Periphery of porcine hepatic lobes has the smallest length density of hepatic sinusoids and bile canaliculi: A stereological histological study with implications for liver biopsies.

BACKGROUND: Porcine liver is widely used in hepatologic research as a large animal model with many anatomical and physiological similarities with humans. However, only limited information on porcine liver spatial microstructure has been published, especially regarding the hepatic sinusoids and bile canaliculi. The aim of our study was to quantify the sinusoidal and bile canalicular network in healthy male and female porcine livers and to map the variability of these structures with heterogenous distribution to improve the evaluability of liver biopsy samples. METHODS: Livers from 12 healthy piglets (6 females and 6 neutered males) were sampled into 36 tissue samples per organ, representing six hepatic lobes and three different regions related to the hepatic vasculature (peripheral, paracaval and paraportal region). Histological sections were processed with a random orientation of the cutting plane. The endothelium and the bile canaliculi were stained using Ricinus communis agglutinin I lectin histochemistry. The length densities of hepatic sinusoids LV(sinusoids,liver), of bile canaliculi LV(bile canaliculi,liver) and volume fraction VV(sinusoids,liver) and surface density SV(sinusoids,liver) of sinusoids were estimated using stereological methods. The newly acquired morphometric data were compared with previously published data on density of porcine hepatocytes and fractions of connective tissue. RESULTS: The peripheral region had smallest LV(sinusoids,liver), smallest LV(bile canaliculi,liver) and greatest VV(sinusoids,liver). The six hepatic lobes had statistically comparable length densities of both sinusoids and bile canaliculi, but the left lateral lobe had smallest VV(sinusoids,liver). Regions with greater LV(sinusoids,liver) had also greater LV(bile canaliculi,liver) and SV(sinusoids,liver) and were accompanied by greater density of smaller hepatocytes. Regions with smaller LV(sinusoids,liver) and LV(bile canaliculi,liver) contained a greater fraction of interlobular connective tissue. CONCLUSIONS: The length density of hepatic sinusoids is smaller in the peripheral regions of the porcine liver than in other regions related to the hepatic vasculature - paracaval and paraportal regions, and smaller in castrated males than in females. Greater length density of liver sinusoids was linked with greater local density of bile canaliculi, with local increase in the density of smaller hepatocytes and, simultaneously, with smaller fractions of hepatic connective tissue. The intrahepatic and inter-sexual variability of the porcine liver morphology needs to be taken into account when designing and interpreting experiments involving the histological quantification of the microvascular network. The complete primary morphometric data describing the distribution of morphometric parameters within porcine liver were made available in a form facilitating the power analysis to justify the minimal number of tissue samples or animals required when designing further histological evaluation studies. The macroscopic map of microvessels and bile canaliculi variability facilitates their assessment in liver biopsies in the pig.

Histological mapping of healing of the small and large intestine - A quantitative study in a porcine model.

BACKGROUND: Gastrointestinal anastomoses are performed in many patients every year. The pathogenesis of aberrant anastomotic healing and the causes of intestinal leakage are not fully understood. The present study gathered and critically evaluated histological quantitative data to deepen current knowledge of anastomotic healing in the small and large intestine and its complications and outline the options for further experimental in vivo research in large porcine animal models. METHODS: Three groups of porcine intestinal anastomoses were compared: small intestine without defect (SI; n = 7), small intestine with an additional defect (SID; n = 8), and large intestine (LI; n = 7). Multilevel sampling (2112 micrographs) and stereological methods were used for histological quantification of proliferation (Ki-67 immunohistochemistry), neutrophil infiltration (myeloperoxidase staining), vascularity (von Willebrand factor) and type I and type III collagen formation (picrosirius red in polarized light) within the region of anastomosis compared to the region outside of anastomosis. RESULTS: Quantitative histological evaluation revealed the following results. i) Proliferation, vascularity, and collagen, but not neutrophils, were more highly expressed within the anastomosis than outside of the anastomosis region. ii) Porcine large and small intestine were not interchangeable based on histological evaluation of surgical experiments. The presence or absence of an additional experimental defect strongly affected healing, but the healing seemed complete after 21 days. iii) The microscopic structure of small intestine segments was more affected by their proximity to the anastomosis than the structure of large intestine segments. CONCLUSIONS: Histological quantification was more laborious than the previously used semiquantitative scoring system evaluating the healing rate of intestinal anastomoses, but it provided detailed maps of biological processes within individual intestine layers. The primary data collected in the study are open and available for power sample analyses to calculate the minimum numbers of samples justified in future experiments on porcine intestines. The porcine intestine is a promising animal model with translational potential for human surgery.

