aputative breakout site for the patient’s ventricular tachycardia: the QRSmorphology during pacing was a match in all 12 leads . A serieOn subsequent induction attempts, another faster VT with a distinct QRSmorphology was induced, which rapidly degenerated into ventricular fibrillation.Cardioversion attempts at 10 J and 20 J (delivered via intracardiac paddles)were unsuccessful; a third cardioversion attempt at 30 J was successful inrestoring sinus rhythm. Once again, several minutes of direct cardiac massagewas performed until return of spontaneous circulation. No further attempts atinduction were performed.The patient underwent placement of an epicardial lead prior to sternotomy repairand chest closure. He was transferred back to the CT-ICU with an IABP in place.He had a protracted recovery complicated by renal failure necessitating dialysisand by ventilator-associated pneumonia necessitating prolonged ventilatorsupport; however, he had no further episodes of VT during his hospital stay, andafter being weaned from ventilator support he ultimately underwent placement ofa Unify 3231-40Q CRT-D biventricular pacemaker/implantablecardioverter-defibrillator  on postoperativeday 68. He was ultimately discharged on postoperative day 84. At 1-, 3-, 6-, and12-month follow-up, the patient had experienced no recurrent episodes ofventricular tachycardia or ventricular fibrillation. Follow-up echocardiographydemonstrated a modest improvement in his left ventricular ejection fraction to40%.59The majority of cardiac ablations for treatment of ventricular tachycardia areperformed via endovascular approaches, although a subset of patients will requireepicardial ablation for successful treatment. When the latter is required,subxiphoid access to the pericardial space via percutaneous techniques or minimallyinvasive surgical access has been demonstrated to be safe and effective, andgenerally does not pose any special challenges from the standpoint of being able toemploy EAM to facilitate mapping and ablation.11 Although EAM was attempted, they noted that their efforts resulted in“distorted maps that were not reliable,” and the operators relied heavily on priorendocardial maps that had been created in 4 out of the 5 patients. The decision toplace the anterior and posterior mapping patches near the axilla to allow forcomplete exposure of the thoracic cavity may have contributed to these distortions.11For a subset of patients such as the one presented herein, a hybrid open chestprocedure via a median sternotomy may be indicated. Patel et al published thelargest such series of patients to date, in which 5 patients with end-stage heartfailure underwent an open chest electrophysiology study with epicardial mappingduring the period of LVAD placement.10 Although endocardial mapping was not performed during the course of thisstudy, the otherwise standard system setup employed should have allowed forconcurrent endocardial geometries and maps to be generated if needed.In this study, the creation of geometries with a high degree of anatomic fidelity andminimal geometric distortion was created by the following: (1) applying all 4 torsomapping patches such that they were rotated 45° along the patient’s craniocaudalaxis from their usual positions, allowing for maintenance of the relative orthogonalrelationship of the patches to each other; (2) only using geometry collected fromthe distal bi-pole of the mapping catheter; and (3) the creation of a stableconductance medium by bathing the exposed pericardial space in saline. A secondarybenefit of the saline bath was the absence of difficulties associated with highimpedances during ablation as reported in other studies.The techniques presented herein will hopefully add to the armament of tools thatelectrophysiologists have at their disposal when tackling otherwise difficult VTablation cases.


Thus, the mechanism by which cystamine inhibits transglutaminase activity in vivo could be due to either cystamine or cysteamine, which depends on the local redox environment. Cystamine inactivates transglutaminases by promoting the oxidation of two vicinal cysteine residues on the enzyme to an allosteric disulphide, whereas cysteamine acts as a competitive inhibitor for transamidation reactions catalyzed by this enzyme. The latter mechanism is likely to result in the formation of a unique biomarker, N-(γ-glutamyl)cysteamine that could serve to indicate how cyst(e)amine acts to inhibit transglutaminases inside cells and the body.Cystamine is commonly used as a transglutaminase inhibitor. This disulphide undergoes reduction  This disulphide is also reduced to cysteamine within the body. Cystamine and cysteamine both inhibit transglutaminases but by different mechanisms. Therefore, the purpose of this discussion is to highlight the redox behavior of cystamine and cysteamine 2O depending on the transglutaminase isozyme or conditions. Thus, subject to the nucleophile, transglutaminases catalyze transamidation, esterification, or