Fresh Grafts Research Articles

Cryopreservation of unrelated donor hematopoietic stem cells: the right answer for transplantations during the COVID-19 pandemic?

Cryopreservation was recommended to ensure continuity of unrelated donor (UD) hematopoietic stem cell transplantation (HSCT) during COVID-19 pandemic. However, its impact on clinical outcomes and feasibility was not well known. We compared 32 patients who underwent UD HSCT using cryopreserved peripheral blood stem cells (PBSC) during the COVID-19 pandemic with 32 patients who underwent UD HSCT using fresh PBSC in the previous period. Median neutrophil engraftment was 17.5 and 17.0 days with cryopreserved and fresh grafts, respectively. Non-significant delays were found in platelet recovery days (25.5 versus 19.0; P = 0.192) and full donor chimerism days (35.0 and 31.5; P = 0.872) using cryopreserved PBSC. The rate of acute graft-versus-host disease at 100 days was 41% (95% CI [21–55%]) in cryopreserved group versus 31% (95% CI [13–46%]) in fresh group (P = 0.380). One-hundred days progression-relapse free survival and overall survival did not differ significantly. During COVID-19 pandemic, six frozen UD donations were not transfused and logistical and clinical issues regarding cryopreservation procedure, packaging, and transporting appeared. In summary, UD HSCT with cryopreserved PBSC was safe during this challenging time. More efforts are needed to ensure that all frozen grafts are transplanted and cryopreservation requirements are harmonized.

Incorporation of anterior iliac crest or calvarial bone grafts in reconstructed atrophied maxillae: A randomized clinical trial with histomorphometric and micro‐CT analyses

BackgroundAutologous bone grafts have been applied successfully to severely atrophied maxilla via a preimplant procedure. Differences in graft incorporation at the microscopic level can be the decisive factor in the choice between anterior iliac crest and calvarial bone.PurposeTo compare conversion of anterior iliac crest bone and calvarial bone 4 months after grafting of the edentulous maxilla.Materials and methodsTwenty consecutive patients were randomly assigned to either anterior iliac crest (n = 10) or calvarial (n = 10) bone harvesting to reconstruct their atrophied maxillae. Biopsies were taken from both fresh bone grafts and reconstructed maxillae after 4 months healing, at time of implant placement. Micro‐CT, histomorphometric and histological analyses were performed.ResultsMicro‐CT analysis revealed that both the anterior iliac crest and calvarial bone grafts retained their volume and bone mass after being incorporated in the maxilla, but with a favor for calvarial bone grafts: calvarial bone grafts had a higher mineral density before and after incorporation. Both bone grafts types were well incorporated after 4 months of healing with preservation of bone volume and mineral density. Although the fresh bone biopsies were similar histomorphometrically, after 4 months of graft incorporation, the osteoid percentage and osteocyte count remained higher in the anterior iliac crest bone whereas the percentage of bone was higher in the calvarial bone grafts compared to the anterior iliac crest bone grafts.ConclusionsBoth donor sites, that is, anterior iliac crest and calvarial bone, are well suited to provide a reliable and stable basis for implant placement 4 months after grafting with mineral density, porosity, and resorption rate in favor of calvarial bone grafts.

Universal Engraftment after Allogeneic Hematopoietic Cell Transplantation Using Cryopreserved CD34-Selected Grafts

