Design, In Vitro Evaluation and In Vivo Biocompatibility of Additive Manufacturing Three-Dimensional Printing of ß beta-Tricalcium Phosphate Scaffolds for Bone Regeneration.

The reconstruction of bone deficiencies remains a challenge due to the limitations of autologous bone grafting. The objective of this study is to evaluate the bone regeneration efficacy of additive manufacturing of tricalcium phosphate (TCP) implants using lithography-based ceramic manufacturing (LCM). LCM uses LithaBone TCP 300 slurry for 3D printing, producing cylindrical scaffolds. Four models of internal scaffold geometry were developed and compared. The in vitro studies included cell culture, differentiation, seeding, morphological studies and detection of early osteogenesis. The in vivo studies involved 42 Wistar rats divided into four groups (control, membrane, scaffold (TCP) and membrane with TCP). In each animal, unilateral right mandibular defects with a total thickness of 5 mm were surgically performed. The animals were sacrificed 3 and 6 months after surgery. Bone neoformation was evaluated by conventional histology, radiology, and micro-CT. Model A (spheres with intersecting and aligned arrays) showed higher penetration and interconnection. Histological and radiological analysis by micro-CT revealed increased bone formation in the grafted groups, especially when combined with a membrane. Our innovative 3D printing technology, combined with precise scaffold design and efficient cleaning, shows potential for bone regeneration. However, further refinement of the technique and long-term clinical studies are crucial to establish the safety and efficacy of these advanced 3D printed scaffolds in human patients.

Early Cytomegalovirus Reactivation in Renal Recipients Is Associated with High Levels of B Cell Maturation Antigen Transcript Expression Prior to Transplantation.

Cytomegalovirus (CMV) infection is the most frequent infection episode in kidney transplant (KT) recipients. Reactivation usually occurs in the first three months after transplantation and is associated with higher cellular and/or antibody-mediated rejection rates and poorer graft performance. CMV induces the expression of BAFF (B-cell-activating factor, a cytokine involved in the homeostasis of B cells), which communicates signals for survival and growth to B cells and virus-specific plasma cells via the R-BAFF (BAFF receptor), TACI (the calcium modulator, the cyclophilin ligand interactor), and BCMA (B cell maturation antigen) receptors. These molecules of the BAFF system have also been suggested as biomarkers for the development of alloantibodies and graft dysfunction. This prospective study included 30 CMV-IgG seropositive KT recipients. The expression levels of the genes BAFF-R, transmembrane activator and CAML interactor (TACI), and B cell maturation antigen (BCMA) in peripheral blood leukocytes (PBL) pre-KT were determined using qPCR. qPCR was also used to monitor CMV reactivation in the first three months following KT. The remainder of the KT recipients were classified as CMV- reactivation, and those with more than 500 copies/mL in at least one sample were classified as CMV+ reactivation. There were no discernible variations in the BAFF-R and TACI transcript expression levels. In the CMV+ group, we examined the relationship between the transcript levels and peak viremia. Peak viremia levels and BCMA transcript levels showed a strong correlation. BAFF-R and TACI expressions showed no measurable differences. In patients with early CMV reactivation, high BCMA receptor expression was associated with increased plasmablast, lymphocyte B cell class-switched levels (LBCS), and viral load. Our findings demonstrate that pre-KT BCMA transcript levels increased in KT recipients with early CMV reactivation. These transcript levels positively correlate with peak viremia and weakly with plasmablast and LBCS levels in PBLs.

All That Glitters in cfDNA Analysis Is Not Gold or Its Utility Is Completely Established Due to Graft Damage: A Critical Review in the Field of Transplantation.

In kidney transplantation, a biopsy is currently the gold standard for monitoring the transplanted organ. However, this is far from an ideal screening method given its invasive nature and the discomfort it can cause the patient. Large-scale studies in renal transplantation show that approximately 1% of biopsies generate major complications, with a risk of macroscopic hematuria greater than 3.5%. It would not be until 2011 that a method to detect donor-derived cell-free DNA (dd-cfDNA) employing digital PCR was devised based on analyzing the differences in SNPs between the donor and recipient. In addition, since the initial validation studies were carried out at the specific moments in which rejection was suspected, there is still not a good understanding of how dd-cfDNA levels naturally evolve post-transplant. In addition, various factors, both in the recipient and the donor, can influence dd-cfDNA levels and cause increases in the levels of dd-cfDNA themselves without suspicion of rejection. All that glitters in this technology is not gold; therefore, in this article, we discuss the current state of clinical studies, the benefits, and disadvantages.

Monitoring of Soluble Forms of BAFF System (BAFF, APRIL, sR-BAFF, sTACI and sBCMA) in Kidney Transplantation.

