Effect of 3 Preservation Methods (Freezing, Cryopreservation, and Freezing + Irradiation) on Human Menisci Ultrastructure: An Ex Vivo Comparative Study With Fresh Tissue as a Gold Standard.

BACKGROUND: Three main meniscus preservation methods have been advocated: freezing (-80°C), freezing with gamma irradiation (-80°C + 25 kGy), and cryopreservation (-140°C). HYPOTHESIS: All preservation methods will result in structural and architectural properties similar to those of fresh meniscus, defined as the gold standard. STUDY DESIGN: Controlled laboratory study. METHODS: Five human intact menisci were collected from 5 patients undergoing total knee arthroplasty. The inclusion criteria were patients <70 years old with primary unilateral (medial) femorotibial knee osteoarthritis and without surgical or traumatic history on the operated knee. Four cubes (9 mm3) were cut inside of the white, or avascular, area of each specimen's middle horn and divided into 4 groups: "fresh" control, frozen (-80°C), cryopreserved (-140°C), and frozen + irradiated (-80°C + 25 kGy). Specimens of the control group were evaluated at day 1, and specimens from the frozen, cryopreserved, and frozen + irradiated groups were evaluated after 1 month of storage. Evaluation was performed with electron microscopy according a validated protocol to analyze (1) mean diameters of the collagen fibers in longitudinal and transverse sections in 5 points per section and (2) validated architectural scores. RESULTS: No significant difference was found between the control and cryopreserved groups regarding mean transverse and longitudinal diameters (transverse: 95.39 ± 15.87 nm vs 99.62 ± 19.23 nm, P = .1; longitudinal: 96.31 ± 13.96 nm vs 94.57 ± 16.42 nm, P = .1). Significant differences were found between the control and frozen groups (transverse: 95.39 ± 15.87 nm vs 70.20 ± 13.94 nm, P < .001; longitudinal: 96.31 ± 13.96 nm vs 71.28 ± 10.64 nm, P < .001) and the control and frozen + irradiated groups (transverse: 95.39 ± 15.87 nm vs 63.1 ± 15.57 nm, P < .001; longitudinal: 96.31 ± 13.96 nm vs 60.9 ± 14.8 nm, P < .001). Regarding architectural score calculation, there were significant differences between the control and frozen groups (4.5 ± 1.3 vs 2.3 ± 1.4, P = .02) and the control and frozen + irradiated groups (4.5 ± 1.3 vs 1.4 ± 0.9, P = .02). CONCLUSION: Cryopreservation is the only method that preserves fresh meniscus architectural specificities. Freezing and freezing + irradiation methods modify histologic properties of meniscal allograft. Irradiation deeply alters diameters and the organization of collagen fibers, and this method should be used with caution to preserve and sterilize meniscus tissue. CLINICAL RELEVANCE: The results of our study exhibited detrimental effects of simple freezing and freezing + irradiation on the collagen network of sample meniscus. If those effects occur in menisci prepared for allograft procedures, important differences could appear on the basis of the preservation procedure in terms of the graft's mechanical properties and, thus, the patient's outcomes.

[Concurrence of acute graft rejection and polyomavirus nephropathy: A clinical case]. / Klinicheskoe nabliudenie sochetaniia ostrogo ottorzheniia transplantata s poliomavirusnoi nefropatiei.

The paper describes a case of diagnosing acute renal graft rejection concurrent with polyomavirus nephropathy. Histochemical and electron microscopic methods were used to examine biopsy specimens, which showed morphological changes occurring in the allograft, the ultrastructural characteristics of polyomavirus and the features of its spread in kidney tissue structures.

Laser Scanning In Vivo Confocal Microscopy of Clear Grafts after Penetrating Keratoplasty.

PURPOSE: To evaluate the changes of keratocytes and dendritic cells in the central clear graft by laser scanning in vivo confocal microscopy after penetrating keratoplasty (PK). METHODS: Thirty adult subjects receiving PK at Shandong Eye Institute and with clear grafts and no sign of immune rejection after surgery were recruited into this study, and 10 healthy adults were controls. The keratocytes and dendritic cells in the central graft were evaluated by laser scanning confocal microscopy, as well as epithelium cells, keratocytes, corneal endothelium cells, and corneal nerves (especially subepithelial plexus nerves). RESULTS: Median density of subepithelial plexus nerves, keratocyte density in each layer of the stroma, and density of corneal endothelium cells were all lower in clear grafts than in controls. The dendritic cells of five (16.7%) patients were active in Bowman's membrane and stromal membrane of the graft after PK. CONCLUSIONS: Activated dendritic cells and Langerhans cells could be detected in some of the clear grafts, which indicated that the subclinical stress of immune reaction took part in the chronic injury of the clear graft after PK, even when there was no clinical rejection episode.