As a result of the COVID-19 pandemic, most centers performing allogeneic hematopoietic cell transplantation (allo-HCT) have switched to the use of cryopreserved grafts. Previous investigators have suggested that cryopreserved allografts may heighten risk of nonengraftment. To date, no study has investigated the effect of cryopreservation of CD34-selected hematopoietic progenitor cells (CD34+ HPCs) used as the sole graft source. In this study, we sought to evaluate outcomes after unrelated donor or matched sibling allo-HCT with cryopreserved CD34+ HPCs. This was a single-center analysis of adult patients with hematologic malignancies who underwent allo-HCT with cryopreserved CD34-selected allo-HCT grafts between January 2010 and June 2017. All patients received ablative conditioning and antirejection prophylaxis with rabbit antithymocyte globulin. G-CSF-mobilized leukapheresis products underwent CD34 selection using the CliniMACS Reagent System. Cells were then cryopreserved in DMSO (final concentration 7.5%) to -90 °C using a controlled-rate freezing system before being transferred to vapor-phase liquid nitrogen storage. In internal validation, this method has shown 92% mean CD34+ cell viability and 99.7% mean CD34+ cell recovery. Engraftment was defined as the first of 3 consecutive days of an absolute neutrophil count of ≥0.5. Platelet recovery was recorded as the first of 7 consecutive days with a platelet count ≥20 K/μL without transfusion. Kaplan-Meier methodology was used to estimate overall survival (OS) and relapse-free survival (RFS), and cumulative incidence functions were used to estimate rates of relapse, nonrelapse mortality (NRM), and acute graft-versus-host disease (GVHD). A total of 64 patients received a cryopreserved CD34-selected graft. The median CD34+ cell count before cryopreservation was 6.6×106/kg (range, 1.4 to 16.1×106/kg), and the median CD3+ cell count was 2.0×103/kg (range, 0 to 21.1×106/kg). All patients were engrafted, at a median of 11 days post-HCT (range, 8 to 14 days). One patient had poor graft function in the setting of cytomegalovirus viremia, necessitating a CD34-selected boost on day +57. The median time to platelet recovery was 16 days (range, 13 to 99 days). The estimated 2-year OS was 70% (95% confidence interval [CI], 58% to 83%) with cryopreserved grafts versus 62% (95% CI, 57% to 67%) with fresh grafts (hazard ratio [HR], 0.86; 95% CI, 0.54 to 1.35; P=.5). The estimated 2-year RFS in the 2 groups was 59% (95% CI, 48% to 74%) versus 56% (95% CI, 51% to 61%; HR, 1.01; 95% CI, 0.68 to 1.51; P > .9). The cumulative incidence of relapse at 2 years was 29% (95% CI, 17% to 41%) versus 23% (95% CI, 19% to 27%; P=.16), and the cumulative incidence of NRM at 2 years was 17% (95% CI, 9% to 28%) versus 23% (95% CI, 19% to 28%; P=.24). The cumulative incidence of grade II-IV acute GVHD by day +100 was 16% with cryopreserved grafts (95% CI, 8% to 26%) and 16% (95% CI, 13% to 20%; P=.97) with fresh grafts. Moderate to severe chronic GVHD by day +365 occurred in only 1 recipient of a cryopreserved graft (2%). Our data show that in patients with hematologic malignancies who received cryopreserved allogeneic CD34+ HPCs, engraftment, GVHD, and survival outcomes were consistent with those seen in recipients of fresh allogeneic CD34+ HPC grafts at our center. Our laboratory validation and clinical experience demonstrate the safety of our cryopreservation procedure for CD34-selected allografts.

662 Single Surgeon Experience Using a Polyactide-based Copolymer Dressing to Over and Secure Split Thickness Skin Grafts in Burn Patients.

Abstract Introduction In both partial thickness burns and skin graft donor sites, coverage with Polylactide-based copolymer dressing (PLBC dressing) has been shown to result in expedited healing and improved pain outcomes when compared to more traditional techniques. These advantages are generally attributed to the way in which PLBC remains as an intact coating over the wound bed throughout the healing process, protecting wounds from the contamination and microtraumas associated with changes more conventional dressings. At our institution, we began selectively utilizing PLBC as a means of securing and protecting fresh skin graft, in hopes that we would find similar benefits in this application. Methods Clinical Protocol-- The PLBC dressing was used at the attending surgeon’s discretion. In these cases, meshed STSG was placed over prepared wound beds. Staples were not utilized. PLBC dressing was then placed over the entirety of the graft surface, securing graft in place by adhering to wound bed through intercises. (Staples were not used.) The graft and PLBC complex was further dressed with a layer of non-adherent cellulose based liner with petroleum based lubricant, and an outer layer of cotton gauze placed as a wrap or bolster. Post operatively, the outer layer (“wrap”) of gauze was replaced as needed for saturation. The PLBC and adherent “inner” liner were left in place until falling off naturally over the course of outpatient follow-up. Retrospective Review-- With IRB approval, patients treated PLBC over STSG between April 2018 to March 2019 were identified via surgeon’s log and pulled for review. Documentation gathered from operative notes, progress notes (inpatient and outpatient) and clinical photography was used to identify demographics, mechanism of injury, depth, total body surface area percentage (TBSA%), size of area treated with PLBC dressing, graft loss, need for re-grafting, signs of wound infection, antibiotic treatment, and length of stay. Results Twenty-two patients had STSG secured and dressed with PLBC. Median patient age was 36.5 years. Median TBSA was 5.1%, and median treated area 375 cm2. Follow up ranged from 21 to 232 days post-operatively, with two patients lost to follow up. All patients seen in outpatient follow up were noted to have “complete graft take” or “minimal” graft. None of the areas treated with PLBC dressing required re-grafting. There were no unplanned readmissions, and no wound infections were diagnosed or treated. Practitioners in in-patient setting and in follow up clinic reported satisfaction with the PLBC dressing. Conclusions The PLBC dressing was a feasible solution for securing and dressings STSGs. Future work is needed to determine whether its use is associated with an improvement in patient outcomes.

