Xenograft Group Research Articles

Experimental study of rabbit bone mesenchymal stem cells seeding onto xenogenic calf cortical bone with partial trabecular bone

To explore the possibility of xenogenic calf cortical bone with partial trabecular bone as cell carriers in tissue engineering, and to provide direct evidence for clinical application of matrix scaffold. Rabbit bone mesenchymal stem cells (BMSCs) were seperated and cultured on the surface of xenogenic calf cortical bone with partial trabecular bone. Relative growth rate was measured and cytotoxicity was graded. The growth, proliferation and matrix secretion of BMSCs in calf cortical strut were observed by scanning electron microscope. Sixty 3-month-old New Zealand rabbits were divided into 3 groups (20 in each group): a BMSCs/bone xenograft group, a simple bone xenograft group and an autogenous iliac bone group. Each group was implanted with the BMSCs induced osteogenic composite calf bone, simple bone xenograft and autogenous iliac bone into the ala of ilium. The expressions of bone morphogenetic protein-2 (BMP-2) mRNA were detected by reverse transcription polymerase chain reaction in 4, 8, 12, 24 weeks after the operation. The toxicity gradation was 0 to 1. BMSCs adhered the surface of calf cortical strut and extended in the cancellous bone, and extracellular matrices were found. The BMP-2 mRNA expression in the simple bone xenograft group was lower than that in the BMSCs/bone xenograft group and the autogenous iliac bone group at all time points (P<0.05),which was lower in the BMSCs/bone xenograft group than that in the autogenous iliac bone group (P<0.05). Rabbit BMSCs combined with calf cortical bonescaffold with partial cancellous bone have good osteogenic capacity in the New Zealand rabbits.

Retrospective Evaluation of Crestal Bone Changes Around Implants With Reduced Abutment Diameter Placed Non‐Submerged and at Subcrestal Positions: The Effect of Bone Grafting at Implant Placement

There is limited information regarding the effect of grafting of the osteotomy after subcrestal implant placement. The primary aim of this study is to retrospectively evaluate the effect of bone grafting of the defect between the bone crest and the coronal aspect of implants with reduced abutment diameter placed non-submerged and at subcrestal positions. Records of 50 consecutive patients treated with subcrestally placed dental implants grafted with a xenograft (Group A) and 50 consecutive patients with subcrestally placed dental implants without any grafting material (Group B) were reviewed. For each implant, the radiographs after placement were compared to images from the last follow-up visit and evaluated regarding the following: 1) degree of subcrestal positioning of the implant, 2) changes of marginal hard-tissue height over time, and 3) whether marginal hard-tissue could be detected on the implant platform at the follow-up visit. The mean marginal loss of hard tissues was 0.11 ± 0.30 mm for Group A and 0.08 ± 0.22 mm for Group B. Sixty-nine percent of the implants in Group A and 77% of the implants in Group B demonstrated hard tissue on the implant platform. There were no statistically significant differences between the groups regarding marginal peri-implant hard-tissue loss. The present study fails to demonstrate that grafting of the remaining osseous wound defect between the bone crest and the coronal aspect of the implant has a positive effect on marginal peri-implant hard-tissue changes.

The Contegra bovine jugular vein graft versus the Shelhigh pulmonic porcine graft for reconstruction of the right ventricular outflow tract: A comparative study

Reconstruction of the right ventricular outflow tract plays a major role in congenital cardiac surgery. With the advent of the Contegra bovine jugular vein graft and the Shelhigh pulmonic xenograft, hopes were high that the lack of availability of homografts would be overcome. The present study evaluated both grafts and investigated the influence of known risk factors for premature graft failure. From December 1999 to September 2008, 84 consecutive patients (mean age, 12 ± 15 years) with a total of 100 implanted conduits (43 Contegra bovine jugular vein grafts and 57 Shelhigh pulmonic xenografts) were included in this study. Primary end points were reintervention, reoperation, and death. The rate of overall conduit replacement was 25% for the Shelhigh pulmonic xenograft versus 26% for the Contegra bovine jugular vein graft. The predominant mode of failure was conduit stenosis for both groups (23% for the Shelhigh pulmonic xenograft vs 19% for the Contegra bovine jugular vein graft), with a mean time to replacement of 18 ± 9 months for the Shelhigh pulmonic xenograft versus 42 ± 4 months for the Contegra bovine jugular vein graft (P = .25). Histopathological analysis revealed a similar chronic inflammatory reaction in both conduits, but it was significantly stronger in the Shelhigh pulmonic xenograft group. The Contegra bovine jugular vein graft showed frequently the formation of a stenotic membrane at the distal anastomosis site. Age of less than 1 year, body surface area, pulmonary stenosis, and conduit size of less than 14 mm could not be identified as risk factors for premature failure. Both conduits fail predominantly because of stenosis and are subject to a chronic inflammatory reaction, although this was stronger in the Shelhigh pulmonic xenograft group. Mean time to replacement was 18 ± 9 months for the Shelhigh pulmonic graft group versus 42 ± 4 months for the Contegra bovine graft group (P = .25). Because there is a trend toward earlier failure in the Shelhigh pulmonic xenograft group, we currently prefer to implant the Contegra bovine jugular vein graft for right ventricular outflow tract reconstruction.