deamidation of glutaminyl residues. Transamidation involving the ε amine of lysyl residues is the reaction most often catalyzed by transglutaminases and results in the formation of εN-(γ-glutamyl)lysine isodipeptide linkages between polypeptide chains (εN-(γ-glutamyl)lysine cross-linked proteins . The inisorders . The preisorders , as wellisorders . These t277 undergo a thiol-disulphide interchange to produce a mixed disulphide that prevents catalysis cysteamine (N-(γ-glutamyl)cysteamine by transglutaminases could account for two puzzling observations pertaining to the metabolism of exogenously supplied cysteamine. The first of these observations is that a significant portion of the administered cysteamine is unaccounted for following analysis of established routes of metabolism. Cysteamine generated endogenously by the catabolism of pantetheine is oxidized to hypotaurine and then taurine . I. IN-, by micromolar amounts of cysteamine. At these concentrations, thiols (RSH) such as cysteamine reduce transition metals , while being oxidized to the corresponding disulphide (RSSR):A novel hypothesis for the metabolism of cysteamine is that it is covalently attached to proteins by intracellular transglutaminases. This hypothesis is supported by the observation that a significant portion of radiolabeled cysteamine administered to animals or cells is covalently bound to proteins, but not by disulphide bonds ,59. ThisO2) to superoxide , which is a mild oxidant at physiological pH values (7.2–7.4):Dismutation of superoxide yields hydrogen peroxide cysteamine. Isopeptide linkages are resistant to proteolysis and consequently transglutaminase-made N-(γ-glutamyl)amines are excised as free N-(γ-glutamyl)amines during proteolysis of proteins bearing these species [N-(γ-glutamyl)amines are present in various body fluids and reflect the levels of active transglutaminases in tissues [S-(2-aminoethyl)l-cysteine ketimine decarboxylated dimer, as well as protein-bound and free N-(γ-glutamyl)cysteamine should provide a comprehensive accounting of the metabolism of exogenous cyst(e)amine, in addition to indicating the mechanism by which cysteamine inhibits intracellular transglutaminases.The above conjecture could be readily tested by investigating the plasma of cysteamine-treated animals or medium of cells in culture treated with cysteamine for the presence of free  species . Free N- tissues ,66,67. I370 or Cys371 form a disulphide in a manner that involves a third cysteinyl residue, Cys230, and ERp57 [370–Cys371 disulphide linkage by thioredoxin activates extracellular transglutaminase 2 [370 and Cys371 cysteamine), cysteamine may be of use in the treatment of celiac disease and other diseases involving transglutaminases.Transglutaminases are fully activated by the binding of three calcium ions per enzyme and reducing conditions that maintain the active site cysteine in a fully reduced state ,2,47. Thminase 2 . The actd Cys371 C. As nottrations . Given ttrations  and the in situ activities of these transglutaminase pools are therefore of interest as possible therapeutics. The evidence presented here indicates that cystamine inhibits extracellular transglutaminases, while its reduced congener – cysteamine – inhibits intracellular transglutaminases. This distinction is important for the design of other transglutaminase inhibitors based on the mechanisms by which cysteamine or cystamine inhibit these enzymes cysteamine) may provide a means of determining the mechanism by which intracellular transglutaminases are inhibited following the administration of cystamine or cysteamine.The activities of intracellular and extracellular transglutaminases contribute to a number of important pathologies. Agents that safely inhibit the ulphiram ). It may


Characterization of regenerated bone reveals osteogenesis of organized, vascularized bone with histologic and mechanical characteristics comparable to native bone. Radiographic and histologic analyses were consistent with patent craniofacial sutures. Lastly, through application of 3D morphometric facial surface analysis, our results support that DIPY-3DPBC scaffolds do not cause premature closure of sutures and preserve normal craniofacial growth. Based on this novel evaluation model, this DIPY-3DPBC scaffold strategy is a promising candidate as a safe, efficacious pediatric bone tissue engineering strategy.This study investigates a comprehensive model of bone regeneration capacity of dypiridamole-loaded 3D-printed bioceramic (DIPY-3DPBC) scaffolds composed of 100% beta-tricalcium phosphate (β –TCP) in an immature rabbit model through the time of facial maturity. The efficacy of this construct was compared to autologous bone graft, the clinical standard of care in pediatric craniofacial reconstruction, with attention paid to volume of regenerated bone by 3D reconstruction, histologic and mechanical properties of regenerated bone, and long-term safety regarding potential craniofacial growth restriction. Additionally, long-term