Analysis of the Chemical Composition, Morphological Characterization, and Porosimetry of Allograft and Comparison with Xenograft for Dental Applications / Análisis de la Composición Química, Caracterización Morfológica y Porosimetría de Aloinjerto y Comparación con Xenoinjerto para Aplicaciones Odontológicas

SUMMARY: Tissue engineering aims to fabricate a scaffold that exhibits a suitable surface topography for a desired cellular response. Therefore, a study analyzing the characteristics of bone grafts is important for future research directions. This work aims to analyze the physical-chemical characteristics of commercially available bone grafts of human and bovine origin for dental use, using morphological analysis of the surface and chemical composition by variable pressure scanning electron microscope (VP-SEM) and energy-dispersive x-ray (EDX) spectrometry. In addition, pore diameter and surface area were analyzed by degassing method using a porosimeter, and particle size by laser diffraction. The analyzed allograft and xenograft particles differ in morphological characteristics and chemical composition. The allograft particles present a cuboidal and prismatic geometric morphology with angled edges and the absence of macropores. On the contrary, the xenograft particles present an irregular morphology with macropores in their structure. There is a statistically significant difference in C, P, and Ca between the xenograft and allografts (p < 0,05). The analyzed composition of allografts showed mainly the presence of C and O. In contrast, the composition of the xenograft was mainly Ca. These differences could influence the osteogenic properties of allografts and xenografts. This analysis provides basic information to understand the physicochemical properties of allografts and xenografts that facilitate cell-graft interaction.

La ingeniería de tejidos tiene como objetivo fabricar un andamio que muestre una topografía de superficie adecuada para una respuesta celular deseada. Por tanto, un estudio que analice las características de los injertos óseos es importante para futuros enfoques de investigación. Este trabajo tiene como objetivo analizar las características físico-químicas de injertos óseos de origen humano y bovino disponibles comercialmente para uso odontológico, mediante análisis morfológico de la superficie y composición química mediante microscopio electrónico de barrido de presión variable (VP-SEM) y x-dispersivo de energía. espectrometría de rayos (EDX). Además, el diámetro de los poros y el área superficial se analizaron mediante el método de desgasificación utilizando un porosímetro y el tamaño de las partículas mediante difracción láser. Las partículas de aloinjerto y xenoinjerto analizadas difieren en características morfológicas y composición química. Las partículas del aloinjerto presentan una morfología geométrica cúbica y prismática con bordes angulados y ausencia de macroporos. Por el contrario, las partículas de xenoinjerto presentan una morfología irregular con macroporos en su estructura. Existe una diferencia estadísticamente significativa en C, P y Ca entre el xenoinjerto y los aloinjertos (p < 0,05). La composición analizada de los aloinjertos mostró principalmente la presencia de C y O. Por el contrario, la composición del xenoinjerto fue principalmente Ca. Estas diferencias podrían influir en las propiedades osteogénicas de los aloinjertos y xenoinjertos. Este análisis proporciona información básica para comprender las propiedades fisicoquímicas de aloinjertos y xenoinjertos que facilitan la interacción célula-injerto.

Comparison of diameter and length of subclavian arteries to external jugular veins in variably sized dogs: A cadaveric study.

OBJECTIVE: To evaluate the length and diameter of a left external jugular vein graft as a substitute for the left subclavian artery in the modified Blalock-Thomas-Taussig shunt (mBTTS) in differently sized dogs. STUDY DESIGN: Cadaveric study. ANIMALS: Dog cadavers of three weight categories (10/group): <9.5 kg, 9.5 to 27 kg, and > 27 kg. METHODS: The length and infused external diameters of harvested vessels were measured with vernier calipers and recorded. A matched-pairs t test was used to test the difference in vessel lengths. The agreement in vessel diameters was assessed by using Lin's concordance correlation coefficient (CCC). Pearson's correlation coefficients (CC) were determined for vessel diameter to weight category and vessel length to weight category. RESULTS: The external jugular vein measured longer than the subclavian artery in all dogs (52.0 ± 20.8 mm and 23.0 ± 8.9 mm, respectively), with a mean difference of 28 ± 14.3 mm (P < .001). The mean external infused subclavian and external jugular diameters measured 7.8 ± 2.2 mm and 8.0 ± 2.5 mm, respectively (P = .32). Lin's CCC was 0.87. Pearson's CC were 0.74 in both vessel diameters (P < .001); they were 0.36 and 0.43, respectively, for subclavian artery and external juglar vein length (P < .001). CONCLUSION: Autologous external jugular vein grafts had an external diameter similar to subclavian artery and a significantly longer length in variably sized dogs. CLINICAL SIGNIFICANCE: External jugular vein grafts may be an acceptable graft choice for mBTTS.