An Assessment of Blood Vessel Remodeling of Nanofibrous Poly(ε-Caprolactone) Vascular Grafts in a Rat Animal Model.

The development of an ideal vascular prosthesis represents an important challenge in terms of the treatment of cardiovascular diseases with respect to which new materials are being considered that have produced promising results following testing in animal models. This study focuses on nanofibrous polycaprolactone-based grafts assessed by means of histological techniques 10 days and 6 months following suturing as a replacement for the rat aorta. A novel stereological approach for the assessment of cellular distribution within the graft thickness was developed. The cellularization of the thickness of the graft was found to be homogeneous after 10 days and to have changed after 6 months, at which time the majority of cells was discovered in the inner layer where the regeneration of the vessel wall was found to have occurred. Six months following implantation, the endothelialization of the graft lumen was complete, and no vasa vasorum were found to be present. Newly formed tissue resembling native elastic arteries with concentric layers composed of smooth muscle cells, collagen, and elastin was found in the implanted polycaprolactone-based grafts. Moreover, the inner layer of the graft was seen to have developed structural similarities to the regular aortic wall. The grafts appeared to be well tolerated, and no severe adverse reaction was recorded with the exception of one case of cartilaginous metaplasia close to the junctional suture.

Nanofiber Fractionalization Stimulates Healing of Large Intestine Anastomoses in Rabbits.

Background: A current topic of ma jor interest in regenerative medicine is the development of novel materials for accelerated healing of sutures, and nanofibers seem to be suitable materials for this purpose. As various studies have shown, nanofibers are able to partially substitute missing extracellular matrix and to stimulate cell proliferation and differentiation in sutures. Therefore, we tested nanofibrous membranes and cryogenically fractionalized nanofibers as potential materials for support of the healing of intestinal anastomoses in a rabbit model. Materials and Methods: We compared cryogenically fractionalized chitosan and PVA nanofibers with chitosan and PVA nanofiber membranes designed for intestine anastomosis healing in a rabbit animal model. The anastomoses were biomechanically and histologically tested. Results: In strong contrast to nanofibrous membranes, the fractionalized nanofibers did show positive effects on the healing of intestinal anastomoses in rabbits. The fractionalized nanofibers were able to reach deep layers that are key to increased mechanical strength of the intestine. Moreover, fractionalized nanofibers led to the formation of collagen-rich 3D tissue significantly exceeding the healing effects of the 2D flat nanofiber membranes. In addition, the fractionalized chitosan nanofibers eliminated peritonitis, significantly stimulated anastomosis healing and led to a higher density of microvessels, in addition to a larger fraction of myofibroblasts and collagen type I and III. Biomechanical tests supported these histological findings. Conclusion: We concluded that the fractionalized chitosan nanofibers led to accelerated healing for rabbit colorectal anastomoses by the targeted stimulation of collagen-producing cells in the intestine, the smooth muscle cells and the fibroblasts. We believe that the collagen-producing cells were stimulated both directly due to the presence of a biocompatible scaffold providing cell adhesion, and indirectly, by a proper stimulation of immunocytes in the suture.

CTNNB1 mutations, TERT polymorphism and CD8+ cell densities in resected hepatocellular carcinoma are associated with longer time to recurrence.