degradation of scaffold constructs was evaluated. At 24 weeks  Cleft palate and craniosynostosis, or premature fusion of the cranial sutures, for instance, are two of the most common congenital causes of craniofacial bone defects/deformities and account for 1 in 600 and 1 in 2000 live births respectively. Affected patients commonly require bony reconstruction at the time of active facial growth and surgical interventions are designed to restore the craniofacial skeleton while limiting impairment to face and skull development. Furthermore, these deformities and required surgical interventions are a source of psychosocial and financial burden not only for patients but families and healthcare systems due to their prevalence and treatment complexity associated with this population4.Defects of the pediatric craniofacial skeleton can occur due to congenital, neoplastic, infective, traumatic, or iatrogenic causes7. Key limitations of autologous bone graft include donor site morbidity, bone resorption, limited quantity and shape of bone stock, as well as difficulty in shaping bone grafts into complex 3-dimensional forms such as those often needed to reconstruct craniofacial defects11. While alternatives to autologous tissues such as allograft or foreign-body implants can ameliorate some of these challenges, they pose their own restrictions including inflammation, infection, extrusion, fragmentations and inability to grow or entirely integrate with the child’s growing skeleton7.Autologous bone grafting remains the standard of care for reconstruction of the pediatric craniofacial skeleton15, an optimal approach has yet to be demonstrated for use in the pediatric population17. Compared with adult patients, the thickness of pediatric bone is thinner18, and there is reduction in the osteoinductive potential of the dura after 12 months19 which impairs native regeneration of bony defects. Moreover, long-term growth and development of the craniofacial skeleton must be carefully considered since even with gold-standard treatment, patients may experience growth restriction and require multiple revision surgeries17. An ideal pediatric bone replacement or regenerative therapeutic agent would be biocompatible, patient specific, bioresorbable, lead to the regeneration of mature, vascularized bone and ultimately restore form and function of the skeleton without impeding facial development18. Due to these unique considerations, efficacious translation of bone tissue engineering strategies in a pediatric context have been limited and investigation into pediatric skeletal tissue engineering strategies remains in its infancy21. We seek to evaluate a novel tissue regeneration strategy that may begin to fulfill these criteria for successful translation to a pediatric craniofacial context by investigating both a cleft and calvarial surgical defect model.Due to the limitations of current reconstructive options, there exists growing interest in bone tissue engineering and regeneration strategies for pediatric craniofacial reconstruction, including the application of bioactive scaffolds, osteogenic pharmacologic agents, and stem cell therapies. Despite relative advances in adult craniofacial bone tissue engineering22. The scaffolds described in this study comprised of 100% beta-tricalcium phosphate (β -TCP) to provide structure and rigidity to the defect, promote osteocyte migration and osteoconduction within scaffold interstices, and demonstrate better biodegradability compared to other commonly used biomaterials such as hydroxyappetite (HA)12, thus leaving uninterrupted regenerated bone.Our approach combines customizable 3D-printed bioceramic (3DPBC) scaffolds and dipyridamole, a novel osteoinductive agent. By utilizing computer aided design/manufacturing (CAD/CAM), scaffolds can be precisely designed and manufactured to the specifications of the patient’s craniofacial defectin vitro23 and in vivo24. Furthermore, dipyridamole has a long history of safe use in pediatric patients, with decades of safe systemic administration exceeding the systemic exposure resulting from local delivery for osteogenesis28.Dipyridamole (DIPY), an indirect adenosine 2A receptor (A2AR) agonist, has been utilized to augment the osteoinductive potential of these bioceramic scaffolds. DIPY blocks cellular adenosine uptake by the purine transporter ENT1, thereby increasing extracellular adenosine. A2AR activation has been shown to stimulate osteoblast proliferation and differentiation in vivo across critically-sized defects compared to unfilled controls, which do not regenerate both in adult and pediatric translational animal models31. Dipyridamole has been shown to significantly augment the osteogenic capacity of 3DPBC scaffolds compared to scaffolds without dipyridamole33. Importantly, optimization studies have identified a dose of 1,000 µM to maximally promote osteogenic regeneration when used in combination with 3DPBC scaffolds in animal models without observed