Pediatric Donor Glomerulopathy Is a Possible Cause of Abnormal Urinalysis in Adults Receiving Small Pediatric Donor Kidneys.

BACKGROUND: Reports about prognosis of adults receiving small pediatric-donor kidneys (PDK) as compared to those receiving elder pediatric or adult donor kidneys (ADKs) are controversial. This study aimed to examine the outcomes of adults receiving small PDK and possible prognostic factors. METHODS: The records of adults who received kidneys from donors < 10 years old at our center from July 1, 2011 to June 30, 2018 were reviewed. RESULTS: A total of 121 adults were small PDK recipients. Twenty-three patients received 29 biopsies or nephrectomy between 6 and 896 days posttransplantation days. Seven patients (30.4%) had pediatric donor glomerulopathy (PDG), which developed from 113 to 615 days posttransplantation. The incidence of proteinuria and hematuria was significantly higher in the PDG group. The characteristic pathological finding in PDG was irregular lamination and splintering of the glomerular basement membrane (GBM). Donor age, donor weight, and donor kidney volume were significantly less in PDG cases compared with the non-PDG cases. For the risk factors of PDG, increasing urinary RBC count during follow-up was an independent predictor, while increasing donor age and body weight were protective factors. PDG was not a significant risk factor for Scr increasing of PDKs. CONCLUSIONS: PDG is a potential cause of abnormal urinalysis in adults receiving small PDKs. The pathological characteristic change of PDG is splitting and lamination of GBM. Persistent hematuria after transplantation in recipients of PDK is a predictor of PDG development.

Impact of adjusted kidney volume measured in the bench surgery on one-year renal function in kidney transplantation.

BACKGROUND: Kidney transplantation is the treatment of choice in patient with end stage chronic kidney disease, offering the best long term survival and greater Quality of Life in this group of patients. Graft volume was correlated with improved renal function in living donor transplantations. The primary aim of this study was to correlate renal volume adjusted to body surface area with renal function one year (estimated glomerular filtration rate; eGFR) after kidney transplantation. METHODS: This single-center, prospective cohort study included 256 patients who underwent kidney transplantation from January 2011 through December 2015 at Hospital das Clínicas de Botucatu-UNESP. We evaluated three kidney measurements during the bench surgery; the final graft volume was calculated using the ellipsoid formula and adjusted to body surface area. RESULTS: In the living donors there was positive correlation between adjusted graft volume and eGFR (r = 0.311, p = 0.008). Multivariate analysis revealed that low rejection rate and increased adjusted graft volume were independent factors correlated with eGFR. In deceased donors, there was no correlation between adjusted kidney volume and eGFR (r = 0.08, p = 0.279) in univariate analysis, but a multivariate analysis indicated that lower kidney donor profile index (KDPI), absence of rejection and high adjusted kidney volume were independent factors for better eGFR. CONCLUSION: Adjusted kidney volume was positively correlated with a satisfactory eGFR at one year after living donor and deceased donor transplantations.

Small hamstring autograft is defined by a cut-off diameter of 7 mm and not recommended with allograft augmentation in single-bundle ACL reconstruction.