BACKGROUND: Hepatocellular carcinoma (HCC) is a fatal disease characterized by early genetic alterations in telomerase reverse transcriptase promoter (TERTp) and ß-catenin (CTNNB1) genes and immune cell activation in the tumor microenvironment. As a novel approach, we wanted to assess patient survival influenced by combined presence of mutations and densities of CD8+ cytotoxic T cells. METHODS: Tissue samples were obtained from 67 HCC patients who had undergone resection. We analysed CD8+ T cells density, TERTp mutations, rs2853669 polymorphism, and CTNNB1 mutations. These variables were evaluated for time to recurrence (TTR) and disease free survival (DFS). RESULTS: TERTp mutations were found in 75.8% and CTNNB1 mutations in 35.6% of the patients. TERTp mutations were not associated with survival but polymorphism rs2853669 in TERTp was associated with improved TTR and DFS. CTNNB1 mutations were associated with improving TTR. High density of CD8+ T-lymphocytes in tumor center and invasive margin correlated with longer TTR and DFS. Combined genetic and immune factors further improved survival showing higher predictive values. E.g., combining CTNNB1 mutations and high density of CD8+ T-lymphocytes in tumor center yielded HRs of 0.12 (0.03-0.52), p = 0.005 for TTR and 0.25 (0.09-0.74), p = 0.01 for DFS. CONCLUSION: The results outline a novel integrative approach for prognostication through combining independent predictive factors from genetic and immune cell profiles. However, larger studies are needed to explore multiple cell types in the tumor microenvironment.

Porcine spleen as a model organ for blunt injury impact tests: An experimental and histological study.

The spleen is a large and highly vascularized secondary lymphatic organ. Spleen injuries are among the most frequent trauma-related injuries in the abdominal region. The aims of the study were to assess the volume fractions of the main splenic tissue components (red pulp, white pulp, trabeculae and reticular fibres) and to determine the severity of splenic injury due to the experimental impact test. Porcine spleens (n = 17) were compressed by 6.22 kg wooden plate using a drop tower technique from three impact heights (50, 100 and 150 mm corresponding to velocities 0.79, 1.24 and 1.58 m/s). The pressure was measured via catheters placed in the splenic vein. The impact velocity was measured using lasers. The severity of induced injuries was analysed on the macroscopic level. The volume fractions of splenic components were assessed microscopically using stereology. The volume fraction of the red pulp was 76.4%, white pulp 21.3% and trabeculae 2.7% respectively. All impact tests, even with the low impact velocities, led to injuries that occurred mostly in the dorsal extremity of the spleen, and were accompanied by bleeding, capsule rupture and parenchyma crushing. Higher impact height (impact velocity and impact energy) caused more severe injury. Porcine spleen had the same volume fraction of tissue components as human spleen, therefore we concluded that the porcine spleen was a suitable organ model for mechanical experiments. Based on our observations, regions around hilum and the diaphragmatic surface of the dorsal extremity, that contained fissures and notches, were the most prone to injury and required considerable attention during splenic examination after injury. The primary mechanical data are now available for the researchers focused on the splenic trauma modelling.

Complex genetic and histopathological study of 15 patient-derived xenografts of aggressive lymphomas.

Non-Hodgkin lymphomas (NHL) represent the most common hematologic malignancies. Patient-derived xenografts (PDXs) are used for various aspects of translational research including preclinical in vivo validation of experimental treatment approaches. While it was repeatedly demonstrated that PDXs keep majority of somatic mutations with the primary lymphoma samples, from which they were derived, the composition of PDX tumor microenvironment (TME) has not been extensively studied. We carried out a comparative genetic and histopathological study of 15 PDX models derived from patients with various types of NHL including diffuse large B-cell lymphoma (DLBCL; n = 7), Burkitt lymphoma (BL; n = 1), mantle cell lymphoma (MCL; n = 2), and peripheral T-cell lymphomas (PTCL; n = 5). Whole exome sequencing (WES) of the PDXs and primary lymphoma cells was implemented in 13 out of 15 cases with available DNA samples. Standard immunohistochemistry (IHC) was used to analyze the composition of PDX TME. WES data confirmed that PDXs maintained the genetic heterogeneity with the original primary lymphoma cells. In contrast, IHC analysis revealed the following recurrently observed alterations in the composition of PDX tumors: more blastoid lymphoma cell morphology, increased proliferation rate, lack of non-malignant cellular components including T cells and (human or murine) macrophages, and significantly lower intratumoral microvessel density and microvessel area composed of murine vessels. In addition, PDX tumors derived from T-NHL displayed additional differences compared to the primary lymphoma samples including markedly lower desmoplasia (i.e., the extent of both reticular and collagen fibrosis), loss of expression of cytotoxic granules (i.e., perforin, TIA, granzyme B), or loss of expression of T-cell specific antigens (i.e., CD3, CD4, CD8). Our data suggest that despite keeping the same genetic profiles, PDX models of aggressive NHL do not recapitulate the microenvironmental heterogeneity of the original lymphomas. These findings have implications on the relevance of PDX models in the context of preclinical research.