negative side-effects including premature suture fusion or exuberant bone formation in the short-term28.Our group has previously demonstrated that 3DPBC scaffolds significantly generate vascularized bone 28. A foremost concern in clinical pediatric craniofacial reconstruction is the effects of interventions on development of the facial skeleton at craniofacial maturity. Longitudinal demonstration of scaffold osteogenesis, favorable degradation over time, and safety from immaturity to skeletal maturity are necessary for successful translation to and adoption in a pediatric craniofacial context. Finally, while scaffold superiority over null interventions has been demonstrated in previous reports31, direct comparison of scaffold osteogenesis compared to standard of care interventions such as autogenous bone graft has yet to be performed.While these pilot studies have demonstrated safety of this approach in the immature craniofacial skeleton, they have been limited to short term analyses of scaffolds compared to unfilled defectsThis study employs two different models of pediatric craniofacial defects and reports a 6-month analysis of the effect of DIPY-3DPBC scaffolds on osteogenic regeneration compared to autogenous bone graft and measures the effects of this bone tissue engineering construct on the development of the growing craniofacial skeleton throughout and beyond the period of skeletal maturity.28  and comparable to volume of bone volume fraction of autogenous bone graft and un-operated calvarium  this was significantly increased compared to bone graft and un-operated bone . In the alveolus, trabecular thickness was lower in regenerated bone compared to bone graft (p < 0.001) and native bone (p = 0.01). Trabecular spacing was also lower in regenerated bone compared to bone graft (p = 0.006) and native bone (p < 0.001). These findings may represent bone regeneration filling the dimensions of the implanted scaffold and increased anabolic activity in response to the higher-load conditions of the alveolus compared to the calvariumNon-decalcified histologic sections of defects stained with Van Giesen’s red revealed bone spanning the entirety of calvarial and alveolar defects Fig. . Bone waCharacterization of bone from corresponding un-operated, contralateral calvarium and alveolus revealed differences in cellular organization of bone. In the calvarium, bone demonstrated primarily trabecular organization characteristic of cancellous bone. The periosteal and dural aspects of the calvarial shelf demonstrated lamellar orientation parallel to external calvarial curvature. Between these plates, spaces with adipose tissue characteristic of diploic bone marrow cavities were observed Fig. . In the Defects repaired with DIPY-3DPBC scaffolds revealed both lamellar organization and formation of trabeculae consistent with cancellous bone maturation as well as maturation of compact bone, with formation of osteons with Haversian canals containing micro vessels stained with Stevene’s blue Fig. . In the Across samples, bone in all conditioned demonstrated characteristics of mature, organized vascular and cellular components of bone Fig. . Histolop = 0.20; Fig. p = 0.50; Table Osteocyte density analysis revealed mean calvarial scaffold osteocyte was statistically equivalent across groups  and hardness (H), acquired by means of nanoindentation displayed an Er of 9.5 ± 1.7 GPa and H of 0.36 ± 0.05 for scaffold-regenerated bone, which yielded to be statistically homogenous with native bone analyzed outside of scaffold interstices  result in opportunities and inspire investigation of alternative strategies for bone tissue engineering. In patients affected by cleft lip and palate, reconstruction of an alveolar (maxillary) cleft can ensure proper tooth eruption, maxillary arch stability, and appropriate midface development. Unfortunately, autogenous bone graft in reconstruction of both the alveolus and calvarium is limited by survival, donor site morbidity and added healthcare cost associated with extended operative time and increased length of stay for pain control associated with bone graft harvest12, which may obviate the need for reoperation in a pediatric population, and 3) use of dipyridamole is safe and preserves normal craniofacial growth in this high-risk population.We propose that DIPY-3DPBC scaffolds offer several advantages to clinical standard of care and published bone tissue engineering strategies alike: 1) custom design via CAD/CAM of β –TCP allows for personalized reconstruction of large defects compared to bone graft or porous biomaterials and also specification of meso and nanostructure to optimize bone regeneration, 2) composition of 100% β –TCP allows for improved degradation of inorganic scaffold component compared to hydroxyapatite (HA) scaffoldsin vivo investigation of DIPY-3DPBC scaffolds to date, and our results bring to bear the above advantages of dipyridamole 3DPBC scaffolds. Our previously published investigations demonstrate