PURPOSE: The present study was to analyze graft failure rates of hamstring tendon (HT) autografts with a cut-off graft diameter of 8 mm or 7 mm, and compare clinical outcomes between augmented small HT with an allograft and non-augmented relatively large HT in single-bundle anterior cruciate ligament reconstruction (ACLR). METHODS: A literature search of PubMed, EMBASE, and the Cochrane Library was performed based on the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-analysis) guidelines. Studies to assess graft failure of autologous HT ACLR were reviewed, and graft failure rates with a cut-off graft diameter of 8 mm or 7 mm were further extracted. Clinical comparative studies of ACLR between augmented small HT with an allograft and non-augmented relatively large HT autografts were also included. Results are presented as risk ratio (RR) for binary data and weighted mean difference for continuous data with 95% confidence intervals (CI). RESULTS: Nine studies with 2243 knees were included. Four studies examined the effect of HT autograft diameter on graft failure and five studies assessed clinical outcomes of allograft augmentation to small HT autografts. No significant difference was noted in graft failure with a cut-off diameter of 8 mm. No significant difference was found between diameters > 7 and ≤ 7 mm, but a significant difference was observed between diameters ≥ 7 and < 7 mm (RR = 0.49; 95% CI 0.26-0.92, I2 = 0%, P = 0.03). A trend towards increased risk of graft failure was noted for allograft-augmented HT compared with non-augmented HT autografts (RR = 0.43; 95% CI 0.18-1.02, I2 = 0%), but no significant differences were noted in IKDC, Lysholm, and Tegner scores between these groups. CONCLUSION: The present study did support the use of 7 mm as a reference for cut-off diameter for small HT autografts, but not allograft augmentation to small HT autografts. These findings would guide clinical application of small HT autografts in single-bundle ACLR. LEVEL OF EVIDENCE: IV.

Hamstring Autograft Too Small: How Much Allograft Do You Need to Supplement to a Desired Hybrid Graft Size?

PURPOSE: To determine a simple rule for choosing supplemental allograft size for hybrid anterior cruciate ligament reconstruction using mathematical and cadaveric models. METHODS: Mathematical and cadaveric models were used to determine the rule. The mathematical model required application of the geometric Pythagorean theorem to add areas of circles. Cadaveric semitendinosus and gracilis tendons were combined in multiple quadrupled hamstring size combinations and then sized using standard surgical techniques to confirm the mathematical model. RESULTS: Geometric measurement, not simple addition, of graft diameters was required to determine the final graft size. Direct comparison of cadaveric and mathematical models showed close relations. If a final graft size of 7 mm is desired, an added diameter of all grafts of approximately 9.5 mm is needed. If a final graft size of 8 mm is desired, an added diameter of all grafts of approximately 11 mm is needed. If a final graft size of 9 mm is desired, an added diameter of all grafts of approximately 12.5 mm is needed. If a final graft size of 10 mm is desired, an added graft diameter of approximately 14 mm is needed. Cadaveric hamstring measurements were similar to the mathematical model. CONCLUSIONS: By use of mathematical and cadaveric models, simple rules for determining the additional size of allograft diameter needed to supplement undersized hamstring autograft were created. CLINICAL RELEVANCE: With the increasing availability of allograft types and sizes, surgeons currently have no guidelines on the size of allograft that is required to supplement an undersized hamstring autograft. Simple rules were created for determining the amount of allograft supplementation required for undersized hamstrings and are easily applied to clinical situations.

Survival Outcomes of Oversized Cardiac Allografts in Pediatric Patients-A Single-Center Experience in Taiwan.

OBJECTIVES: An oversized cardiac allograft may have a negative impact on survival outcomes according to previous studies; however, due to the shortage of pediatric donor hearts, the use of oversized cardiac allografts is sometimes inevitable. In this study, we reported the survival outcomes of pediatric patients in relation with the donor-recipient weight ratio. METHODS: Twenty-eight children, aged 3 months to 17 years, with dilated cardiomyopathy underwent primary cardiac transplantation at the National Taiwan University Hospital between 1995 and 2012. We analyzed these patients according to the donor-recipient weight ratio: group 1 (n = 19) with donor-recipient weight ratio <2.5 (median 1.1, interquartile range 1.0-1.6), and group 2 (n = 9) with donor-recipient weight ratio ≥2.5 (median 3.0, inter-quartile range 2.87-3.5). RESULTS: The 30-day survival rate was 100% for both group 1 and group 2 (P = 1). The survival rates for group 1 and group 2 were 95% vs 100% at 1 year, 84% vs 89% at 5 years, and 73% vs 61% at 10 years. The median survival was 14.4 years vs 12.9 years (P = .6313). CONCLUSION: In this cohort, the use of oversized cardiac allograft in pediatric patients for dilated cardiomyopathy did not have a negative effect on short-term and long-term survival.