T- and B-Cells in the Inner Invasive Margin of Hepatocellular Carcinoma after Resection Associate with Favorable Prognosis.

In this retrospective study on 67 patients with hepatocellular carcinoma (HCC), after tumor resection, we evaluated the significance of CD3+ and CD8+ T-lymphocytes and CD20+ B-lymphocytes in tumor and non-tumor liver for time to recurrence (TTR), disease-free survival (DFS) and overall survival. After immunohistochemical staining, the density of nucleated lymphocyte profiles (QA) was estimated stereologically in the tumor center (TC), inner margin (inn M), outer margin (out M), peritumor and non-tumor liver. In TC, intermediate and high QA of CD8+ cells predicted longer TTR, whereas CD3+ and CD20+ were predictive only at high QA. DFS was predicted by high QA of CD3+, CD8+ and CD20+ cells in TC. The inn M harbored smaller QA of CD3+, CD8+ and CD20+ lymphocytes than out M. In contrast to out M, high T-cells' QA and intermediate and high B-cell QA in inn M predicted longer TTR and DFS. High inn M/out M QA ratios of CD3+ and CD20+ cells were associated with longer TTR and DFS, whereas high inn M/out M QA ratio of CD8+ was predictive only for DFS. Patients with intermediate-high QA of combined CD8+ and CD20+ cells in inn M showed longer TTR and DFS, compared to CD8+-high or CD20+-high alone. Our findings highlight overall heterogeneity of the tumor invasive margin, the importance of inn M, and the predictive role of B-cells.

Are ovine and porcine carotid arteries equivalent animal models for experimental cardiac surgery: A quantitative histological comparison.

BACKGROUND: Coronary artery bypass grafting (CABG) is a common cardiac surgery. Manufacturing small-diameter (2-5 mm) vascular grafts for CABG is important for patients who lack first-choice autologous arterial, or venous conduits. Ovine and porcine common carotid arteries (CCAs) are used as large animal models for in vivo testing of newly developed tissue-engineered arterial grafts. It is unknown to what extent these models are interchangeable and whether the left and right arteries of the same subjects can be used as experimental controls. Therefore, we compared the microscopic structure of paired left and right ovine and porcine CCAs in the proximodistal direction and compared these animal model samples to samples of human coronary arteries (CAs) and human internal thoracic arteries (ITAs). METHODS: We compared the histological composition of whole CCAs of sheep (n = 22 animals) with whole porcine CCAs (n = 21), segments of human CAs (n = 21), and human ITAs (n = 21). Using unbiased sampling and stereological methods, we quantified the fractions of elastin, total collagen, type I collagen, type III collagen, smooth muscle actin (SMA) and chondroitin sulfate (CS) A, B, and C. We also quantified the densities and distributions of nuclear profiles, nervi vasorum and vasa vasorum as well as the thickness of the intima-media and total wall thickness. RESULTS: The differences between the paired samples of left and right CCAs in sheep were substantially greater than the differences in laterality in porcine CCAs. The right ovine CCAs had a smaller fraction of elastin (p < 0.001), greater fraction of SMA (p < 0.01), and greater intima-media thickness (p < 0.001) than the paired left side CCAs. In pigs, the right CCAs had a greater fraction of elastin (p < 0.05) and a greater density of vasa vasorum in the media (p < 0.001) than the left-side CCAs. The fractions of elastin and CS decreased and the fraction of SMA increased in the proximodistal direction in both the ovine (p < 0.001) and porcine (p < 0.001) CCAs. Ovine CCAs had a muscular phenotype along their entire length, but porcine CCAs were elastic-type arteries in the proximal segments but muscular type arteries in middle and distal segments. The CCAs of both animals differed from the human CAs and ITAs in most parameters, but the ovine CCAs had a comparable fraction of elastin and CS to human ITAs. CONCLUSIONS: From a histological point of view, ovine and porcine CCAs were not equivalent in most quantitative parameters to human CAs and ITAs. Left and right ovine CCAs did not have the same histological composition, which is limiting for their mutual equivalence as sham-operated controls in experiments. These differences should be taken into account when designing and interpreting experiments using these models in cardiac surgery. The complete morphometric data obtained by quantitative evaluation of arterial segments were provided to facilitate the power analysis necessary for justification of the minimum number of samples when planning further experiments. The middle or distal segments of ovine and porcine CCAs remain the most realistic and the best characterized large animal models for testing artificial arterial CABG conduits.