Contrast enhanced maximum intensity projection ultrasound imaging for assessing angiogenesis in murine glioma and breast tumor models: A comparative study

The purpose of this study was to prospectively compare noninvasive, quantitative measures of vascularity obtained from four contrast enhanced ultrasound (US) techniques to four invasive immunohistochemical markers of tumor angiogenesis in a large group of murine xenografts. Glioma (C6) or breast cancer (NMU) cells were implanted in 144 rats. The contrast agent Optison (GE Healthcare, Princeton, NJ) was injected in a tail vein (dose: 0.4 ml/kg). Power Doppler imaging (PDI), pulse-subtraction harmonic imaging (PSHI), flash-echo imaging (FEI), and Microflow imaging (MFI; a technique creating maximum intensity projection images over time) was performed with an Aplio scanner (Toshiba America Medical Systems, Tustin, CA) and a 7.5 MHz linear array. Fractional tumor neovascularity was calculated from digital clips of contrast US, while the relative area stained was calculated from specimens. Results were compared using a factorial, repeated measures ANOVA, linear regression and z-tests. The tortuous morphology of tumor neovessels was visualized better with MFI than with the other US modes. Cell line, implantation method and contrast US imaging technique were significant parameters in the ANOVA model ( p < 0.05). The strongest correlation determined by linear regression in the C6 model was between PSHI and percent area stained with CD31 ( r = 0.37, p < 0.0001). In the NMU model the strongest correlation was between FEI and COX-2 ( r = 0.46, p < 0.0001). There were no statistically significant differences between correlations obtained with the various US methods ( p > 0.05). In conclusion, the largest study of contrast US of murine xenografts to date has been conducted and quantitative contrast enhanced US measures of tumor neovascularity in glioma and breast cancer xenograft models appear to provide a noninvasive marker for angiogenesis; although the best method for monitoring angiogenesis was not conclusively established.

Measurement of reoxygenation during fractionated radiotherapy in head and neck squamous cell carcinoma xenografts

Hypoxic tissues lack adequate oxygenation and it has been long established that tumours commonly exhibit hypoxia and that hypoxia is a factor contributing towards resistance to radiotherapy. To develop computer models and make predictions about the affects of tumour hypoxia on treatment outcome, quantitative tumour oxygenation and reoxygenation data from in vivo systems is required. The aim of this study was to investigate the timing and degree of reoxygenation during radiotherapy in a human head and neck squamous cell carcinoma xenograft mouse model (FaDu). Mice were immobilised using a novel restraining system and exposed unanaesthetised in 3 or 5 Gy fractions, up to a maximum of 40 Gy. Partial pressures of oxygen (pO2) measurements were recorded at six time points throughout the 2 week course of radiotherapy, using a fibre optic system. Tumours receiving 0-30 Gy did not exhibit an increase in pO2. However, the mean pO2 after 2 weeks of accelerated fractionated radiotherapy (40 Gy) was significantly increased (P<0.01) compared to the mean pO2 of tumours not receiving the full schedule (0-30 Gy). These results lead to the conclusion of an average reoxygenation onset time of 2 weeks in this group of xenografts. A relatively large range of pO2 values measured at each dose point in the study indicate a large inter-tumour variation in oxygenation among the tumours. Data from this experimental work will be used to define the range of reoxygenation onset times implemented in a Monte Carlo computer model, simulating hypoxic head and neck cancer growth and radiotherapy.

Detection of oesophageal cancer biomarkers by plasma proteomic profiling of human cell line xenografts in response to chemotherapy