Ovine model of burn wounds grafted with ovine cadaver skin

Shortage in autograft to cover burn wounds involves a frequent use of cadaver skin (CS) as a temporary cover to prevent infection, dehydration and preparation of wounds for subsequent autografting.We aimed to establish an ovine model of burn wound healing using ovine CS (OCS). Quality and efficacy of fresh and frozen OCS overlaid on to excised 3rd degree flame burn wounds in sheep were evaluated in comparison to autograft.Histologically, autografted wounds maintained normal skin structure at different time points. Wounds overlaid with fresh OCS graft showed signs of rejection starting from day 7. At day 14, the epidermis was mostly rejected. The rejection was completed by day 20 with signs of immunoreaction and presence of many immune cells. Frozen OCS was rejected in the same pattern. Immediately prior to grafting, the thickness was comparable between freshly prepared and frozen OCS for 10 or 40 days. Significant reduction in viability was detected in OCS frozen for 40 days.Both fresh or frozen ovine OCS were rejected within 10 days that mimics CS rejection time in humans (∼8.4 days), suggesting that ovine model of burn wound grafted with OCS can successfully be used in burn wound research mimicking clinical scenario.

Frozen versus fresh corneal graft carriers in Boston keratoprosthesis surgery: 10-year outcomes

ObjectiveTo compare 10-year clinical outcomes of frozen versus fresh corneal graft carriers for the Boston Keratoprosthesis type 1 (KPro). DesignProspective, non-masked randomized controlled trial. ParticipantsNineteen eyes of 19 patients having undergone Boston KPro type 1 implantation using a fresh or frozen graft carrier. MethodsAll patients that underwent Boston KPro type 1 implantation by a single experienced surgeon using an allograft carrier between October 2008 and March 2010 at the Centre Hospitalier de l'Université de Montréal were considered. Patients were excluded if they had a history of prior KPro implantation in the same eye. A subset of the patient cohort enrolled in the initial study protocol of 24 months continued follow-up to 120 months. Participants were randomized to receive either a fresh or frozen corneal graft carrier depending on tissue availability from the eye bank on the day of KPro implantation. ResultsNineteen eyes of 19 patients were included, with 11 in the fresh group and 8 in the frozen group. At 10 years, in the fresh and frozen groups respectively, device retention was 91% and 75%; mean best corrected visual acuity increased from counting fingers preoperatively to 20/300 and 20/125; and incidence of complications per patient was 2.36 and 2.37. There were no statistically significant differences between groups for any of these outcome measures (p > 0.05 for all analyses). ConclusionsFresh and frozen corneal graft carriers offer similar clinical outcomes for KPro implantation in terms of device retention, change in visual acuity, and rate of complications at 10 years.

Frozen Fat Grafts Maintain Vascular Endothelial Growth Factor Expression and Mediate Angiogenesis During Adipose-Derived Stem Cell Enrichment for Soft Tissue Augmentation.

Fresh fat grafts are commonly used in both esthetic and reconstructive surgeries, but the graft resorption rate varies. Cryopreservation of unused fat for later touch-up is one option to resolve this variation. In our previous studies, we found that fat cryopreservation may be a practical strategy for storing fat tissue. To explore the cryopreservation method, we evaluated the role of vascular endothelial growth factor (VEGF) in human frozen fat grafts. The concentration of VEGF in human frozen fat grafts subjected to different preservation times was determined using Western blotting and enzyme-linked immunosorbent assay. The angiogenic effect of frozen fat grafts was evaluated using a chorioallantoic membrane assay. Furthermore, the impact of adding human adipose-derived stem cells (hADSCs) or different concentrations of avastin (bevacizumab) to frozen fat grafts on angiogenesis was assessed. The viability of frozen fat grafts with or without hADSCs was evaluated using a nude mouse implantation study. Explanted fat tissues were examined on days 1, 4, 7, 14, 28, and 90, and morphological and histological analyses, immunohistochemistry, and enzyme-linked immunosorbent assay (VEGF concentration) were carried out. No significant difference in VEGF concentration between fresh and frozen fat was observed with respect to preservation duration. In the chorioallantoic membrane assay, frozen fat grafts with hADSCs displayed significantly enhanced angiogenesis. Avastin was found to decrease angiogenesis in frozen fat grafts. However, in the nude mouse implantation study, frozen fat grafts displayed VEGF maintenance, with the highest concentration observed on day 7. Adding hADSCs to the graft further increased the VEGF concentration and CD31 expression. Fat graft viability was found to be higher in the frozen fat grafts containing hADSCs than in grafts without hADSCs. Human fat grafts can maintain VEGF expression under frozen conditions for at least 12 months. The addition of hADSCs to the frozen fat graft could further enhance angiogenesis, VEGF expression, and fat cell viability.