The consequence of biologic graft processing on blood interface biocompatibility and mechanics.

Processing ex vivo derived tissues to reduce immunogenicity is an effective approach to create biologically complex materials for vascular reconstruction. Due to the sensitivity of small diameter vascular grafts to occlusive events, the effect of graft processing on critical parameters for graft patency, such as peripheral cell adhesion and wall mechanics, requires detailed analysis. Isolated human umbilical vein sections were used as model allogenic vascular scaffolds that were processed with either: 1. sodium dodecyl sulfate (SDS), 2. ethanol/acetone (EtAc), or 3. glutaraldehyde (Glu). Changes in material mechanics were assessed via uniaxial tensile testing. Peripheral cell adhesion to the opaque grafting material was evaluated using an innovative flow chamber that allows direct observation of the blood-graft interface under physiological shear conditions. All treatments modified the grafts tensile strain and stiffness properties, with physiological modulus values decreasing from Glu 240±12 kPa to SDS 210±6 kPa and EtAc 140±3 kPa, P<.001. Relative to glutaraldehyde treatments, neutrophil adhesion to the decellularized grafts increased, with no statistical difference observed between SDS or EtAc treatments. Early platelet adhesion (% surface coverage) showed no statistical difference between the three treatments; however, quantification of platelet aggregates was significantly higher on SDS scaffolds compared to EtAc or Glu. Tissue processing strategies applied to the umbilical vein scaffold were shown to modify structural mechanics and cell adhesion properties, with the EtAc treatment reducing thrombotic events relative to SDS treated samples. This approach allows time and cost effective prescreening of clinically relevant grafting materials to assess initial cell reactivity.

Inosculation of blood vessels allows early perfusion and vitality of bladder grafts--implications for bioengineered bladder wall.

Bioengineered bladder tissue is needed for patients with neurogenic bladder disease as well as for cancer. Current technologies in bladder tissue engineering have been hampered by an inability to efficiently initiate blood supply to the graft, ultimately leading to complications that include graft contraction, ischemia, and perforation. To date, the biological mechanisms of vascularization on transplant have not been suitably investigated for urologic tissues. To better understand the mechanisms of neovascularization on bladder wall transplant, a chimeric mouse model was generated such that angiogenesis and vasculogenesis could be independently assessed in vivo. Green fluorescence protein (GFP) transgenic mice received bone marrow transplants from ß-galactosidase (LacZ) transgenic animals and then subsequent bladder wall transplants from wild-type donor mice. Before euthanization, the aorta was infused with fluorescent microbeads (fluorospheres) to identify perfused vessels. The contributions of GFP (angiogenesis) and LacZ (vasculogenesis) to the formation of CD31-expressing blood vessels within the wild-type graft were evaluated by immunohistochemistry at different time points and locations within the graft (proximal, middle, and distal) to provide a spatiotemporal analysis of neovascularization. The GFP index, a measure of angiogenic host ingrowth, was significantly higher at proximal versus mid or distal regions in animals 2-16 weeks post-transplant. However, GFP index did not increase over time in any area. Within 7 days post-transplant, perfusion of primarily wild-type, donor blood vessels in the most distal areas of the graft was observed by intraluminal fluorospheres. In addition, chimeric host-donor (GFP-wild type) blood vessels were evident in proximal areas. The contribution of vasculogenesis to vascularization of the graft was limited, as LacZ cells were not specifically associated with the endothelial cells of blood vessels, but rather found primarily in areas of inflammation. The data suggest that angiogenesis of host blood vessels into the proximal region leads to inosculation between host and donor vessels and subsequent perfusion of the graft via pre-existing graft vessels within the first week after transplant. As such, the engineering of graft blood vessels and the promotion of inosculation might prevent graft contraction, thereby potentiating the use of bioengineered bladder tissue for transplantation.

Pathological changes in primary cilia: A novel mechanism of graft cholangiopathy caused by prolonged cold preservation in a rat model of orthotopic liver transplantation.