Background:The incidence of oesophageal adenocarcinoma is increasing worldwide but survival remains poor. Neoadjuvant chemotherapy may improve survival, but targeting treatment to patients who respond to chemotherapy could be improved by the availability of markers of response. This study sought proteomic markers of therapeutic response using an adenocarcinoma xenograft model.Methods:Epirubicin, cisplatin or 5-fluorouracil was administered to severe combined immune-deficient mice bearing OE19 oesophageal adenocarcinoma xenografts. Murine plasma samples from treated and untreated xenografts were analysed by surface-enhanced laser desorption/ionisation time-of-flight mass spectroscopy, and panels of peaks were found using class prediction models that distinguished treatment groups. Proteins in these peaks were identified by mass spectroscopy in tryptic digests of purified fractions. Five paired samples from oesophageal cancer patients before and after chemotherapy were analysed using the same methodology.Results:Plasma protein peaks were identified that differed significantly (P<0.05, ANOVA) between the treated xenograft and control groups. Marker panels predicted treated vs untreated xenografts with sensitivities of 100%, specificities of 86–100% and test efficiencies of 89–100%. Three of the proteins identified in these panels, apolipoprotein A-I, serum amyloid A and transthyretin were confirmed in the clinical samples.Conclusion:Plasma protein markers can be detected in response to chemotherapy in oesophageal adenocarcinoma xenografts and in clinical samples, and have the potential to monitor response and guide chemotherapy in oesophageal adenocarcinoma.

Gene array studies in orthotopic pig to baboon cardiac xenotransplantation

Xenograft survival is a balance between immunological rejection and adaptive changes in the graft. Immunohistochemical analysis of vascular immunoglobulin deposition in heterotopic and orthotopic pig to primate cardiac xenografts shows vascular antibody deposition as early as 30 min after reperfusion and throughout the transplant period in both GT+ and GTKO hearts. Despite the abundance of antibody, graft contractility in heterotopic transplants and the ability of the graft to support orthotopic recipients remained high, with little evidence of microvascular thrombosis for extended periods of time. This suggests that the graft is actively adapting to chronic antibody exposure to forestall thrombosis.To investigate the effects of chronic antibody exposure we used an Affymetrix porcine gene array to study the changes in gene expression in orthotopic transplant recipients. Four sets of mRNA were studied, Group 1: normal porcine hearts were used as controls (n = 2), Group 2: long surviving pig‐to‐primate orthotopic xenografts (n = 5), Group 3: orthotopic xenografts which failed in the perioperative period to support the recipient (n = 7) and Group 4: orthotopic xenografts that were rejected (n = 3). RNA from each group was used to produce cDNA for hybridization to an Affymetrix porcine gene array (?900624). This array contains 23 935 probes. We used a previously reported extended annotation database which provides annotations for 21 122 gene titles [1]. The raw fluorescence was normalized and each probe was subjected to a present/absent test using the Affymetrix MAS 5.0 algorithm. A Student's t‐test was used to compare expression of each probe in the experimental groups (Groups 2–4) to Group 1 the control hearts. The DAVID Bioinformatics database [2] was used to identify enrichments in Kegg pathways and in Gene Ontogeny attributes.Orthotopic cardiac xenograft recipients survived for 14, 23, 34, 40 and 57 days without rejection. Recipients died due to sudden death for unknown cause, atrial hemorrhage, pulmonary hemorrhage, pneumonitis and sepsis respectively. Histologic analysis of these hearts at necropsy showed minimal injury (10%) in three recipients and moderate damage (35–45%) in two recipients. In no instance was death due to heart failure. Group 3 hearts failed within 48 hours of transplant. These hearts initially exhibited good cardiac function, based on echocardiography but could not maintain that function as they were weaned from inotropic support. Histology of the failed hearts showed vascular antibody deposition but only rarely was complement deposition (C5b) detected. There was moderate ischemic injury to the myocardium but little evidence of hemorrhage. In no instance was heart failure due to hyperacute rejection. The rejected orthotopic xenografts failed on days 9, 14 and 25. Histology showed widespread vascular antibody staining and complement deposition.Real time PCR validation of the gene array data was performed with 58 probes using RNA from the long surviving xenograft (Group 2). There was greater than 90% correlation. Groups 2–4 showed a similar number of probes which passed the exclusion test (16 006 Group 2, 16 991 Group 3 and 16 214 Group 4), however, rejected hearts (Group 4) showed the fewest number of probes with significant deviation from the control samples. Genes with highly altered levels of expression (more that 3 standard deviations from the controls) were variably regulated between Groups 2–4. For example, long surviving orthotopic hearts (Group 2) exhibited an equal proportion of genes with highly increased or decreased expression. In rejected hearts (Group 4) genes with highly altered expression were overwhelming due to decreased gene expression. All experimental groups showed increased expression of genes commonly associated with myocardial injury.Bioinformatics analysis detected enrichment in genes involved in KEGG pathways for focal adhesion, extracellular matrix receptor interactions and cell communication in the long surviving orthotopic hearts (Group 2). Additionally there was enrichment in several metabolic pathways. The changes in metabolic gene expression were also evident, to lesser degree, in the rejected hearts but not observed in hearts which failed during the perioperative period (Group 3). These results suggest that long surviving orthotopic cardiac xenografts maybe undergo extracellular remodeling and have altered metabolic activity. This analysis suggests that chronic antibody exposure may effect endothelial cell functions beyond haemostasis which promote cardiac remodeling and altered metabolism and may contribute to graft failure.This work was supported by NIH grant #U01 AI066310