Variation of BMP2 Concentration and Its Activity in Bone Grafts Obtained from Patients Undergoing Hip Replacement Surgery.

Bone morphogenetic protein 2 (BMP2) is a member of a subgroup of the transforming growth factor beta superfamily and triggers various signaling events which in turn stimulate chondrogenesis, osteogenesis, angiogenesis and extracellular matrix remodeling leading to fracture healing. In this study, we quantified the concentration of BMP2 in fresh human bone grafts obtained from 40 patients undergoing hip replacement surgery. Besides the concentration, the activity of the detected BMP2 was also investigated. In this study, the concentration of BMP2 in fresh human bone grafts obtained from 40 patients undergoing hip replacement surgery was quantified. Human BMP2 enzyme-linked immunosorbent assays and bicinchoninic acid quantification was used to determine the total concentration of protein present in each sample. To determine the activity of the BMP2 found in each bone sample, alkaline phosphatase activity was measured by colorimetric assay. The amount of BMP2 seemed to vary slightly between the patients. Taking into consideration the patient's gender, we observed that male patients presented slightly more BMP2 in comparison with females. When analyzing the activity of BMP2, we observed that in female patients, the activity was slightly higher in comparison to males. This variation may be caused by a number of factors, including but not limited to gender, age, osteoporosis and previous diseases. This information shows that the osteogenic potential of different bone graft samples is not consistent. The activity of BMP2 in femur heads obtained from patients undergoing total hip replacement surgery showed significant variation according to gender and age. The measurement of bone proteins activity might be promising as a qualitative method in bone banks and should be further investigated.

Fresh osteochondral graft. Indications, surgical technique and scientific evidence

Las lesiones condrales de la rodilla de espesor completo suponen un desafío para el cirujano ortopédico cuando se presentan en pacientes jóvenes y son sintomáticas. El aloinjerto osteocondral en fresco es un procedimiento válido para tratar grandes defectos osteocondrales de rodilla en pacientes jóvenes y activos, en los que la cirugía protésica no está indicada. Esta técnica quirúrgica ofrece la posibilidad de restaurar de manera inmediata la estructura histológica condral y ósea original sin añadir comorbilidad a la zona donante. Puede ser también utilizada en casos complejos de reconstrucción articular en el contexto de osteonecrosis, osteocondritis disecante, mala unión, artritis postraumática o en procedimientos de revisión. Los bancos de células y tejidos han mejorado la disponibilidad de este tipo de aloinjerto gracias a la optimización de las técnicas de procesado y de almacenamiento. Asimismo, la aplicación de protocolos estrictos de cribado de los donantes mediante pruebas serológicas aseguran un riesgo de transmisión de enfermedades mínimo. Para asegurar la supervivencia del injerto, es necesario realizar una adecuada selección del paciente, así como evaluar las condiciones de malalineación, estabilidad ligamentosa y déficit meniscal. El objetivo de este trabajo es revisar las indicaciones y los aspectos más destacados de la técnica quirúrgica, además de describir la tasa de supervivencia del aloinjerto osteocondral en fresco en distintas localizaciones de la rodilla.