Using virtual microscopy for the development of sampling strategies in quantitative histology and design-based stereology.

Only a fraction of specimens under study are usually selected for quantification in histology. Multilevel sampling or tissue probes, slides and fields of view (FOVs) in the regions of interest (ROIs) are required. In general, all parts of the organs under study should be given the same probability to be taken into account; that is, the sampling should be unbiased on all levels. The objective of our study was to provide an overview of the use of virtual microscopy in the context of developing sampling strategies of FOVs for stereological quantification. We elaborated this idea on 18 examples from multiple fields of histology, including quantification of extracellular matrix and muscle tissue, quantification of organ and tumour microvessels and tumour-infiltrating lymphocytes, assessing osseointegration of bone implants, healing of intestine anastomoses and osteochondral defects, counting brain neurons, counting nuclei in vitro cell cultures and others. We provided practical implications for the most common situations, such as exhaustive sampling of ROIs, sampling ROIs of different sizes, sampling the same ROIs for multiple histological methods, sampling more ROIs with variable intensities or using various objectives, multistage sampling and virtual sampling. Recommendations were provided for pilot studies on systematic uniform random sampling of FOVs as a part of optimizing the efficiency of histological quantification to prevent over- or undersampling. We critically discussed the pros and cons of using virtual sections for sampling FOVs from whole scanned sections. Our review demonstrated that whole slide scans of histological sections facilitate the design of sampling strategies for quantitative histology.

Vancomycin-Loaded Collagen/Hydroxyapatite Layers Electrospun on 3D Printed Titanium Implants Prevent Bone Destruction Associated with S. epidermidis Infection and Enhance Osseointegration.

The aim of the study was to develop an orthopedic implant coating in the form of vancomycin-loaded collagen/hydroxyapatite layers (COLHA+V) that combine the ability to prevent bone infection with the ability to promote enhanced osseointegration. The ability to prevent bone infection was investigated employing a rat model that simulated the clinically relevant implant-related introduction of bacterial contamination to the bone during a surgical procedure using a clinical isolate of Staphylococcus epidermidis. The ability to enhance osseointegration was investigated employing a model of a minipig with terminated growth. Six weeks following implantation, the infected rat femurs treated with the implants without vancomycin (COLHA+S. epidermidis) exhibited the obvious destruction of cortical bone as evinced via a cortical bone porosity of up to 20% greater than that of the infected rat femurs treated with the implants containing vancomycin (COLHA+V+S. epidermidis) (3%) and the non-infected rat femurs (COLHA+V) (2%). The alteration of the bone structure of the infected COLHA+S. epidermidis group was further demonstrated by a 3% decrease in the average Ca/P molar ratio of the bone mineral. Finally, the determination of the concentration of vancomycin released into the blood stream indicated a negligible systemic load. Six months following implantation in the pigs, the quantified ratio of new bone indicated an improvement in osseointegration, with a two-fold bone ingrowth on the COLHA (47%) and COLHA+V (52%) compared to the control implants without a COLHA layer (27%). Therefore, it can be concluded that COLHA+V layers are able to significantly prevent the destruction of bone structure related to bacterial infection with a minimal systemic load and, simultaneously, enhance the rate of osseointegration.