Abstract B27: A preclinical mouse model of orthotopic pleural cancer that facilitates noninvasive quantitative monitoring of tumor progression and therapy response

Abstract Purpose: The purpose of our study is to (a) establish a reproducible mouse model of orthotopic pleural cancer that recapitulates the pathobiology of human pleural malignancy (including primary malignant pleural mesothelioma, lung and metastatic breast cancers), and (b) validate the use of bioluminescence imaging (BLI) and serum biomarker assessment to noninvasively monitor tumor progression and therapy response in preclinical therapeutic trials. Experimental Design and Results: Human mesothelioma and lung cancer cell lines with variable expression levels of the tumor-associated antigen, mesothelin, were retrovirally transduced to stably express GFP-Firefly Luciferase, to facilitate BLI. Orthotopic xenograft mice were established by direct tumor cell inoculation into the pleural space with &amp;gt; 95% engraftment in both athymic nude and SCID-beige mice. The pathobiologic resemblance of the xenograft tumors to human disease was confirmed by demonstrating: (a) visceral and parietal pleural tumor growth 58 pattern, mediastinal lymph node involvement, and diaphragmatic invasion by magnetic resonance imaging (MRI) and histology, (b) strong, sustained expression of tumor markers at advanced stage of disease by WT-1, Calretinin, Mesothelin, and TTF-1 immunohistochemistry, and (c) extensive tumor lymphangiogenesis by CD-34 and LYVE-1 immunofluorescence. Tumor burden correlated with BLI signal (total flux photons/s) and soluble mesothelin-related peptide, SMRP (Mesomark® ELISA assay) both in vitro and in vivo (Pearson's correlation r=0.998, p&amp;lt;0.0001 and r=0.89, p=0.04, respectively). We determined the accuracy of BLI and SMRP (range 0.4–23nM) in monitoring tumor progression by serial assessment of xenograft mice (n=36, with initial tumor burden of 1×103–1×106 cells). In the orthotopic pleural xenografts, BLI signal correlated with SMRP (r=0.56, p=0.0001); no such correlation was observed in subcutaneous flank or intraperitoneal xenografts. To further demonstrate the reliability of BLI and SMRP to noninvasively monitor therapeutic response, groups of pleural mesothelioma xenografts (n=28) were treated with either: (a) Cisplatin (4mg/kg IP weekly), (b) thoracic radiation (20Gy total in 5 fractions), (c) combination chemoradiation, or (d) no treatment. BLI, SMRP, and survival were followed serially. Therapy response was demonstrated by decreased BLI intensity in mice treated with chemoradiation compared to controls (3.7×107 vs 2.0×108 photons/s, respectively, p=0.04) as well as decreased SMRP (p=0.03) which correlated with improved survival by Kaplan-Meier analysis (64 vs 34.5 days, p=0.0001). Conclusions: Pleural involvement by solid tumors remains a significant problem in clinical oncology affecting nearly 250,000 patients annually in the U.S. with no validated preclinical model for therapeutic investigations. We have validated a clinically relevant, reproducible orthotopic model of pleural malignancy that recapitulates human disease and allows for noninvasive quantitative monitoring of pleural tumor burden, progression and therapeutic response. Our xenograft model provides a novel platform to investigate targeted therapies for pleural malignancies. Citation Information: Cancer Res 2009;69(23 Suppl):B27.

Medium Term Outcome of Laparoscopic Sacrocolpopexy With Xenografts Compared to Synthetic Grafts

We compared the medium term anatomical and subjective outcome, and graft related complications after sacrocolpopexy for apical vaginal prolapse using xenogenic or polypropylene grafts. We prospectively studied 50 consecutive patients who underwent laparoscopic sacrocolpopexy with porcine grafts of small intestinal submucosa (21) or dermal collagen (29). We compared these patients to 100 consecutive controls in whom polypropylene was used, and underwent surgery before (50) or after (50) the cases. The primary outcome was anatomical cure (stage I or less at any compartment). Secondary outcomes were graft related complications, subjective cure and quality of life evaluated by clinical examination, standardized interview and prolapse specific questionnaire, respectively. At study closure 129 (86%) patients were available for functional evaluation and 104 (67%) were available for anatomical evaluation. Mean postoperative followup (+/-33 months) and baseline characteristics except age were comparable. The overall anatomical failure rate was comparable (49% vs 34%, p = 0.053) but failures at the vault (21% vs 3%, p <0.01) and posterior compartment (36% vs 19%, p <0.05) were more frequent in the xenograft group. There were 6 reoperations including secondary laparoscopic sacrocolpopexy (5) and cystocele repair (1), all confined to the xenograft group (p <0.01). Graft related complications were equally frequent (11%) in the xenograft and polypropylene groups. The reoperation rate for graft related complications was not different (xenograft 3% vs controls 11%, p = 0.20). There were no differences in functional outcome and quality of life between xenografts and controls. While overall anatomical failure was comparable, sacrocolpopexy using xenograft was associated with more apical failures and reoperations for prolapse than with polypropylene without differences in functional outcome. The use of xenografts did not decrease the number of graft related complications.