Feasibility of using marginal liver grafts in living donor liver transplantation.

Liver transplantation (LT) is one of the most effective treatments for end-stage liver disease caused by related risk factors when liver resection is contraindicated. Additionally, despite the decrease in the prevalence of hepatitis B virus (HBV) over the past two decades, the absolute number of HBsAg-positive people has increased, leading to an increase in HBV-related liver cirrhosis and hepatocellular carcinoma. Consequently, a large demand exists for LT. While the wait time for patients on the donor list is, to some degree, shorter due to the development of living donor liver transplantation (LDLT), there is still a shortage of liver grafts. Furthermore, recipients often suffer from emergent conditions, such as liver dysfunction or even hepatic encephalopathy, which can lead to a limited choice in grafts. To expand the pool of available liver grafts, one option is the use of organs that were previously considered "unusable" by many, which are often labeled "marginal" organs. Many previous studies have reported on the possibilities of using marginal grafts in orthotopic LT; however, there is still a lack of discussion on this topic, especially regarding the feasibility of using marginal grafts in LDLT. Therefore, the present review aimed to summarize the feasibility of using marginal liver grafts for LDLT and discuss the possibility of expanding the application of these grafts.

Survival Outcomes in Split Compared With Whole Liver Transplantation.

Split-liver transplantation (SLT) should be cautiously considered because the right trisection (RTS) graft can be a marginal graft in adult recipients. Herein, we analyzed the outcomes of RTS-SLT in Korea, where >75% of adult liver transplantations are performed with living donor liver transplantation. Among 2462 patients who underwent deceased donor liver transplantations (DDLTs) from 2005 to 2014, we retrospectively reviewed 86 (3.5%) adult patients who received a RTS graft (RTS-SLT group). The outcomes of the RTS-SLT group were compared with those of 303 recipients of whole liver (WL; WL-DDLT group). Recipient age, laboratory Model for End-Stage-Liver Disease (L-MELD) score, ischemia time, and donor-to-recipient weight ratio (DRWR) were not different between the 2 groups (P > 0.05). However, malignancy was uncommon (4.7% versus 36.3%), and the donor was younger (25.2 versus 42.7 years) in the RST-SLT group than in the WL-DDLT group (P < 0.05). The technical complication rates and the 5-year graft survival rates (89.0% versus 92.8%) were not different between the 2 groups (P > 0.05). The 5-year overall survival (OS) rate (63.1%) and graft-failure-free survival rate (63.1%) of the RTS-SLT group were worse than that of the WL-DDLT group (79.3% and 79.3%; P < 0.05). The factors affecting graft survival rates were not definite. However, the factors affecting OS in the RTS-SLT group were L-MELD score >30 and DRWR ≤1.0. In the subgroup analysis, OS was not different between the 2 groups if the DRWR was >1.0, regardless of the L-MELD score (P > 0.05). In conclusion, a sufficient volume of the graft estimated from DRWR-matching could lead to better outcomes of adult SLTs with a RTS graft, even in patients with high L-MELD scores.

Living Donor Liver Transplantation: Technical Innovations.

Living donor liver transplantation (LDLT) has found a place to serve the end-stage liver disease community as the donor safety and recipient suitability has been elucidated. Donor safety is of paramount importance and transplant programs must continue endeavors to maintain the highest possible standards. At the same time, adequacy of grafts based on recipient clinical status via their model for end-stage liver disease (MELD) score and volumetric studies to achieve a GRBWR >0.8, along with special attention to anatomic tailoring and portal venous flow optimization are necessary for successful transplantation. Technical innovations have improved sequentially the utility and availability of LDLT.