BAFF system plays an essential role in B cells homeostasis and tolerance, although it has widely not been tested in transplantation with doubtful results. The main purpose was to study the BAFF soluble forms and their correlation with acute rejection (AR) and donor-specific antibodies production. Serum levels of BAFF, APRIL, and soluble forms of their receptors were analyzed in renal recipients with and without acute rejection (AR/NAR) appearance. All molecules were evaluated at pre- and post-transplantation. sTACI showed a significant correlation with BAFF and sR-BAFF levels, and sBCMA also showed a positive correlation with sAPRIL levels. A significant increase in sAPRIL levels in patients suffering AR was also found, and ROC curves analysis showed an AUC = 0.724, a concentration of 6.05 ng/ml (sensitivity: 66.7%; specificity: 73.3%), the best cutoff point for predicting AR. In the post-transplant dynamics of sAPRIL levels in the longitudinal cohort, we observed a significant decrease at 3 and 6 month post-transplantation compared to pretransplantation status. We also observed that recipients with high pre-transplant levels of sAPRIL generated antibodies earlier than those with lower sAPRIL levels, although their long-term post-transplantation was not different. Our results show that elevated serum levels of APRIL may be helpful as a biomarker for the diagnosis of AR, although the longitudinal study shows that it is not helpful as a prognostic biomarker.

Personalized Medicine for Kidney Transplantation: Association of Graft Survival and Acute Transplant Rejection with Genetic Variation in B Cell Activating Factor System Signaling.

Kidney transplantation (KT) clinical outcomes are highly variable across patients and would benefit from predictive biomarkers to achieve personalized/precision medicine. The B cell activating factor (BAFF) system signaling plays an essential role in B lymphocytes' homeostasis, and is implicated in activation and survival of B lymphocytes. Single nucleotide polymorphisms (SNPs) in BAFF system genes are therefore strong candidates to identify the genetic mechanisms underpinning variable clinical outcomes in KT. We report here new findings on BAFF system genetic polymorphisms in KT patients in relation to two key phenotypes of clinical interest: graft survival and acute rejection (AR). A total of 168 KT patients, of which 29 suffered AR, participated in this study. The BAFF system polymorphisms in five genes TNFSF13B, TNFSF13, TNFRSF13C, TNFRSF13B, and TNFRSF17 were characterized using TaqMan SNP genotyping. Patients with KT who had an AA genotype in polymorphism rs3803800 of the TNFSF13 gene had a higher risk of suffering AR (p = 0.046; odds ratios = 3.38, 95% CI: 1.02-11.2). Moreover, patients with AA genotype (rs3803800) in the TNFSF13 gene had a significantly lower AR-free time than the GG/GA genotypes (69.2% vs. 85.7%; p = 0.037). Of importance, bioinformatics analysis showed that the polymorphism rs3803800 could alter splicing regulation and affect the proliferation-inducing ligand (APRIL) expression levels. The analysis of graft survival did not show a significant association with the polymorphisms analyzed in this study. In conclusion, the rs3803800 genetic polymorphism from this study of BAFF system genes appears to display importance in AR-free time for KT patients, and thus, warrants further research in independent populations as a putative predictive biomarker of AR. These findings also inform future personalized/precision medicine efforts and functional genomic studies in KT patients.

MicroRNA Expression Changes in Kidney Transplant: Diagnostic Efficacy of miR-150-5p as Potential Rejection Biomarker, Pilot Study.

BACKGROUND: The kidney allograft biopsy is considered the gold standard for rejection diagnosis but is invasive and could be indeterminate. Several publications point to the role of miRNA expression in suggesting its involvement in the acceptance or rejection of organ transplantation. This study aimed to analyze microRNAs involved in the differentiation and activation of B and T lymphocytes from kidney transplant (KT) patients' peripheral blood leukocytes to be used as biomarkers of acute renal rejection (AR). METHODS: A total of 15 KT patients with and without acute rejection (AR/NAR) were analyzed and quantified by miRNA PCR array. A total of 84 miRNAs related to lymphocyte differentiation and activation B and T were studied. The functions and biological pathways were analyzed to predict the potential targets of differential expressed miRNAs. RESULTS: Six miRNA were increased in the AR group (miR-191-5p, miR-223-3p, miR-346, miR-423-5p, miR-574-3p, and miR-181d) and miR-150-5p was increased in the NAR group. In silico studies showed a total of 2603 target genes for the increased miRNAs in AR, while for the decrease miRNA, a total of 1107 target-potential genes were found. CONCLUSIONS: Our results show that KT with AR shows a decrease in miR-150-5p expression compared to NAR, suggesting that the decrease in miR-150-5p could be related to an increased MBD6 whose deregulation could have clinical consequences.

PCR Array Technology in Biopsy Samples Identifies Up-Regulated mTOR Pathway Genes as Potential Rejection Biomarkers After Kidney Transplantation.