To study the impairment of cholangiocyte primary cilia caused by prolonged cold preservation and its correlation with graft cholangiopathy after orthotopic liver transplantation (OLT). Subjects were 60 male Wistar rats that were divided into 2 groups: a control group (n = 30) receiving a donor liver preserved for 1 h and a study group (n = 30) receiving a donor liver preserved for 12 h. A two-cuff method was used to establish the OLT model, and the hepatic artery and bile ducts were reconstructed using stents. Samples were collected 2, 8, and 16 weeks after surgery, and 5 samples were collected from each group at each time point. Serum biochemical indicators were measured, morphological changes in intrahepatic bile ducts and cholangiocyte primary cilia were observed using an optical microscope and scanning electronic microscope, respectively, and the ciliary marker (α-tubulin) and membrane proteins (PC-1, TRPV4, and P2Y12) were detected using immunofluorescence analysis and Western blotting. In the study group, phlogocytes infiltrated around bile ducts and bile ducts proliferated markedly at 8 weeks. At 16 weeks, the biliary structures were indistinct and some bile ducts disappeared, a large amount of collagen was deposited, numerous phlogocytes infiltrated around ducts, some biliary epithelial cells (BECs) were deformed or dead, and primary cilia disappeared. In the control group, the intrahepatic bile ducts and BECs were nearly intact and the primary cilia were present. In the study group, the expression of α-tubulin, polycystin-1 (PC-1), TRPV4, and P2Y12 in bile ducts disappeared completely after 8 weeks. In the control group, expression of the marker and proteins decreased at 2 weeks and increased slightly after 8 weeks. These results suggest that the study group had dysfunctional primary cilia at the start of OLT and that this dysfunction was irreversible. In the control group, the primary cilia defects and subsequent biliary injury were temporary. Thus, prolonged cold preservation of a donor liver may cause graft cholangiopathy by altering the integrity and functions of cholangiocyte primary cilia.

Schwann-like cells seeded in acellular nerve grafts improve nerve regeneration.

BACKGROUND: This study evaluated whether Schwann-like cells (SLCs) induced from bone marrow-derived mesenchymal stem cells (BM-MSCs) transplanted into acellular nerve grafts (ANGs) could repair nerve defects compared with nerve isografts and ANGs with BM-MSCs. METHODS: BM-MSCs extracted, separated and purified from the bone marrow of rats, and some of the BM-MSCs were cultured with mixed induction agents that could induce BM-MSCs into SLCs. Either SLCs or BM-MSCs were seeded onto 10-mm ANGs, and the isografts were chosen as the control. The walking-track test, tibialis anterior muscle weight measurement, electrophysiological examination, toluidine blue staining, transmission electron micrographs and immunostaining of S-100 and VEGF in these three groups were evaluated in a 10-mm rat sciatic injury-repair model. RESULTS: The walking-track test, tibialis anterior muscle weight measurement and electrophysiological examination of the sciatic nerve suggested the groups of ANGs with SLCs and isografts obtained better results than the BM-MSC group (P<0.05). Meanwhile, the results of the SLCs and isograft groups were similar (P>0.05). All the histomorphometric analyses (toluidine blue staining, transmission electron micrographs and immunostaining of S-100 and VEGF) showed that there were more regenerating nerve fibers in the group of ANGs with SLCs than the BM-MSCs (P<0.05), but there was no significant difference between the SLC and isograft groups (P>0.05). CONCLUSIONS: SLCs seeded in ANGs and isografts show better functional regeneration compared with BM-MSCs seeded in ANGs. Additionally, SLCs combined with ANGs present almost the same outcome as the isografts. Therefore, SLCs with ANGs can be a good choice in nerve defect repairs.

Assessment of in vivo behavior of polymer tube nerve grafts simultaneously with the peripheral nerve regeneration process using scanning electron microscopy technique.

In this study, scanning electron microscopy (SEM) has been applied for instantaneous assessment of processes occurring at the site of regenerating nerve. The technique proved to be especially useful when an artificial implant should have been observed but have not yet been extensively investigated before for assessment of nerve tissue. For in vivo studies, evaluation of implant's morphology and its neuroregenerative properties is of great importance when new prototype is developed. However, the usually applied histological techniques require separate and differently prepared samples, and therefore, the results are never a 100% comparable. In our research, we found SEM as a technique providing detailed data both on an implant behavior and the nerve regeneration process inside the implant. Observations were carried out during 12-week period on rat sciatic nerve injury model reconstructed with nerve autografts and different tube nerve grafts. Samples were analyzed with haematoxylin-eosin (HE), immunocytochemical staining for neurofillament and S-100 protein, SEM, TEM, and the results were compared. SEM studies enabled to obtain characteristic pictures of the regeneration process similarly to TEM and histological studies. Schwann cell transformation and communication as well as axonal outgrowth were identified, newly created and matured axons could be recognized. Concurrent analysis of biomaterial changes in the implant (degradation, collapsing of the tube wall, migration of alginate gel) was possible. This study provides the groundwork for further use of the described technique in the nerve regeneration studies.