Comparison of novel xenograft (bovine fetal growth plate) and allograft effects on experimental bone defect healing in rabbit

Large bone defects resulting from trauma, tumors, osteitis, implant loosening, or corrective osteotomies require surgical therapy because spontaneous regeneration is limited to relatively small defects. Currently, transplantation of autografts or allografts, mineral bone substitutes, and callus distraction are the most commonly used techniques for skeletal reconstruction. Each method has significant limitations, e.g., availability and biological or biomechanical reasons. This study was designed to evaluate allograft and new xenograft (bovine fetal growth plate) effects on the bone healing process. Twenty male New Zealand White rabbits were used in this study. In the allograft group, the defect was filled by fresh allogeneic cortical graft; in the xenograft group, the defect was filled by a segment of bovine fetal growth plate and was fixed by cerclage wire. Radiological, histopathological, and biomechanical evaluations were performed and results were scored and analyzed statistically. Statistical tests did not support significant differences between the two groups radiographically at the 14th postoperative day (P > 0.05). There was a significant difference in bone formation at the 28th, 42nd, and 56th postoperative days. There were significant radiological differences for bone union and remodeling by the 42nd day postoperatively (P < 0.05). The xenograft was superior to the allograft by the 56th postoperative day for radiological bone formation (P < 0.03); histopathological and biomechanical evaluation revealed no significant differences between the two groups. It can be concluded that the superior bone healing process in the xenograft group was due to the presence of some osteoinduction proteins in bovine fetal growth plate.

Natural Bovine Anorganic Apatite and Collagen Presents Osteoconductivity and Contribute to Bone Repair of Rat Calvaria Critical Size Defect

The aim of this study was verify the biological efficacy of the use of a xenograft for bone loss therapy. Blood clot, particulate autogenous bone or anorganic bovine xenograft filled critical size defects (CSD) in rat calvaria (8mm diameter). After 0, 7, 30 and 90 days the animals were killed and macroscopic, radiographic and histopathological analysis were conducted. Although no treatment promoted the total closure of bone defect, autogenous bone group had better bone repair after 90 days, followed by xenograft group that exhibited direct bone neoformation onto, and around, the particles confirming its osteoconductivity. In conclusion, the xenograft tested in vivo showed biocompatibility, biodegradability and osteoconductive properties in rat calvaria CSD.

Effect of bovine fetal growth plate as a new xenograft in experimental bone defect healing: radiological, histopathological and biomechanical evaluation

Bone grafting is used to enhance healing in osteotomies, arthrodesis, and multifragmentary fractures and to replace bony loss resulting from neoplasia or cysts. They are source of osteoprogenitor cells and induce bone formation and provide mechanical support for vascular and bone ingrowth. Autografts are used commonly but quantity of retrieved bone is limit. This study was designed to evaluate autograft and new xenograft (Bovine fetal growth plate) effects on bone healing process. Twenty male White New Zealand rabbits were used in this study. In autograft group the defect was filled by fresh autogenous cortical graft, in xenograft group the defect was filled by a segment of bovine fetal growth plate and was fixed by cercelage wire. Radiological, histopathological and biomechanical evaluations were performed blindly and results scored and analyzed statistically. Statistical tests did not support significant differences between two groups at the 14th and 28th postoperative day radiographically (P > 0.05). There was a significant difference for remodeling at the 42nd postoperative radiologically (P < 0.05). Xenograft was superior to autograft at the 56th postoperative day for radiological bone formation (P < 0.03). Histopathological and biomechanical evaluation revealed no significant differences between two groups. The results of this study indicate that satisfactory healing occurred in rabbit radius defect filled with calf fetal growth plate. Complications were not identified and healing was faster than cortical autogenous grafting. It was concluded that the use of calf fetal growth plate as a new xenograft is an acceptable alternative to cortical autogenous graft and could reduce the morbidity associated with harvesting autogenous graft during surgery.