Clinical and Radiographic Evaluation of Fresh Socket Grafting using Beta-tri-calcium Phosphate with Collagen and Platelet-Rich Fibrin for Recieving Dental Implant

Aim: To compare the clinical and 3D volumetric socket bone preservation using Beta tri calcium pohosphate plus collagen (B-TCP/Col) versus Platelet Rich Fibrin (PRF) assessed by 3D cone beam computed tomography.Patients and methods: 20 patients were treatment planned for extraction of mandibular and maxillary teeth, leaving 20 sockets for the study. Sockets were divided into 2 groups, each group consisting of 10 sockets. Sockets were grafted as follows: Group (I) sockets were grafted with B-TCP/col, Group (II) sockets were grafted with PRF. Cone beam computed tomography (CBCT) were done immediately and 3 months postoperatively, then collected data were statistically analyzed.Results: After 3 months, Bone height (mm) showed significant difference between the tested groups where Group A (B-TCP) produced the highest mean Bone height followed by Group B (PRF) . Bone width (mm) showed insignificant differences between the tested groups. Bone density showed insignificant differences between the tested groups.

Transplantation of allogeneic cryopreserved hematopoietic cell grafts during the Covid-19 pandemic: A National Marrow Donor Program perspective.

Transplantation of allogeneic cryopreserved hematopoietic cell grafts during the Covid-19 pandemic: A National Marrow Donor Program perspective.

Impact of Cryopreservation of Donor Grafts on Outcomes of Allogeneic Hematopoietic Cell Transplant (HCT)

Introduction: Cryopreservation of peripheral blood stem cell (PBSC) or bone marrow (BM) grafts is rarely performed, thus information about the effect of cryopreservation on graft characteristics and HCT outcomes is limited. Given the global pandemic leading to changing practices for donor graft collection and increasing utilization of cryopreservation, we evaluated the outcomes of HCT recipients who received either fresh or cryopreserved allogeneic BM or PBSC grafts. The primary endpoint was engraftment. Secondary endpoints were incidence of acute graft-vs.-host disease (aGVHD), relapse, transplant related mortality (TRM), disease free survival (DFS), and overall survival (OS). Methods: We included 7397 patients transplanted between 2013 and 2018. 1883 cryopreserved graft recipients were divided into three cohorts based on graft source and donor type: matched related (MRD) PBSC (n=1,051), matched unrelated (MUD) PBSC (n=678), and matched related or unrelated bone marrow donors (n=154). All patients received conventional calcineurin-based GVHD prophylaxis strategies. Patients who received cryopreserved grafts were matched with 5514 patients who received fresh adjusting for graft type (BM vs. PB), donor source (MRD vs. 8/8 MUD), Disease Risk Index (DRI, low risk vs. intermediate risk vs. high risk), recipient age (within 5-years) and propensity score (within 1 standard deviation from pooled sample). The propensity score was based on age, Karnofsky score, diagnosis, disease risk index, HCT-comorbidity index, and conditioning intensity. The level of statistical significance for the main analyses was p<0.01 due to multiple comparisons. The reason for cryopreservation was available on a subset of URD product recipients. Results: Baseline characteristics for all cohorts are shown in Table 1. CD34+ doses reflect enumeration at infusion. In univariate analyses, rates of graft failure and neutrophil recovery at day 28 were similar for cryopreserved and fresh grafts for BM or related PBSC recipients but differed in cryopreserved vs. fresh MUD PBSC (5% vs 2%, p<0.001 and 87 vs 94%, p<0.001, respectively). Rates of platelet recovery within 28 days were similar in marrow cohorts, but lower with cryopreserved related (92% vs 96%, p<0.001) and MUD PBSC (87 vs 94%, p<0.001) grafts. In matched pair multivariable analyses, there were no significant differences in any outcomes with cryopreserved vs fresh BM grafts, irrespective of donor type. With related donor PBSC, cryopreservation was associated with decreased platelet recovery (HR=0.73, CI=0.68-0.78, p<0.001) and an increased risk of both grade II-IV (HR=1.27, CI=1.09-1.48, p=0.002) and grade III-IV (HR=1.48, CI=1.19-1.84, p<0.001) aGVHD, but had no impact on other outcomes. In contrast, with MUD PBSC grafts, cryopreservation was associated with delayed engraftment of neutrophils (HR=0.77, CI=0.71-0.84, p<0.001) and platelets (HR=0.61, CI=0.56-0.66, p<0.001), an increased risk of TRM (HR=1.4, CI=1.18-1.66, p<0.001) and relapse (HR=1.32, CI=1.11-1.58, p=0.002), and decreased DFS (HR=1.36, CI=1.20-1.55, p<0.001) and OS (HR=1.38, CI=1.22-1.58, p<0.001) but did not affect the incidence of aGVHD. In a subset analysis of 299 URD recipients, the most common reason for cryopreservation was delays due to patient-related events, eg., additional chemotherapy, infection. These recipients had decreased OS compared to products cryopreserved for non-patient reasons (HR=0.65, CI=0.44-0.96, p=0.029). Conclusions: The retrospective nature of this analysis, and the fact that cryopreservation is likely to have been performed in recipients with a different (higher) risk profile compared to those receiving fresh products limits our ability to draw firm confusions. Despite these limitations, we conclude that cryopreservation in some patient populations may have a negative impact on engraftment and transplant outcomes. The decision to cryopreserve donor grafts, particularly PBSC grafts, should be carefully considered and highlights the need for further investigation of the effect of cryopreservation of allogeneic grafts where patient factors can be controlled for. Disclosures Farhadfar: CSL Behring: Research Funding; Incyte pharmaceutical: Other: Member of GVHD advisory forum. Frey:Kite Pharma: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Syntax: Consultancy, Honoraria. Devine:Magenta Therapeutics: Consultancy. Shaw:Orca Bio: Consultancy. Wingard:Shire: Other: Personal Fees - serve on data monitoring committee for clinical trial; Ansun: Other: Personal Fees - serve on data monitoring committee for clinical trial; Merck: Other: Personal Fees - serve on data monitoring committee for clinical trial; Cidara: Other: Personal Fees - serve on data monitoring committee for clinical trial; ReViral: Other: Personal Fees - serve on data monitoring committee for clinical trial.