that DIPY-3DPBC scaffolds regenerate defects that are critically-sized, or otherwise have been demonstrated to prevent spontaneous bony healing27. Here, we add to prior studies to demonstrate that dipyridamole-coated 3DPBC tissue engineering construct can generate vascularized bone with comparable volumetric density as autogenous bone graft, the clinical standard of care, as well as native bone. We used radiographic assessment as our primary endpoint, as this would be the clinical means of evaluating this technology, and with it we were able to determine that scaffold degradation kinetics demonstrated progressive absorption of the scaffold and replacement with regenerated bone. We then applied human bone tissue engineering assessments with regards to vascular, cellular, and biomechanical criteria for mature, successful bone regeneration, which further underscored viability of this approach compared to native bone and gold-standard treatment after 24 weeks in vivo40.We present a novel, multimodal investigation including radiographic, histologic, biomechanical, and facial symmetry analyses to directly develop and assess these clinically-translatable bone-engineering advantages for pediatric patients. To our knowledge, the current study is the longest 41. Morphometric analysis has previously been used to describe patterns of growth, shape and development in unaffected and dysmorphic faces in syndromic human pediatric populations43. Application of this to animal study provided a means to concurrently analyze size and form of developing rabbit skulls. Previous animal models of experimentally-induced unilateral craniofacial suture restriction results in facial asymmetry between affected an un-affected sides44. Further, investigation of unilateral coronal synostosis and anterior synostotic plagiocephaly in humans suggests that premature closure of cranial and midface sutures can result in asymmetry of the calvarium as well as the midface47. In our model, even with close proximity of the surgical defects to developing sutures, DIPY-3DPBC scaffolds do not produce an increased craniofacial asymmetry compared to those reconstructed with bone graft or un-operated controls. It is notable that previous studies utilizing the same growing rabbit model demonstrated premature fusion of the cranial suture and restricted cranial growth when cranial defects were treated with BMP-248. BMP-2 is a commonly used osteogenic agent in pediatric craniofacial reconstruction despite growing evidence that its use can result in premature suture fusion and facial growth disruption50. As noted previously, growth restriction is a serious complication of pediatric craniofacial reconstruction that challenges autogenous reconstruction, autogenous implants, and bioceramic tissue engineering alike38. The lack of facial asymmetry demonstrated in this sample, suggests that DIPY-3DPBC scaffolds do not negatively affect facial suture patency or craniofacial development. Our 3D morphometric analysis is consistent with 2D reference measurements used clinically in evaluation of the maxilla and calvarium, supporting the novel application of this 3D modeling strategy for assessment of facial symmetry in a translational animal model.Most importantly, no adverse effects on cranial sutures or craniofacial growth were noted in the study animals, which developed through the time of craniofacial skeletal maturity. Physiologic and clinical relevance was of critical importance in the application this model to an often difficult to assess population. Facial maturity in our model was defined conservatively at 28–30 weeks of ageOur surgical injury protocol was applied to two distinct surgical sites, to model the various complex clinical applications and to acknowledge the functionally different considerations of calvarial and alveolar reconstruction. We selected alveolar and calvarial reconstruction as they represent a significant clinical burden for pediatric craniofacial patients as well as areas of active tissue engineering research. Craniofacial growth and development is affected by complex genomic, metabolic, and mechanical factors — often patients require multiple craniofacial procedures before skeletal maturity. We believe that the success of our model at both defect sites supports the efficacy and clinical translatability of this technology.12.For example, this study employed a simplified model of bone graft in approximating the complexity and size of calvarial defects treated in the clinical arena. Cranial vault remodeling often requires significant resection and reconstruction in irregular geometries that may rely on alloplastic reconstruction given limited stock and shape of donor bone. Further, skull thickness and regenerative capacity may vary based on anatomic location of cranial reconstruction. It is unlikely that in a clinical scenario, an autograft with the precise dimensions and curvature of the reconstructed defect would be used in the fashion described in this