Defining regional infusion treatment strategies for extremity melanoma: comparative analysis of melphalan and temozolomide as regional chemotherapeutic agents

Five different human melanoma xenografts were used in a xenograft model of extremity melanoma to evaluate the variability of tumor response to regionally administered melphalan or temozolomide and to determine if various components of pertinent drug resistance pathways for melphalan [glutathione S-transferase (GST)/glutathione] and temozolomide [O(6)-alkylguanine DNA alkyltranferase (AGT)/mismatch repair (MMR)] could be predictive of tumor response. Xenograft-bearing rats underwent regional isolated limb infusion with either melphalan (90 mg/kg) or temozolomide (2,000 mg/kg). The levels of AGT activity, GST activity, glutathione level, and GST/AGT expression were examined in this group of xenografts and found to be quite heterogeneous. No correlation was identified between melphalan sensitivity and the GST/glutathione cellular detoxification pathway. In contrast, a strong correlation between the levels of AGT activity and percentage increase in tumor volume on day 30 (r = 0.88) was noted for tumors treated with temozolomide. Regional therapy with temozolomide was more effective when compared with melphalan for the xenograft with the lowest AGT activity, whereas melphalan was more effective than temozolomide in another xenograft that had the highest AGT activity. In three other xenografts, there was no significant difference in response between the two chemotherapy agents. This study shows that AGT activity may be useful in predicting the utility of temozolomide-based regional therapy for advanced extremity melanoma tumors. Our observations also point out the limited ability of analysis of the GST/glutathione pathway to predict response to chemotherapies like melphalan whose resistance is primarily mediated through a complex mechanism of detoxification.

Restore Orthobiologic Implant

Following repairs of large-to-massive tears of the rotator cuff, the rates of tendon retears are high and often involve tissue deficiency. Animal studies of the Restore Orthobiologic Implant, a collagen-based material derived from the small intestine mucosa of pigs, have indicated that it might be used to help overcome such problems. We carried out a study to determine whether patients who received this xenograft to augment a rotator cuff repair exhibited greater shoulder strength, shoulder function, and/or resistance to retearing. We compared data from a group of patients who had undergone conventional rotator cuff repair with xenograft augmentation (the xenograft group) with data from a group in whom a repair had been done by the same surgeon without augmentation (the controls). The groups were matched for gender, mean age, and mean size of the rotator cuff tear. All subjects completed a pain and function questionnaire and were given a systematic clinical shoulder examination preoperatively and at three, six, and twenty-four months postoperatively. The twenty-four-month visit included magnetic resonance imaging to determine whether a retear had occurred. Four patients who had received a xenograft had a severe postoperative reaction requiring surgical treatment. At two years after the surgery, six of the ten tendons repaired with a xenograft and seven of the twelve control tendons had retorn, as documented by magnetic resonance imaging. The patients with a xenograft had significantly less lift-off strength, as measured with a dynamometer, and significantly less strength in internal rotation and adduction than the controls at two years after the surgery (all p < 0.05). Also, the xenograft group had significantly more impingement in external rotation, a slower rate of resolution of pain during activities, more difficulty with hand-behind-the-back activities, and less sports participation (all p < 0.05). Two years after surgical repair of a large rotator cuff defect supplemented with a xenograft, patients had several persisting deficits and no recognizable benefit as compared with the results in a control group. In view of these findings, together with the unsatisfactorily high proportion of patients with a severe inflammatory reaction to the xenograft, we do not recommend use of the Restore Orthobiologic Implant in its present form. Therapeutic Level III. See Instructions to Authors for a complete description of levels of evidence.

Restore Orthobiologic Implant

Background: Following repairs of large-to-massive tears of the rotator cuff, the rates of tendon retears are high and often involve tissue deficiency. Animal studies of the Restore Orthobiologic Implant, a collagen-based material derived from the small intestine mucosa of pigs, have indicated that it might be used to help overcome such problems. We carried out a study to determine whether patients who received this xenograft to augment a rotator cuff repair exhibited greater shoulder strength, shoulder function, and/or resistance to retearing. Methods: We compared data from a group of patients who had undergone conventional rotator cuff repair with xenograft augmentation (the xenograft group) with data from a group in whom a repair had been done by the same surgeon without augmentation (the controls). The groups were matched for gender, mean age, and mean size of the rotator cuff tear. All subjects completed a pain and function questionnaire and were given a systematic clinical shoulder examination preoperatively and at three, six, and twenty-four months postoperatively. The twenty-four-month visit included magnetic resonance imaging to determine whether a retear had occurred. Results: Four patients who had received a xenograft had a severe postoperative reaction requiring surgical treatment. At two years after the surgery, six of the ten tendons repaired with a xenograft and seven of the twelve control tendons had retorn, as documented by magnetic resonance imaging. The patients with a xenograft had significantly less lift-off strength, as measured with a dynamometer, and significantly less strength in internal rotation and adduction than the controls at two years after the surgery (all p < 0.05). Also, the xenograft group had significantly more impingement in external rotation, a slower rate of resolution of pain during activities, more difficulty with hand-behind-the-back activities, and less sports participation (all p < 0.05). Conclusions: Two years after surgical repair of a large rotator cuff defect supplemented with a xenograft, patients had several persisting deficits and no recognizable benefit as compared with the results in a control group. In view of these findings, together with the unsatisfactorily high proportion of patients with a severe inflammatory reaction to the xenograft, we do not recommend use of the Restore Orthobiologic Implant in its present form. Level of Evidence: Therapeutic Level III. See Instructions to Authors for a complete description of levels of evidence.