Free margin length and coaptation surface area in normal tricuspid aortic valve: an anatomical study.

OBJECTIVES: Aortic cusp free margins are a central target in most aortic valve repair operations to optimize valve coaptation. The objective of this anatomical study was to analyse the normal dimensions of free margin length (FML) and coaptation surface and to analyse their relationship with other valve and root dimensions in normal tricuspid aortic valves. METHODS: We analysed 25 aortic root homografts. Eight valve and root measurements were obtained from fresh specimens including the length of the free margin while applying appropriate tension on the structures. The valves were then fixed with formalin in the diastolic position under pressure to allow measurement of the coaptation surface. In addition to normal values, we analysed the correlations and ratios between the different measures. RESULTS: The mean FML was 34.3 ± 3.1 mm. The FML was similar between the 3 cusps and correlated with all other valve and root measures. The ratio of the FML to the geometric height was 1.81, and the free edge length/sinotubular junction was 1.29. The mean coaptation surface was 122 ± 21 mm2 per cusp and corresponded to 41% of the cusp surface. The central coaptation length was 3.3 ± 0.8 mm, and the lateral coaptation length was 5.9 ± 0.6 mm. The total coaptation surface per specimen was 184 ± 32 mm2. CONCLUSIONS: We described the normal dimensions of the FML and coaptation surface in the tricuspid aortic valve. These measurements will serve in the further development of an objective method of free margin shortening based on intraoperative measurements of the FML to treat cusp prolapse and low coaptation after valve-sparing surgery.

Small-for-size grafts increase recurrence of hepatocellular carcinoma in liver transplantation beyond milan criteria.

Living donor liver transplantation (LDLT) has been reported to have high rates of hepatocellular carcinoma (HCC) recurrence compared with deceased donor liver transplantation (DDLT). This has been assumed to be due to the frequent use of small-for-size grafts (SFSGs) in LDLT rather than DDLT, but the relationship between graft size and prognosis remains controversial. This study aimed to clarify the effect of SFSGs on the oncologic outcomes of patients with HCC who underwent LDLT. Between January 2005 and December 2015, 597 consecutive patients underwent LDLT. Among these patients, those with HCC who underwent LDLT were randomly matched at a 1:3 ratio (graft-to-recipient body weight ratio [GRWR] < 0.8%:GRWR > 0.8%) according to propensity score. HCC recurrence and patient survival were analyzed using the Kaplan-Meier method and log-rank test. In addition, stratified subgroup analysis based on the Milan criteria was performed. SFSG was defined as a GRWR < 0.8%. Using propensity score matching, 82 patients with GRWR < 0.8% and 246 patients with GRWR ≥ 0.8% were selected. For patients with HCC within the Milan criteria, no significant difference of HCC recurrence (P = 0.82) and patient survival (P = 0.95) was found based on GRWR. However, for patients with HCC beyond the Milan criteria, 1-, 3-, and 5-year recurrence-free survival rates were 52.4%, 49.3%, and 49.3%, respectively, for patients with GRWR < 0.8%, and 76.5%, 68.3%, and 64.3%, respectively, for patients with GRWR ≥ 0.8% (P = 0.049). The former group exhibited poor patient survival rates (P = 0.047). In conclusion, for patients with HCC within the Milan criteria, no significant difference in oncologic outcomes was found based on liver graft size. However, among the patients with HCC beyond the Milan criteria, SFSG recipients showed poor oncologic outcomes. Because extended criteria are frequently used in LDLT for HCC, a recipient's prognosis can be improved if a liver graft of appropriate size is carefully selected during donor selection. Liver Transplantation 24 35-43 2018 AASLD.

Comparable short- and long-term outcomes in deceased-donor and living-donor liver retransplantation.