report. As such, the calvarial bone graft results achieved in our investigation may represent idealized outcomes of results for cranial reconstruction. As a result, the equivalence of DIPY-3DPBC scaffold reconstruction to this idealized bone graft model in our study underscores the potential advantages of DIPY-3DPBC scaffolds, as an inherent benefit of the technology is the ability to design and implement custom-fitted implants tailored to complex reconstructive challenges52. Given our demonstration of successful regeneration in this model, the investigation of bony regeneration in a congenital cleft animal model and finally the study of regeneration in syndromic clefts remain important further areas of investigation.One limitation of our model is that we do not directly investigate the repair of congenital facial clefts —instead, we employ a surgically created cleft model in order to approximate the treatment of congenital clefts commonly presenting in pediatric patients. Our aim was to investigate bony regeneration of surgical defects in this pediatric model, which we believe remains relevant in the treatment of congenital cleft patients, as the opposing alveolar ridge may be trimmed in preparation for filling by bone graft in congenital cleft patientsin vitro 3D culture models.Future studies will also be aimed at long-term analysis of bone quality following the completion of scaffold resorption in order to accurately estimate scaffold degradation kinetics and the continued viability and morphology of regenerated bone. Also, we acknowledge that rodent models typically represent idealized models of bone regeneration technologies due to favorable bone metabolism and scale, this is a limitation inherent to translational research, especially given important concerns about safety in pediatric clinical trials. As such, we also intend to investigate the use of these biomaterials in reconstructing larger defects in a swine animal model as well human Ultimately, we uniquely demonstrate that DIPY-3DPBC scaffolds generate vascularized bone with radiographic, histologic, and mechanical properties similar to native bone as defined by clinically-relevant outcomes — all without impediment to normal craniofacial skeletal development. Thus, we believe the DIPY-3DPBC scaffolds presented in this report may overcome limitations of autologous bone graft and begin to fulfill context-specific requirements of an effective pediatric bone tissue engineering strategy.All experiments were approved by the NYU School of Medicine Institutional Animal Care and Use Committee (IACUC). Experiments were performed in accordance with the NYU School of Medicine Institutional Animal Care and Use Committee (IACUC) standards. Sixteen (n = 8 per group) four-week-old  New Zealand White rabbits underwent surgical creation of two unilateral right-sided defects: in the calvarium and alveolus (see supplementary information for study groups). Defects were repaired with either custom DIPY-3DPBC scaffolds or autogenous bone graft.28. First, a 13 mm skin incision was made on posterior scalp to the right of midline in order to visualize the rabbit’s calvarium. Soft tissue and periosteum were dissected to expose calvarial bone. Defects were then created uniformly, located 2 mm posterior and lateral to the coronal and sagittal sutures, respectively, using at 10 mm diameter trephine 53 via anesthetic barbiturate overdose. Secondary confirmation of euthanasia was performed via decapitation in accordance with NYU School of Medicine IUCAC standards.Buprenorphine (0.01 mg/kg) and enrofloxacin (5 mg/kg) were administered subcutaneously every 12-hours for up to 48-hours post-op. Animals were given food, 24. Ink preparation requires ~2 hours. Calvarial scaffolds were printed layer-by-layer in paraffin oil as 10 mm-diameter cylindrical lattices  , and constructed using a custom-built 3D direct-write micro-printer system . Colloid gel ink was formulated to achieve solid volume fraction of 46% by combining ceramic powder, ammonium polyacrylate, deionized water, hydroxypropyl methylcellulose, and polyethlenimine55. This DIPY concentration has been demonstrated to result in favorable bone regeneration in previous studies29.Scaffolds were loaded by immersion in 2% bovine collagen solution  as a carrier and then loaded with 1,000 µM DIPYen bloc, cleared of surrounding soft tissue, and imaged using micropositron emission tomography computed tomography  scanner at 86 µm slice resolution. CT scans were used to reconstruct animal skulls for evaluation of facial symmetry.Rabbit heads were removed Amira  to create 3D digital models of the region of interest (ROI) including scaffold or regenerated bone. Bone regeneration, scaffold degradation, and maxillary suture patency were calculated using µCT reconstruction. As previously described28, 3D reconstructions were uniformly thresholded for bone, scaffold, and soft tissue or empty space using consistent values for bone and scaffold which demonstrate