Mesenchymal Stromal Cell Therapy in Novel Porcine Model of Diffuse Liver Damage Induced by Repeated Biliary Obstruction.

In liver surgery, biliary obstruction can lead to secondary biliary cirrhosis, a life-threatening disease with liver transplantation as the only curative treatment option. Mesenchymal stromal cells (MSC) have been shown to improve liver function in both acute and chronic liver disease models. This study evaluated the effect of allogenic MSC transplantation in a large animal model of repeated biliary obstruction followed by partial hepatectomy. MSC transplantation supported the growth of regenerated liver tissue after 14 days (MSC group, n = 10: from 1087 ± 108 (0 h) to 1243 ± 92 mL (14 days); control group, n = 11: from 1080 ± 95 (0 h) to 1100 ± 105 mL (14 days), p = 0.016), with a lower volume fraction of hepatocytes in regenerated liver tissue compared to resected liver tissue (59.5 ± 10.2% vs. 70.2 ± 5.6%, p < 0.05). Volume fraction of connective tissue, blood vessels and bile vessels in regenerated liver tissue, serum levels of liver enzymes (AST, ALT, ALP and GGT) and liver metabolites (albumin, bilirubin, urea and creatinine), as well as plasma levels of IL-6, IL-8, TNF-α and TGF-ß, were not affected by MSC transplantation. In our novel, large animal (pig) model of repeated biliary obstruction followed by partial hepatectomy, MSC transplantation promoted growth of liver tissue without any effect on liver function. This study underscores the importance of translating results between small and large animal models as well as the careful translation of results from animal model into human medicine.

Double-layered Nanofibrous Patch for Prevention of Anastomotic Leakage and Peritoneal Adhesions, Experimental Study.

BACKGROUND/AIM: Anastomotic leakage is a feared complication in colorectal surgery. Postoperative peritoneal adhesions can also cause life-threatening conditions. Nanofibrous materials showed their pro-healing properties in various studies. The aim of the study was to evaluate the impact of double-layered nanofibrous materials on anastomotic healing and peritoneal adhesions formation. MATERIALS AND METHODS: Two versions of double-layered materials from polycaprolactone and polyvinyl alcohol were applied on defective anastomosis on the small intestine of healthy pigs. The control group remained with uncovered defect. Tissue specimens were subjected to histological analysis and adhesion scoring after 3 weeks of observation. RESULTS: The wound healing was inferior in the experimental groups, however, no anastomotic leakage was observed and the applied material always kept covering the defect. The extent of adhesions was larger in the experimental groups. CONCLUSION: Nanofibrous materials may prevent anastomotic leakage but delay healing.

Quantitative In Vivo Monitoring of Hypoxia and Vascularization of Patient-Derived Murine Xenografts of Mantle Cell Lymphoma Using Photoacoustic and Ultrasound Imaging.