BACKGROUND AND PURPOSE: There is a big controversy over liver retransplantation, the only life-saving treatment for patients with a failing or failed liver graft. This retrospective study tried to determine if living-donor liver retransplantation (re-LDLT) is a justifiable alternative to deceased-donor liver retransplantation (re-DDLT). METHODS: Anonymous data of liver transplant patients from January 2000 to April 2016 were reviewed. Recipients of retransplantation were divided into the re-DDLT and re-LDLT groups. The groups were compared in demographic characteristics, pre-retransplant and intraoperative details, and short- and long-term outcomes. Risk for living donors was examined. RESULTS: Twenty-nine patients had 33 re-DDLTs and 15 patients received re-LDLT. The re-LDLT group had lighter grafts (525 vs. 1295 g, p ≤ 0.001), a smaller ratio of graft weight to recipient standard liver volume (56.98 vs. 107.7%, p ≤ 0.001), and shorter cold ischemia (106 vs. 451 min, p ≤ 0.001). The groups were otherwise comparable. Two patients in the re-DDLT group had Grade-5 complication. The groups were similar in patient survival (p = 0.326) and graft survival (p = 0.102). No living donors died, but three of them developed Grade-1 complications. CONCLUSION: With the required expertise, re-LDLT can produce results comparable to those of re-DDLT while keeping donor risk at bay. In places where the demand for deceased-donor liver grafts far outstrips supply, re-LDLT can be considered as an alternative to re-DDLT if the expertise is available and if the potential recipient benefits can balance out the potential donor risks.

Effects of recipient size and allograft type on pediatric liver transplantation for biliary atresia.

The majority of pediatric patients with end-stage liver disease receive a transplant with a whole liver (WL) allograft. However, smaller recipients with biliary atresia (BA) may have improved outcomes with deceased donor partial liver (DDPL) or living donor allografts. This study compares the national outcomes for liver transplantation in BA, with attention to the interaction between liver allograft type and recipient size. From January 2, 2002 to December 30, 2014, 2123 pediatric patients underwent a primary liver transplant for BA. The majority of transplants (53%) were performed with a WL allograft. Utilization of a WL allograft increased from 42% of recipients weighing ≤ 7 kg to 74% of recipients weighing > 14 kg. The 1-, 5-, and 10-year graft survival in recipients weighing ≤7 kg was significantly superior for living donor liver transplantation (LDLT) (91%, 88%, 84%) and DDPL allografts (90%, 84%, 77%) compared with WL allografts (79%, 75%, 74%; P = 0.005). The 1-, 5-, and 10-year graft survival in recipients weighing >14 kg trended toward being inferior in recipients of DDPL allografts (85%, 85%, 71%) compared with WL allografts (96%, 91%, 86%; P = 0.06). Furthermore, the incidence of vascular thrombosis was highest in WL (13%) compared with LDLT (6%) and DDPL (5%) recipients ≤ 7 kg (P = 0.002). Liver retransplantation was also highest in WL (16%) compared with LDLT (9%) and DDPL (9%) recipients ≤ 7 kg (P = 0.02). In conclusion, strong consideration should be given to the use of technical variant allografts in small recipients with BA requiring liver transplantation. Liver Transplantation 23 221-233 2017 AASLD.

Allograft selection for distal femur through cutting contour registration.

Allograft reconstruction is an acceptable procedure for the recovery of normal anatomy after the bone tumor resection. During the past few years, several automated methods have been proposed to select the best anatomically matching allograft from the virtual donor bone bank. The surface-based automated method uses the contralateral healthy bone to obtain the normal surface shape of the diseased bone, which could achieve good matching of the defect and the selected allograft. However, the surface-based method focuses on the matching of the whole bone so that the matching of the contact surface between the allograft and the recipient bone may not be optimal. To deal with the above problem, we propose a cutting contour based method for the allograft selection. Cutting contour from the recipient bone could reflect the structural information of the defect and is seldom influenced by tumor. Thus the cutting contour can be used as the matching template to find the optimal alignment of the recipient bone and the allograft. The proposed method is validated using the data of distal femurs where bone transplantation is commonly performed. Experimental results show that the proposed method generally outperforms the surface-based method within modest extra time. Overall, our contour-based method is an effective complementary technique for allograft selection in the virtual bone bank.

Lyophilized allografts without pre-treatment with glutaraldehyde are more suitable than cryopreserved allografts for pulmonary artery reconstruction.