different µCT intensity in Houndsfield units. Then, volume of each component was calculated as a proportion of total volume of the defect (BV/TV or SV/TV). Un-implanted scaffolds also underwent µCT scanning and reconstruction via this method in order to establish a t = 0 time point to evaluate scaffold degradation.High-resolution micro computed tomography  images were acquired at 18 µm slice resolution for specific areas of interest. These µCT sections were then reconstructed in ImageJ56 and analyzed for trabecular thickness (Tb.Th) and trabecular spacing (Tb.Sp) using BoneJ Thickness function58. Using ROI manager, region of interest with uniform dimensions of trabecular bone were selected across reconstruction type and thresholded for scaffold, bone, and empty space. Regions of interest were adjusted to nearby anatomical landmarks in order to account for sample orientation and ensure analysis of comparable trabecular regions. µCT analysis was conducted by a single, blinded investigator (MW).µCT images were imported into Following whole-face CT imaging, samples were partitioned to isolate region surrounding the calvarial defects. Samples were then dehydrated in a series of alcohol solutions ranging from 70–100% ethanol (EtOH) and embedded in methyl methacrylate resin. The embedded blocks were cut into 250 μm-thick sections using a diamond saw  with individual slices glued onto acrylic slides and ground on a grinding machine  under continuous water irrigation with a series of SiC abrasive paper until a thickness of 100 μm were achieved, after which samples were stained in Stevenel’s blue and Van Gieson picro fuchsin to differentiate between bone, scaffold and soft tissue. Sample sections were imaged at low magnification  using pathology slide scanner  and evaluated for suture patency and bone organization including trabecular organization and creation of diploic spaces in the calvarium Fig. . Samples40. In both the alveolus and calvarium, bone regenerated by scaffolds was analyzed by isolating regions between scaffold struts. Bone graft was analyzed by selecting regions on the corresponding side of animals that underwent bone graft reconstruction. Native bone was analyzed by selecting regions from contralateral, un-operated sides of the calvarium and contralateral alveoli.At high magnification , samples underwent further histologic characterization to compare organization and vascularization of native, bone graft, and scaffold-regenerated bone including previously-defined features that would indicate maturity and remodeling of boneImageJ. 3.5 × 2.0 mm sections of bone were assessed for vessel density (vessel/mm2). Vessels were identified by morphologic appearance of endothelial cells, clear vessel lumen, or presence of intra-vascular erythrocytes. Average vessel diameter was calculated to assess degree of neo-angiogenesis by measuring the widest dimension of the innermost lumen of each identified vessel  was calculated by measuring the number of osteoblasts divided by unit area; osteoblasts were identified by strong cytoplasmic Stevenel’s blue staining, location adjacent to osteoid, and cuboidal cell morphology . was calculated by measuring the number of osteoclasts per unit area; osteoclasts were identified as multinucleated cells with ruffled border, and location in ‘Howship’s lacunae’  polishing as necessary for scratch removal. The nanoindentation testing (n = 16/specimen) was performed using a nanoindenter  equipped with a Berkovich diamond three-sided pyramid probe. The scaffold samples were subject to peak load of 300 µN at a rate of 60 µN/s applied with a holding period of 10 s and an unloading period of 2 s. For each sample, n = 16 indentation points were collected on regenerated bone within scaffold interstices and n = 16 indentations were collected on un-operated bone outside the defect area. Regions of bone were chosen by visualization with light microscope, with scaffold bone selected between regions of scaffold struts and regions of native bone selected distal to the scaffold region and proximal to the maxillary suture in the alveolus, or lateral to the scaffold region in the calvarium. Indentation protocol generated force-displacement curves which was used by indenter software to calculate reduced elastic modulus (Er) and hardness (H) using the following formulae:hc) is the contact area at peak load (Pmax) and S is stiffness27.Nanoindentation was used to assess mechanical properties of regenerated bone61. Single-surface images of rabbit skulls were isolated and prepared from reconstructions of gross CT scans with Meshlab© and Blender©. Using Facemark©, rabbit faces were then annotated by one individual (MW) with 78 anatomically consistent landmarks as previously reported in studies of rabbit craniofacial morphology64. A 3D dense-surface model was built using DSMBuilder©, applying the Procrustes algorithm to compute mean landmarks and thin-plate splines so that mean surfaces could be warped to the mean landmarks