Background: Antibody-mediated rejection (AMR) is the major cause of kidney transplant rejection. The donor-specific human leukocyte antigen (HLA) antibody (DSA) response to a renal allograft is not fully understood yet. mTOR complex has been described in the accommodation or rejection of transplants and integrates responses from a wide variety of signals. The aim of this study was to analyze the expression of the mTOR pathway genes in a large cohort of kidney transplant patients to determine its possible influence on the transplant outcome. Methods: A total of 269 kidney transplant patients monitored for DSA were studied. The patients were divided into two groups, one with recipients that had transplant rejection (+DSA/+AMR) and a second group of recipients without rejection (+DSA/-AMR and -DSA/-AMR, controls). Total RNA was extracted from kidney biopsies and reverse transcribed to cDNA. Human mTOR-PCR array technology was used to determine the expression of 84 mTOR pathway genes. STRING and REVIGO software were used to simulate gene to gene interaction and to assign a molecular function. Results: The studied groups showed a different expression of the mTOR pathway related genes. Recipients that had transplant rejection showed an over-expressed transcript (≥5-fold) of AKT1S1, DDIT4, EIF4E, HRAS, IGF1, INS, IRS1, PIK3CD, PIK3CG, PRKAG3, PRKCB (>12-fold), PRKCG, RPS6KA2, TELO2, ULK1, and VEGFC, compared with patients that did not have rejection. AKT1S1 transcripts were more expressed in +DSA/-AMR biopsies compared with +DSA/+AMR. The main molecular functions of up-regulated gene products were phosphotransferase activity, insulin-like grown factor receptor and ribonucleoside phosphate binding. The group of patients with transplant rejection also showed an under-expressed transcript (≥5-fold) of VEGFA (>15-fold), RPS6, and RHOA compared with the group without rejection. The molecular function of down-regulated gene products such as protein kinase activity and carbohydrate derivative binding proteins was also analyzed. Conclusions: We have found a higher number of over-expressed mTOR pathway genes than under-expressed ones in biopsies from rejected kidney transplants (+DSA/+AMR) with respect to controls. In addition to this, the molecular function of both types of transcripts (over/under expressed) is different. Therefore, further studies are needed to determine if variations in gene expression profiles can act as predictors of graft loss, and a better understanding of the mechanisms of action of the involved proteins would be necessary.

Computational Prediction of Biomarkers, Pathways, and New Target Drugs in the Pathogenesis of Immune-Based Diseases Regarding Kidney Transplantation Rejection.

Background: The diagnosis of graft rejection in kidney transplantation (KT) patients is made by evaluating the histological characteristics of biopsy samples. The evolution of omics sciences and bioinformatics techniques has contributed to the advancement in searching and predicting biomarkers, pathways, and new target drugs that allow a more precise and less invasive diagnosis. The aim was to search for differentially expressed genes (DEGs) in patients with/without antibody-mediated rejection (AMR) and find essential cells involved in AMR, new target drugs, protein-protein interactions (PPI), and know their functional and biological analysis. Material and Methods: Four GEO databases of kidney biopsies of kidney transplantation with/without AMR were analyzed. The infiltrating leukocyte populations in the graft, new target drugs, protein-protein interactions (PPI), functional and biological analysis were studied by different bioinformatics tools. Results: Our results show DEGs and the infiltrating leukocyte populations in the graft. There is an increase in the expression of genes related to different stages of the activation of the immune system, antigenic presentation such as antibody-mediated cytotoxicity, or leukocyte migration during AMR. The importance of the IRF/STAT1 pathways of response to IFN in controlling the expression of genes related to humoral rejection. The genes of this biological pathway were postulated as potential therapeutic targets and biomarkers of AMR. These biological processes correlated showed the infiltration of NK cells and monocytes towards the allograft. Besides the increase in dendritic cell maturation, it plays a central role in mediating the damage suffered by the graft during AMR. Computational approaches to the search for new therapeutic uses of approved target drugs also showed that imatinib might theoretically be helpful in KT for the prevention and/or treatment of AMR. Conclusion: Our results suggest the importance of the IRF/STAT1 pathways in humoral kidney rejection. NK cells and monocytes in graft damage have an essential role during rejection, and imatinib improves KT outcomes. Our results will have to be validated for the potential use of overexpressed genes as rejection biomarkers that can be used as diagnostic and prognostic markers and as therapeutic targets to avoid graft rejection in patients undergoing kidney transplantation.

High expression of CD38, CD69, CD95 and CD154 biomarkers in cultured peripheral T lymphocytes correlates with an increased risk of acute rejection in liver allograft recipients.