Tumor oxygenation and vascularization are important parameters that determine the aggressiveness of the tumor and its resistance to cancer therapies. We introduce dual-modality ultrasound and photoacoustic imaging (US-PAI) for the direct, non-invasive real-time in vivo evaluation of oxygenation and vascularization of patient-derived xenografts (PDXs) of B-cell mantle cell lymphomas. The different optical properties of oxyhemoglobin and deoxyhemoglobin make it possible to determine oxygen saturation (sO2) in tissues using PAI. High-frequency color Doppler imaging enables the visualization of blood flow with high resolution. Tumor oxygenation and vascularization were studied in vivo during the growth of three different subcutaneously implanted patient-derived xenograft (PDX) lymphomas (VFN-M1, VFN-M2 and VFN-M5 R1). Similar values of sO2 (sO2 Vital), determined from US-PAI volumetric analysis, were obtained in small and large VFN-M1 tumors ranging from 37.9 ± 2.2 to 40.5 ± 6.0 sO2 Vital (%) and 37.5 ± 4.0 to 35.7 ± 4.6 sO2 Vital (%) for small and large VFN-M2 PDXs. In contrast, the higher sO2 Vital values ranging from 57.1 ± 4.8 to 40.8 ± 5.7 sO2 Vital (%) (small to large) of VFN-M5 R1 tumors corresponds with the higher aggressiveness of that PDX model. The different tumor percentage vascularization (assessed as micro-vessel areas) of VFN-M1, VFN-M2 and VFN-M5 R1 obtained by color Doppler (2.8 ± 0.1%, 3.8 ± 0.8% and 10.3 ± 2.7%) in large-stage tumors clearly corresponds with their diverse growth and aggressiveness. The data obtained by color Doppler were validated by histology. In conclusion, US-PAI rapidly and accurately provided relevant and reproducible information on tissue oxygenation in PDX tumors in real time without the need for a contrast agent.

Blunt injury of liver: mechanical response of porcine liver in experimental impact test.

OBJECTIVE: The liver is frequently injured in blunt abdominal trauma caused by road traffic accidents. The testing of safety performance of vehicles, e.g. belt usage, head support, seat shape, or air bag shape, material, pressure and reaction, could lead to reduction of the injury seriousness. Current trends in safety testing include development of accurate computational human body models (HBMs) based on the anatomical, morphological, and mechanical behavior of tissues under high strain. APPROACH: The aim of this study was to describe the internal pressure changes within porcine liver, the severity of liver injury and the relation between the porcine liver microstructure and rupture propagation in an experimental impact test. Porcine liver specimens (n = 24) were uniformly compressed using a drop tower technique and four impact heights (200, 300, 400 and 500 mm; corresponding velocities: 1.72, 2.17, 2.54 and 2.88 m s-1). The changes in intravascular pressure were measured via catheters placed in portal vein and caudate vena cava. The induced injuries were analyzed on the macroscopic level according to AAST grade and AIS severity. Rupture propagation with respect to liver microstructure was analyzed using stereological methods. MAIN RESULTS: Macroscopic ruptures affected mostly the interface between connective tissue surrounding big vessels and liver parenchyma. Histological analysis revealed that the ruptures avoided reticular fibers and interlobular septa made of connective tissue on the microscopic level. SIGNIFICANCE: The present findings can be used for evaluation of HBMs of liver behavior in impact situations.

The time has come to extend the expiration limit of cryopreserved allograft heart valves.

Despite the wide choice of commercial heart valve prostheses, cryopreserved semilunar allograft heart valves (C-AHV) are required, and successfully transplanted in selected groups of patients. The expiration limit (EL) criteria have not been defined yet. Most Tissue Establishments (TE) use the EL of 5 years. From physiological, functional, and surgical point of view, the morphology and mechanical properties of aortic and pulmonary roots represent basic features limiting the EL of C-AHV. The aim of this work was to review methods of AHV tissue structural analysis and mechanical testing from the perspective of suitability for EL validation studies. Microscopic structure analysis of great arterial wall and semilunar leaflets tissue should clearly demonstrate cells as well as the extracellular matrix components by highly reproducible and specific histological staining procedures. Quantitative morphometry using stereological grids has proved to be effective, as the exact statistics was feasible. From mechanical testing methods, tensile test was the most suitable. Young's moduli of elasticity, ultimate stress and strain were shown to represent most important AHV tissue mechanical characteristics, suitable for exact statistical analysis. C-AHV are prepared by many different protocols, so as each TE has to work out own EL for C-AHV.

Influence of Mesenchymal Stem Cell Administration on The Outcome of Partial Liver Resection in a Porcine Model of Sinusoidal Obstruction Syndrome.