Human esophageal cancer endothelial cells increase tumor growth by incorporating with mouse endothelium

Current in vivo investigations of tumor angiogenesis mainly rely on the results obtained from engrafted models in mice. In the present study, we attempt to assess the potential of human tumor endothelium to form neovasculature in different engrafted tumor models. The tumor endothelial cells were isolated from human esophageal squamous cell carcinoma, and then identified by anti-VEGFR1/2 immunoreactions and tube formation assay. Esophageal and lung cancer cells were subcutaneously inoculated into nude mice with human esophageal cancer endothelial cells (HECECs), respectively. The human umbilical vein endothelial cells (HUVECs) were also co-inoculated into mice with esophageal cancer cells as a control. The engrafted tumor growth was significantly promoted by co-inoculation of HECECs in comparison with injection of esophageal tumor cells alone. Immunohistochemistry of anti-CD31 and anti-huCD31 was performed to detect the micro-vessels in the engrafted tumors which revealed that the HECECs formed humanized micro-vessels and significantly increased the micro-vessel density in engrafted tumors comparing with the tumors without HECECs. However, HUVEC cells could not enhance the esophageal tumor growth and the growth of lung tumors could not be increased by HECECs, either. Few humanized blood vessels were found in these two groups of xenografts. These results suggest that the specific interaction between HECECs and esophageal tumor cells contributes to the neovasculature construction and esophageal tumor growth in xenografts.

Decellularized heart valve as a scaffold for in vivo recellularization: Deleterious effects of granulocyte colony-stimulating factor

Autologous recellularization of decellularized heart valve scaffolds is a promising challenge in the field of tissue-engineered heart valves and could be boosted by bone marrow progenitor cell mobilization. The aim of this study was to examine the spontaneous in vivo recolonization potential of xenogeneic decellularized heart valves in a lamb model and the effects of granulocyte colony-stimulating factor mobilization of bone marrow cells on this process. Decellularized porcine aortic valves were implanted in 12 lambs. Six lambs received granulocyte colony-stimulating factor (10 microg x kg(-1) x d(-1) for 7 days, granulocyte colony-stimulating factor group), and 6 received no granulocyte colony-stimulating factor (control group). Additionally, nondecellularized porcine valves were implanted in 5 lambs (xenograft group). Angiographic and histologic evaluation was performed at 3, 6, 8, and 16 weeks. Few macroscopic modifications of leaflets and the aortic wall were observed in the control group, whereas progressive shrinkage and thickening of the leaflets appeared in the granulocyte colony-stimulating factor and xenograft groups. In the 3 groups progressive ovine cell infiltration (fluorescence in situ hybridization) was observed in the leaflets and in the adventitia and the intima of the aortic wall but not in the media. Neointimal proliferation of alpha-actin-positive cells, inflammatory infiltration, adventitial neovascularization, and calcifications were more important in the xenograft and the granulocyte colony-stimulating factor groups than in the control group. Continuous re-endothelialization appeared only in the control group. Decellularized xenogeneic heart valve scaffolds allowed partial autologous recellularization. Granulocyte colony-stimulating factor led to accelerated heart valve deterioration similar to that observed in nondecellularized xenogeneic cardiac bioprostheses.