thus enabling the set of surfaces to be closely aligned. Hence the points on a selected face could be mapped to the closest points on each face enabling the induction of a dense surface correspondence of thousands of points that enabled computation of the mean surface. The differences between corresponding points on each surface and the mean surface were used to obtain the principle components that accounted for 99% of variation and which were then used to build the dense-surface model43. For each craniofacial surface, a mirror-image was created and the difference between each sample’s surface and its mirror image calculated an asymmetry index (AI) using previously published methods65. Briefly, a reflected form of each surface was generated and left-right landmarks were re-labelled using Facemark©. DSMBuilder© was then used to generate a DSM of the original and reflected surface. The asymmetry index was calculated from the Euclidean distances between the DSM of the original and reflected surfaces. Both Facemark©and DSMBuilder© were developed by Prof. Hammond and Dr. Suttie61.Morphometric analysis was predicated on the assumption that aberrant craniofacial development secondary to DIPY-3DPBC scaffolds, implanted on the right side of the skull and maxilla, would manifest as asymmetrical growth of the face. Facial development and symmetry at facial maturity was evaluated using 3D dense-surface modeling as previously utilized to characterize patterns of dysmorphic faces in neonatal and pediatric populationsAdditionally, in an effort to detect localized asymmetry resulting from disruption of cranial or maxillary sutures, two targeted sub-models were created using the above method: an isolated midface model and an isolated calvarial model. The isolated midface model was generated by selectively including landmarks anterior to the intraorbital midline (landmark 5), and the isolated calvarial model was generated by including landmarks posterior to the intraorbital midline (landmark 5). Asymmetry Indices were computed by the method described above.Facial symmetry analysis was further validated with bilateral cephalometric measurements. Bilateral maxillary (Maxillary Length (ML): anterior corpus ossis incicivi - anterior maxillary premolar; Maxillary Height (MH): anterior corpus ossis incicivi - posterior alveoli I3) and calvarial measurements : coronal intercept – occipital intercept; Calvarial Width (CW): bregma - coronal intercept;) were taken for each subject. Each measurement was compared between operated and un-operated sides. Ratio between operated and un-operated sides were compared across group conditions including comparison to un-operated age-matched controls.a priori using power analysis based on preliminary 8-week experiments. Analysis revealed n=6/group was necessary to achieve power >80% to detect 25% mean differences in bone volume regeneration of scaffolds compared to bone graft with standard deviation of 15%. Alpha (α) was set to 0.05.Sample size was determined 3D quantification of bone volume at 24 weeks and histologic osteocyte count analyses were analyzed between scaffold, bone graft, and native bone groups using one-way ANOVA with post-hoc comparison of group means with Tukey’s correction.Bone volume fraction and scaffold volume fraction analysis between scaffold and bone graft-repaired defects was performed using generalized linear mixed model (GLMM).Trabecular analysis as well as histologic counts including vessel density, vessel diameter, osteocyte, osteoblast, and osteocyte density were analyzed using one-way ANOVA with post-hoc comparison of groups performed with Tukey’s correction.27.For nanoindentation, reduced elastic modulus and hardness of newly formed bone was compared to internal control bone by using a GLMMFacial growth was independently assessed through morphometric analysis of asymmetry indices and cephalometrics; both analyses were conducted using one-way ANOVA with post-hoc comparison of groups performed with Tukey’s correction.µCT volumetric data, histologic quantification, morphometric asymmetry indices, and nanoindentation data are presented as mean value with corresponding 95% CI.All statistical analyses were performed in SPSS . All graphs were generated using GraphPad Prism .in vivo evaluation of dipyridamole-loaded 3D-printed β –TCP scaffolds in regenerating craniofacial bone in a pediatric rabbit model. Previous investigations of this technology were limited to short-term studies compared to un-filled defects. This is the first study to compare to autogolous bone graft, the standard of care and to directly assess facial symmetry through the age of facial maturity. Using a multi-model evaluation approach, the results of this study favor the osteogenic efficacy, favorable degradation and facial-growth safety of this technology to be translated for craniofacial reconstruction specifically in a pediatric context.In this study, we present what is to our knowledge the longest Supplmentary Table 1