Various methods are available for preservation of vascular grafts for pulmonary artery (PA) replacement. Lyophilization and cryopreservation reduce antigenicity and prevent thrombosis and calcification in vascular grafts, so both methods can be used to obtain vascular bioprostheses. We evaluated the hemodynamic, gasometric, imaging, and macroscopic and microscopic findings produced by PA reconstruction with lyophilized (LyoPA) grafts and cryopreserved (CryoPA) grafts in dogs. Eighteen healthy crossbred adult dogs of both sexes weighing between 18 and 20 kg were used and divided into three groups of six: group I, PA section and reanastomosis; group II, PA resection and reconstruction with LyoPA allograft; group III, PA resection and reconstruction with CryoPA allograft. Dogs were evaluated 4 weeks after surgery, and the status of the graft and vascular anastomosis were examined macroscopically and microscopically. No clinical, radiologic, or blood-gas abnormalities were observed during the study. The mean pulmonary artery pressure (MPAP) in group III increased significantly at the end of the study compared with baseline (P=0.02) and final [P=0.007, two-way repeat-measures analysis of variance (RM ANOVA)] values. Pulmonary vascular resistance of groups II and III increased immediately after reperfusion and also at the end of the study compared to baseline. The increase shown by group III vs group I was significant only if compared with after surgery and study end (P=0.016 and P=0.005, respectively, two-way RM ANOVA). Microscopically, permeability was reduced by ≤75% in group III. In conclusion, substitution of PAs with LyoPA grafts is technically feasible and clinically promising.

Lyophilized allografts without pre-treatment with glutaraldehyde are more suitable than cryopreserved allografts for pulmonary artery reconstruction

Various methods are available for preservation of vascular grafts for pulmonary artery (PA) replacement. Lyophilization and cryopreservation reduce antigenicity and prevent thrombosis and calcification in vascular grafts, so both methods can be used to obtain vascular bioprostheses. We evaluated the hemodynamic, gasometric, imaging, and macroscopic and microscopic findings produced by PA reconstruction with lyophilized (LyoPA) grafts and cryopreserved (CryoPA) grafts in dogs. Eighteen healthy crossbred adult dogs of both sexes weighing between 18 and 20 kg were used and divided into three groups of six: group I, PA section and reanastomosis; group II, PA resection and reconstruction with LyoPA allograft; group III, PA resection and reconstruction with CryoPA allograft. Dogs were evaluated 4 weeks after surgery, and the status of the graft and vascular anastomosis were examined macroscopically and microscopically. No clinical, radiologic, or blood-gas abnormalities were observed during the study. The mean pulmonary artery pressure (MPAP) in group III increased significantly at the end of the study compared with baseline (P=0.02) and final [P=0.007, two-way repeat-measures analysis of variance (RM ANOVA)] values. Pulmonary vascular resistance of groups II and III increased immediately after reperfusion and also at the end of the study compared to baseline. The increase shown by group III vs group I was significant only if compared with after surgery and study end (P=0.016 and P=0.005, respectively, two-way RM ANOVA). Microscopically, permeability was reduced by ≤75% in group III. In conclusion, substitution of PAs with LyoPA grafts is technically feasible and clinically promising.

Computed Tomography and Optical Imaging of Osteogenesis-angiogenesis Coupling to Assess Integration of Cranial Bone Autografts and Allografts.