Evolution of aortic valve replacement in children: A single center experience

Background The availability of an ideal prosthesis for aortic valve replacement (AVR) in children remains controversial due to an early degeneration of xenografts and the potential risks related to anticoagulation with mechanical prostheses. This has led many surgeons to the Ross procedure. This study outlines the evolution of our 30-year experience with AVR in children. Methods One hundred and forty-seven children, aged 10 days to 18 years (mean 11.9 ± 5.7 years), underwent AVR between 1974 and June 2005. Preoperative diagnosis included aortic insufficiency ( n = 39), aortic stenosis ( n = 14), combined aortic stenosis with insufficiency ( n = 78), and complex left ventricular outflow tract obstruction ( n = 16). Xenografts ( n = 11) and mechanical prostheses ( n = 47) were used in 58 patients. The remaining 89 patients had placement of homografts ( n = 8) or underwent a Ross procedure ( n = 81). Of the 147 patients, 87 (59%) had previous procedures. Results Overall early and late mortality was 7.5% (11 / 147 pts). Overall survival estimated by the Kaplan–Meier method, including early mortality, was 94% at 1 year, and 93% at 5, 10, and 20 years. Univariate and multivariate analysis identified date of operation (before 1980) as a risk factor for death ( p = 0.002). Follow-up was complete in 136 patients (5 lost to follow-up), with a total follow-up of 2433.72 patient-years. The overall study group aortic valve-related reoperation rate was 20% (20 / 138 pts), the reoperation rate was highest in xenograft group (60%); followed by mechanical valves group (16%), homograft group (14%), and the Ross procedure group (9%). Ross patients showed significant increase of the annulus diameter ( p = 0.002) and the aortic sinus diameter ( p = 0.01) at the last follow-up. The actuarial rate for freedom from aortic valve-related reoperation was 99% at 1 year, 94% at 5 years, 88% at 10 years, and 85% at 20 years. Univariate and multivariate analysis identified the presence of a xenograft as a risk factor for aortic valve-related reoperation ( p = 0.001). Conclusion AVR in children can be performed with acceptable mortality and minimal mid-term morbidity. The Ross procedure, although more complicated, has the advantage of not requiring anticoagulation. Pulmonary autograft, in our series, has demonstrated growth with no structural degeneration. The potential for development of significant autograft insufficiency and ascending aortic aneurysmal dilatation is small but warrants annual follow-up. Our data supports that the Ross procedure is the AVR of choice in children.

Anti-CD154 mAb Treatment But Not Recipient CD154 Deficiency Leads to Long-Term Survival of Xenogeneic Islet Grafts

The aim of the study was to evaluate the role of recipient CD40 and CD154 in the rejection process of concordant and discordant islets xenotransplantation (Tx). Diabetic C57BL/6 mice, CD40- or CD154-knockout (KO) and complement C3-deficient (C3-/-) mice were transplanted with either rat or human islets. Group 1, C57BL/6 Tx without therapy; Group 2, C57BL/6 Tx with anti-CD154 monoclonal antibody (mAb) (MR1) therapy; Group 3, CD40-KO; and Group 4, CD154-KO Tx without therapy; Group 5, C3-/- Tx without therapy and Group 6, C3-/-Tx with MR1 therapy. Mixed lymphocyte reactions (MLR) were performed. Compared to Group 1, MR1 induced long-term survival of xenografts in Group 2, but not in Group 6, survival of islets was not prolonged significantly in Groups 3 and 4. MLR responses in Group 2 were reduced approximately 50% compared to Group 1. In Groups 3, 4 and 6, MLR responses were not modified by the absence of CD40 or CD154 molecules, or MR1 and were similar to Group 1. Improved graft survival and reduced MLR responses in Group 2, but not in Group 6, could be explained by specific targeting of activated T cells with inactivation by complement- or cellular-mediated mechanisms. Rejection of xenografts and strong MLR responses in Groups 3 and 4 are possible through efficient activation of alternate pathways of costimulation.

Elicited non-anti-αGAL antibodies may cause acute humoral rejection of hDAF pig organs transplanted in baboons

The combination of immunosuppression and GAS 914, a polylysine containing αGal trisaccharide type 2 (TRI 2), has been associated with the prevention of acute humoral xenograft rejection (AHXR) in human decay accelerating factor (hDAF) pig-to-baboon xenotransplants. The aim of this study was to investigate the role of immunosuppression and GAS 914 to neutralize xenoantibodies before and after xenotransplantation. Eight baboons underwent heteropic heart xenotransplantation with hDAF transgenic pig organs, receiving GAS 914 before and after transplantation. Six baboons (Group A) were treated with an immunosuppression protocol that included cyclophosphamide (CyP), Neoral, ERL, and steroids. The other 2 baboons (Group B) were treated with the same immunosuppression but with a 50% reduction in the doses of CyP. No xenograft from Group A underwent acute humoral xenograft (median survival, 27 days), whereas the 2 in Group B experienced rejection (median survival, 6 days). GAS 914 depleted both immunoglobulin (Ig)M and IgG anti-αGAL disaccharide (DI), trisaccharide type 2 (TRI 2), and trisaccharide type 6 (TRI 6), before and after transplantation in Groups A and B. However, cytotoxic antibodies with other anti-pig specificities were elicited by the xenografts in Group B leading to AHXR.