The mayor goal still outstanding into the solid organ transplantation field involves the search of surrogate biomarkers able to predict several clinical events, such as acute rejection (AR) or opportunistic infection. In the present multicenter study, a series of interesting surface antigens with important activator or inhibitory immune functions on cultured peripheral T cells were monitored in liver transplant recipients drawn at baseline and up to one year after transplantation. Sixty-four patients were included in the multicenter study during 3 years. Pre- and post-transplantation surface antigens levels displayed significant differences between AR and non acute rejection (NAR) groups, and also this differential expression was used to construct a risk predictive model based on a composite panel of outcome biomarkers (CD38, CD69, CD95 and CD154). The model was able to stratify these patients at high risk of AR. These preliminary results could provide basic information to improve the immunosuppressive treatment and it might better help to predict AR episodes.

Clinical outcomes after the use of complete autologous oral mucosa equivalents: preliminary cases.

OBJECTIVE: Previously, we reported how to obtain complete autologous oral mucosa equivalents (CAOMEs) composed of an autologous plasma scaffold and fibroblasts together with immature keratinocytes able to build an oral epithelium with a structure similar to that of the oral mucosa. In this study, we present the clinical outcomes after applying our CAOMEs as grafts. STUDY DESIGN: Four patients who needed a CAOME to restore a defect of oral mucosa were selected. Two of the patients suffered from ankyloglossia, and the other 2 required a restoration of the keratinized gum of the alveolar rim. To assess the outcomes, the scale designed by Ewers et al. was used. RESULTS: Clinical and functional improvements were achieved in the patients with ankyloglossia. In cases of gum restoration, the mucosa was regenerated and a prosthetic restoration with implants was achieved. CONCLUSIONS: The results obtained points to the potential use of CAOME in intraoral lining.

Low median fluorescence intensity could be a nonsafety concept of immunologic risk evaluation in patients with shared molecular eplets in kidney transplantation.

Human leukocyte antigen (HLA) antibodies are usually "epitope" and not "antigen" specific. This work presents an interesting case concerning Luminex median fluorescence intensity (MFI) levels in antibodies considered low risk (<1,000), but producing humoral rejection. These low-titer antibodies could play an important role in transplantation. A 42-year-old woman was retransplanted with a deceased donor with negative complement-dependent cytotoxicity cross-matching. Our patient was pretransplant (PrT) sensitized to HLA antigens (single antigens (SA) = 31%) for 1 previous transplant. Thus, the formerly detected sensitized antigens were A32, A30, A31, cross-reacting group 5C, and DQ3 with a MFI(max) ≈ 4,127. In the posttransplantation period (PTP), the patient exhibited important instability in renal function and we detected an increased SA percentage (61%) with MFI(max) = 15,029 (A*32) with other antigens (detected with a low PrT MFI [<1,000]) as anti-A*03 (MFI(max) = 13,301) and anti-A*11 (MFI(max) = 13,714) specificities. Anti-A*03 was a donor-specific antibody (DSA). Renal biopsy was compatible with humoral rejection. The patient was pulsed with methylprednisolone, plasmapheresis, and intravenous immunoglobulin without improvement. Thus, we added anti-CD20 and the initial clinical response was highly favorable. Biopsies resulted in suggestive rejection reversion. MFI A*03 DSA decreased to 6,908 and later to MFI(max) = 5,505. After a 6-month PTP, the patient is well with MFI(max) = 3,124. It was possible to define exactly the potential immunizing epitope eplets whose recognition determined the specific antibody production. A*32:01, A*30:01, A*31:01 (detected PrT), A*11:01, and A*03:01 (detected PTP) alleles have several shared eplets (62QE, 70AQS, and 76VGT), with 62QE being the only eplet present on all alleles. In conclusion, low MFI levels in antibodies considered low risk could be important in posttransplant humoral rejection, although the patient's renal function can be restored. Thus, specific shared eplets should always be investigated with respect to previous transplant mismatches.

Oral carcinoma associated with implant-supported overdenture trauma: a case report.

With the advent of predictable implant support and retention, the implant-supported overdenture (IOD) has become an accepted treatment modality, but some complications could be observed. We present a case report of squamous cell carcinoma developed in an edentulous ridge in relation to bar retaining IOD chronic trauma, in a patient with no history of previous oral cancer. To our knowledge, this is the first case described in the literature and suggests that primary malignancy can appear as a chronic traumatic ulcer and that a high degree of vigilance is required in the follow up of these patients.

Pre-formed donor-specific alloantibodies (DSA) detected only by luminex technology using HLA-coated microspheres and causing acute humoral rejection and kidney graft dysfunction.

The different methodologies for the detection of HLA sensitization could have discrepant results. At the moment, luminex technology seems--in our opinion--to be the most sensitive and safest method for antibody detection, and it should be taken into account during the transplantation process and as a means of indicating immunossuppresive regimen modulation.