A major parameter determining the success of a bone-grafting procedure is vascularization of the area surrounding the graft. We hypothesized that implantation of a bone autograft would induce greater bone regeneration by abundant blood vessel formation. To investigate the effect of the graft on neovascularization at the defect site, we developed a micro-computed tomography (µCT) approach to characterize newly forming blood vessels, which involves systemic perfusion of the animal with a polymerizing contrast agent. This method enables detailed vascular analysis of an organ in its entirety. Additionally, blood perfusion was assessed using fluorescence imaging (FLI) of a blood-borne fluorescent agent. Bone formation was quantified by FLI using a hydroxyapatite-targeted probe and µCT analysis. Stem cell recruitment was monitored by bioluminescence imaging (BLI) of transgenic mice that express luciferase under the control of the osteocalcin promoter. Here we describe and demonstrate preparation of the allograft, calvarial defect surgery, µCT scanning protocols for the neovascularization study and bone formation analysis (including the in vivo perfusion of contrast agent), and the protocol for data analysis. The 3D high-resolution analysis of vasculature demonstrated significantly greater angiogenesis in animals with implanted autografts, especially with respect to arteriole formation. Accordingly, blood perfusion was significantly higher in the autograft group by the 7(th) day after surgery. We observed superior bone mineralization and measured greater bone formation in animals that received autografts. Autograft implantation induced resident stem cell recruitment to the graft-host bone suture, where the cells differentiated into bone-forming cells between the 7(th) and 10(th) postoperative day. This finding means that enhanced bone formation may be attributed to the augmented vascular feeding that characterizes autograft implantation. The methods depicted may serve as an optimal tool to study bone regeneration in terms of tightly bounded bone formation and neovascularization.

Effects of FK-506 and CTLA4--Ig on nerve allografts in mice.

The purpose of this study is to evaluate the effects of FK-506 and cytotoxic T lymphocyte-associated antigen-4 immunoglobulin fusion protein (CTLA4--Ig) on nerve allografts. Adult male inbred C3H mice (mean weight, 25 g) surgically received 8 mm posterior tibial nerve defects, and donor nerve allografts from donor male C57BL mice were implanted. The experimental animals were divided into five groups of 12 animals each that were distinguished from each other by administration of FK-506 and CTLA4--Ig, that is, isografts interposed in the gap between contralateral posterior tibial nerves (group A, positive control); allografts from C57BL mice implanted without administering any treatment (group B, negative control); allografts from C57BL mice implanted and FK-506 injections administered (group C); allografts from C57BL mice implanted and CTLA4--Ig injections administered (group D); and allografts from C57BL mice implanted and FK-506 and CTLA4--Ig injections administered (group E). Postoperative walking-track functional analysis and histomorphometric studies were then conducted. Data were statistically analysed using the Kruskal-Wallis test. Compared with the negative control (group B), mice treated with both FK-506 and CTLA4--Ig (group E) demonstrated better results at 3 weeks post operation. Similar values were observed in all groups, and there were no statistical differences at 6 weeks post operation. Our results demonstrate that a co-administration of FK-506 and CTLA4--Ig results in functional and histomorphometric recovery that is superior to that seen in the absence of these medications.

In vitro evaluation of demineralized freeze-dried bone allograft in combination with enamel matrix derivative.

BACKGROUND: Preclinical and clinical studies suggest that a combination of enamel matrix derivative (EMD) with demineralized freeze-dried bone allograft (DFDBA) may improve periodontal wound healing and regeneration. To date, no single study has characterized the effects of this combination on in vitro cell behavior. The aim of this study is to test the ability of EMD to adsorb to the surface of DFDBA particles and determine the effect of EMD coating on downstream cellular pathways such as adhesion, proliferation, and differentiation of primary human osteoblasts and periodontal ligament (PDL) cells. METHODS: DFDBA particles were precoated with EMD or human blood and analyzed for protein adsorption patterns via scanning electron microscopy. Cell attachment and proliferation were quantified using a commercial assay. Cell differentiation was analyzed using real-time polymerase chain reaction for genes encoding Runx2, alkaline phosphatase, osteocalcin, and collagen 1α1, and mineralization was assessed using alizarinred staining. RESULTS: Analysis of cell attachment revealed no significant differences among control, blood-coated, and EMD-coated DFDBA particles. EMD significantly increased cell proliferation at 3 and 5 days after seeding for both osteoblasts and PDL cells compared to control and blood-coated samples. Moreover, there were significantly higher messenger ribonucleic acid levels of osteogenic differentiation markers, including collagen 1α1, alkaline phosphatase, and osteocalcin, in osteoblasts and PDL cells cultured on EMD-coated DFDBA particles at 3, 7, and 14 days. CONCLUSION: The results suggest that the addition of EMD to DFDBA particles may influence periodontal regeneration by stimulating PDL cell and osteoblast proliferation and differentiation.