Bridging the Gap in Access to Transplant for Underserved Minority Patients Using Mismatched Unrelated Donors and Post-Transplant Cyclophosphamide: A National Marrow Donor Program/be the Match (NMDP/BTM) Initiative

Bridging the Gap in Access to Transplant for Underserved Minority Patients Using Mismatched Unrelated Donors and Post-Transplant Cyclophosphamide: A National Marrow Donor Program/be the Match (NMDP/BTM) Initiative

Clinical and Radiographic Evaluation of Fresh Socket Grafting using Beta-tri-calcium Phosphate with Collagen and Platelet-Rich Fibrin for Receiving Dental Implant

Clinical and Radiographic Evaluation of Fresh Socket Grafting using Beta-tri-calcium Phosphate with Collagen and Platelet-Rich Fibrin for Receiving Dental Implant

Histomorphometric and micro-CT analyses of calvarial bone grafts used to reconstruct the extremely atrophied maxilla.

BackgroundCalvarial bone grafts are successful in the reconstruction of the severely atrophied maxilla as a pre‐implant procedure. However, not much is known about graft incorporation at the microscopic level.PurposeThis study aimed to assess calvarial bone conversion 4 months after being grafted in the edentulous maxillary bone.Materials and methodsIn 13 patients (age:65.3 ± 8.7 years) the atrophic maxilla was reconstructed with autologous calvarial bone. Biopsies were taken from fresh calvarial bone grafts and from the reconstructed maxillae after 4 months of healing. Micro‐CT, histomorphometric, and histological analysis were performed. From three patients biopsies were obtained after 9, 11, or 45 months.ResultsThe micro‐CT analysis revealed that in the maxilla the calvarial bone was well preserved even after 45 months. Histology showed progressive incorporation of grafted bone within a maxillary bone. Osteoid and osteocytes were present in all biopsies indicating new bone formation and vital bone. Histomorphometrically, the percentage of grafted bone volume over total volume decreased from 79.8% (IQR78.7‐83.3) in fresh calvarial grafts to 59.3% (IQR44.8‐64.6) in healed grafts. The biopsies were taken after 9, 11, and 45 months showed similar values.ConclusionsCalvarial bone grafts result in stable and viable bone, good incorporation into native maxillary bone, and a minor decrease in bone volume after healing. Consequently, they provide a solid base for implant placement in severely atrophied edentulous maxillary bone.

Doxycycline preserves chondrocyte viability and function in human and calf articular cartilage ex vivo.