BACKGROUND: In patients with colorectal liver metastases, the possibility for radical liver resection can be limited by oxaliplatin-induced sinusoidal obstruction syndrome (SOS). This study investigates the potential of mesenchymal stem cells (MSC) to improve the outcome of liver resections in pigs with SOS. MATERIALS AND METHODS: SOS was induced in all animals (n=20) on day 0. Animals in the experimental group (n=8) received allogeneic MSC on day 7. Liver resection was performed in all animals on day 14 and the animals were observed until day 28. Ultrasound volumetry, biochemical analysis and histological examination of liver parenchyma was performed during the follow-up period. RESULTS: Six animals from the control group died prematurely, while all animals survived in the experimental group. According to histology, biochemical analysis and ultrasound volumetry, there were no significant differences between the groups documenting the effect of MSC. CONCLUSION: Single dose allogeneic MSC administration improved survival of animals with SOS undergoing partial liver resection. Further experiments with different timing of liver resection and MSC administration should be performed to investigate the effect of MSC in more detail.

Hydrogel Containing Anti-CD44-Labeled Microparticles, Guide Bone Tissue Formation in Osteochondral Defects in Rabbits.

Hydrogels are suitable for osteochondral defect regeneration as they mimic the viscoelastic environment of cartilage. However, their biomechanical properties are not sufficient to withstand high mechanical forces. Therefore, we have prepared electrospun poly-ε-caprolactone-chitosan (PCL-chit) and poly(ethylene oxide)-chitosan (PEO-chit) nanofibers, and FTIR analysis confirmed successful blending of chitosan with other polymers. The biocompatibility of PCL-chit and PEO-chit scaffolds was tested; fibrochondrocytes and chondrocytes seeded on PCL-chit showed superior metabolic activity. The PCL-chit nanofibers were cryogenically grinded into microparticles (mean size of about 500 µm) and further modified by polyethylene glycol-biotin in order to bind the anti-CD44 antibody, a glycoprotein interacting with hyaluronic acid (PCL-chit-PEGb-antiCD44). The PCL-chit or PCL-chit-PEGb-antiCD44 microparticles were mixed with a composite gel (collagen/fibrin/platelet rich plasma) to improve its biomechanical properties. The storage modulus was higher in the composite gel with microparticles compared to fibrin. The Eloss of the composite gel and fibrin was higher than that of the composite gel with microparticles. The composite gel either with or without microparticles was further tested in vivo in a model of osteochondral defects in rabbits. PCL-chit-PEGb-antiCD44 significantly enhanced osteogenic regeneration, mainly by desmogenous ossification, but decreased chondrogenic differentiation in the defects. PCL-chit-PEGb showed a more homogeneous distribution of hyaline cartilage and enhanced hyaline cartilage differentiation.

Experimental fortification of intestinal anastomoses with nanofibrous materials in a large animal model.

Anastomotic leakage is a severe complication in gastrointestinal surgery. It is often a reason for reoperation together with intestinal passage blockage due to formation of peritoneal adhesions. Different materials as local prevention of these complications have been studied, none of which are nowadays routinely used in clinical practice. Nanofabrics created proved to promote healing with their structure similar to extracellular matrix. We decided to study their impact on anastomotic healing and formation of peritoneal adhesions. We performed an experiment on 24 piglets. We constructed 3 hand sutured end-to-end anastomoses on the small intestine of each pig. We covered the anastomoses with a sheet of polycaprolactone nanomaterial in the first experimental group, with a sheet of copolymer of polylactic acid with polycaprolactone in the second one and no fortifying material was used in the Control group. The animals were sacrificed after 3 weeks of observation. Clinical, biochemical and macroscopic signs of anastomotic leakage or intestinal obstruction were monitored, the quality of the scar tissue was assessed histologically, and a newly developed scoring system was employed to evaluate the presence of adhesions. The material is easy to manipulate with. There was no mortality or major morbidity in our groups. No statistical difference was found inbetween the groups in the matter of level of peritoneal adhesions or the quality of the anastomoses. We created a new adhesion scoring system. The material appears to be safe however needs to be studied further to prove its' positive effects.