Prolonging chondrocyte survival is essential to ensure fresh osteochondral (OC) grafts for treatment of articular cartilage lesions. Doxycycline has been shown to enhance cartilage growth, disrupt terminal differentiation of chondrocytes, and inhibit cartilage matrix degradation. It is unknown whether doxycycline prolongs chondrocyte survival in OC grafts. We hypothesized that doxycycline protects against chondrocyte death and maintains function of articular cartilage. To test this hypothesis, we employed human and calf articular cartilages, and incubated chondrocytes isolated from cartilage or cartilage plugs with doxycycline (0, 1 or 10 μg/ml) at either 37°C or 4°C. Chondrocyte viability, apoptosis, glycosaminoglycan (GAG), collagen, and mechanical test in cartilage plugs were measured. We found that reduced chondrocyte viability, increased chondrocyte apoptosis, reduced GAG contents, and impaired equilibrium modulus in cartilage plugs were observed in a time‐dependent manner at both 37°C and 4°C. Chondrocyte viability was further reduced when the plugs were cultured at 4°C as compared to 37°C. Doxycycline prolonged viability and reduced apoptosis of chondrocytes during culture of cartilage plugs. Functionally, doxycycline protected against reduced production of GAG and collagen II as well as impaired mechanical properties in cartilage plugs during culture. Mechanistically, doxycycline increased mitochondrial respiration in cultured chondrocytes. In conclusion, preservation at 37°C is beneficial for maintaining chondrocyte viability in cartilage plugs compared to 4°C. Incubation of doxycycline protects against chondrocyte apoptosis, reduced extracellular matrix, and impaired mechanical properties in cartilage plugs. The findings provide a potential approach using doxycycline at 37°C to preserve chondrocyte viability in fresh OC grafts for treatment of articular cartilage lesions.

DONOR HLA GENOTYPING BY NEXT GENERATION SEQUENCING OF DNA FROM URINE-DERIVED DONOR CELLS OF RENAL ALLOGRAFT RECIPIENTS

Introduction: Antibody-mediated rejection (AMR), dominantly induced by anti-HLA antibody, remains the most important cause of long-term graft loss after kidney transplantation. Donor-specific anti-HLA antibody (DSA) can’t often be determined at a specific allele level since historical donor DNA sample is not available and the existing donor HLA typing information is incomplete or even missing. Here, we developed a novel non-invasive technique by selectively expanding donor cells from urine of renal transplant recipients, and thus generating unlimited donor DNA to perform reliable allele-level HLA typing with next generation sequencing method for all HLA loci. Materials and Methods: This study enrolled 28 renal transplant recipients. The fresh midstream urine from each recipient was collected. After urine sample was centrifuged, the supernatant was discarded and washed twice with PBS. The resulting sediment was resuspended in the culture medium. The cells were plated and non-adherent cells were removed. DNA was extracted from the urine-derived donor cells (UDDCs). NGS sequencing is carried out to determine the HLA loci. Results: UDDCs from 26 patients were successfully isolated. High-resolution HLA results were unambiguously determined in 25 out of 26 DNA samples (25/26, 96%) and they are 100% concordant with donor HLA. We showed five HLA sequencing cases of UDDCs (Fig 1). The HLA typing of UDDCs is completely matched with HLA of donor peripheral blood. We further isolated UDDCs from a patient whose donor HLA information is missing at 10 years after kidney transplantation. Based on the UDDCs HLA typing and LSA results, DQA1*05:01 DQB1*02:01 were established as the culprit DSA loci of this patient (Fig 2). Discussion: DNA could be extracted from fresh kidney graft tissues for HLA genotyping; however, this approach is invasive and may aggravate graft injury. The amount of tissues obtained may be minimal and cannot yield sufficient quantity of DNA for HLA typing[1]. Other investigators extract DNA from paraffin embedded tissues or urinary sediments of KTx patients, and mismatched donor antigens could be deduced by subtracting the recipient’s HLA from the allograft HLA[2,3]. However, robust high-resolution HLA typing data can not be obtained by these methods. With an ex vivo expandable source of exfoliated cells, we provide a novel noninvasive urine-based molecular diagnostic method for donor HLA genotyping. Conclusion: The UDDCs-based HLA genotyping strategy successfully establishes complete donor DNA genotypes and provides a feasible and clinically relevant molecular diagnostic method that help to determine DSA loci for kidney transplant patients with historically insufficient donor HLA information.

A Prospective Randomized Study to Compare Surgical Outcome Using Wet and Dry Temporalis Fascia Graft in Type I Tympanoplasty.

Temporalis fascia is being used as graft material for tympanic membrane repair for over six decades. Though cartilage with or without perichondrium is also being used for different situations, still temporalis fascia graft is being considered as an ideal graft for tympanoplasty. Earlier the dried temporalis fascia was used as a graft but now freshly taken wet temporalis fascia is increasingly being used as a more 'physiological/ live' graft. The present study was planned to compare the results of use of wet and dry temporalis fascia in chronic otitis media mucosal disease. The prospective study was conducted on 100 patients of chronic suppurative otitis media mucosal disease. Chronic ear survey questionnaire were filled to note the impact on quality of life. Otoscopic, oto-endoscopic and examination under microscope performed. Patients were allocated into two groups alternately. Freshly taken wet temporalis graft was used in group I whereas dry temporalis fascia was used in group II. Hearing evaluation by pure tone audiogram was done preoperatively and at 6th months post-operatively. Pre-operative and post-operative hearings in both the groups were compared. Air bone gap closures after surgery in between both the groups were compared to see for any statistical difference. We found similar graft uptake in both the groups after 6months of surgery. The air bone gap closure in group I was 10.80 ± 5.74 and group II was 9.28 ± 4.81, there was no significant difference. However senior authors noted that in some of the cases in group I with fresh wet graft placement neo-tympanum formation was noticed early showing good circumferential leash of blood vessels all around signifying early vascularisation.

Abstract 226: Recellularization of Xenograft Heart Valves Reduces the Xenoreactive Immune Response in an In Vivo Rat Model

Background: Current xenograft valve constructs provoke an intense immune response that may lead to valve dysfunction. Our aim was to address the role of autologous mesenchymal stem cell (MSC) recellularization of xenogenic valves on the activation of the xenoreactive immune response in an in-vivo rat model. Methods: Explanted aortic valve constructs from female Hartley guinea pigs were procured and decellularized, followed by recellularization with syngeneic Sprague-Dawley rat MSCs. The recellularized aortic valve xenografts were then implanted into the infrarenal aorta of recipient female Sprague-Dawley rats. Grafts were implanted as either syngeneic grafts, non-decellularized (fresh), decellularized and recellularized xenografts. Rats were euthanized after 7-days, exsanguinated and the grafts explanted. Total serum immunoglobulin was quantified and histological analysis perfomed to analyze the immune response. Results: Overall survival to endpoint was significantly lower in the decellularized xenograft group (67%; 4/6), compared to fresh (100%; 6/6) and recellularized grafts (100%; 6/6). Similarly grafts in the decellularized group were more likely to have completely thrombosed (50%; 2/4), compared to fresh (33%; 2/6) and recellularized grafts (0%; 0/6). Decellularized guinea pig xenografts, when implanted into rats in-vivo , result in significantly reduced total serum immunoglobulin production and significantly reduced graft cellular infiltrate when compared to fresh xenografts. Moreover, when decellularized guinea pig xenografts were recellularized with syngeneic rat MSCs there was an additional decrease in total serum immunoglobulin production and graft cellular infiltrate when compared to both fresh and decellularized xenografts. Importantly, recellularized guinea pig xenografts had an equivalent total immunoglobulin production and graft cellular infiltrate when compared to syngeneic rat aortic valve controls. Conclusions: Autologous MSC recellularization of xenogenic valves reduces the xenoreactive immune response in an in-vivo rat model and may be an effective approach to decrease the progression of xenograft valve dysfunction.

Long-term observation of airway reconstruction using decellularized tracheal allografts in micro-miniature pigs at growing stage.

IntroductionDecellularized tissue exhibits cell matrix-like properties, along with reduced antigenicity. We explored the potential of decellularized allogeneic trachea to restore the upper respiratory tract, focusing on pediatric application. This study specifically aimed at long-term observation of tissue regeneration using a micro-miniature pig model.MethodsArtificial defects (15 × 15 mm) in the subglottis and trachea of micro-miniature pigs were repaired by transplantation of either allogeneic decellularized or fresh (control) tracheal patches. Pigs were evaluated in situ, by bronchoscopy, every three months, and sacrificed for histological examination at six and twelve months after transplantation.ResultsNo airway symptom was observed in any pig during the observation period. Bronchoscopy revealed the tracheal lumen to be restored by fresh grafts, showing an irregular surface with remarkable longitudinal compression; these changes were mild after restoration with decellularized grafts. Histologically, while fresh graft patches were denatured and replaced by calcified tissue, decellularized patches remained unchanged throughout the observation period. There were regeneration foci of cartilage adjacent to the grafts, and some foci joined the decellularized graft uniformly, suggesting the induction of tracheal reconstitution.ConclusionAllogeneic decellularized tracheal tissue could serve as a promising biomaterial for tracheal restoration, especially for pediatric patients at the growing stage.