In vivo efficacy of bone-marrow-coated polycaprolactone scaffolds for the reconstruction of orbital defects in the pig

Alloplastic materials offer a number of advantages over bone autografts in the reconstruction of craniofacial defects. These include: lack of donor site morbidity, unlimited quantities of available material, and the possibility to conform exactly to the defect. An ideal bioresorbable material would degrade slowly, and have osteoconductive properties to allow replacement and remodeling by osseous tissue. This is seldom observed, the materials instead being replaced by fibrous tissue. Polycaprolactone (PCL), an FDA-approved bioresorbable polymer, has several properties that might make it suitable for reconstruction of craniofacial defects. The technique of fused deposition modeling (FDM) allows for the fabrication of highly reproducible bioresorbable 3D scaffolds. The nature of the fully interconnected pore network might enhance vascular ingrowth and osteoconductive properties. It was hypothesized that coating the scaffolds in bone marrow might enhance bone formation due to the osteoinductive nature of the bone-marrow mesenchymal cells. This study aimed to test these hypotheses in the pig model. Defects measuring 2 x 2 cm were surgically created in each orbit of eight Yorkshire pigs. The orbits were divided into three groups: Group 1 (n=4), no reconstruction (control); Group 2 (n=6), reconstruction with no coated PCL scaffolds; and Group 3 (n=6) reconstruction with bone-marrow-coated PCL scaffolds. The results were evaluated at 3 months by histological and histomorphometric analyses. The defects in Group 1 were covered with fibrous scar tissue. The shape of the reconstructed area was insufficient. The defects in Groups 2 and 3 were reconstructed correctly. In Group 2 the noncoated scaffolds showed 4.5% of new bone formation compared with 14.1% in Group 3, which is statistically significant (p<0.05). The entirely interconnected 3D polycaprolactone scaffold seems to be a promising material. It induces the bone ingrowth required for reconstructing craniofacial and orbital defects. Further long-term evaluations of these PCL scaffolds must be made in order to confirm these conclusions.     

Identification of cyclophilin A from human decidual and placental tissue in the first trimester of pregnancy

The protein patterns of tissue homogenates from human deciduaand placenta of first trimester pregnancies were investigated.Particular attention was paid to the low molecular weight componentsof these tissues, since substantial evidence has accumulatedthat some of these smaller proteins show a characteristic cyclicand pregnancy expression. Two specific bands were purified fromhomogenates of first trimester decidua and placenta using gelfiltration and anion exchange chromatography. These bands wereseparated by gel electrophoresis and blotting onto polyvinylidendifluoridemembrane. Partial amino acid sequencing of both proteins revealedsequences identical to human cyclophilin A. One protein wassequenced V-N-P-T-V-F-F-D-I-A-V-D-G-E-P-L-G-R-(X)-S-F-E-L-F-A-D-K-V-Pand identified as the 17 kDa isoform of cyclophilin A. The otherprotein was sequenced V-N-P-T-V-F-F-D-I-A and identified asthe 18 kDa isoform of cyclophilin A. cyclophilin A/decidua/placenta/progesterone/progesterone receptor     

Recent insights into the signals that control αβ/γδ-lineage fate

Summary:  During thymopoiesis, two major types of mature T cells are generated that can be distinguished by the clonotypic subunits contained within their T-cell receptor (TCR) complexes: αβ T cells and γδ T cells. Although there is no consensus as to the exact developmental stage where αβ and γδ T-cell lineages diverge, γδ T cells and precursors to the αβ T-cell lineage (bearing the pre-TCR) are thought to be derived from a common CD4⁻CD8⁻ double-negative precursor. The role of the TCR in αβ/γδ lineage commitment has been controversial, in particular whether different TCR isotypes intrinsically favor adoption of the corresponding lineage. Recent evidence supports a signal strength model of lineage commitment, whereby stronger signals promote γδ development and weaker signals promote adoption of the αβ fate, irrespective of the TCR isotype from which the signals originate. Moreover, differences in the amplitude of activation of the extracellular signal-regulated kinase- mitogen-activated protein kinase-early growth response pathway appear to play a critical role. These findings will be placed in context of previous analyses in an effort to more precisely define the signals that control T-lineage fate during thymocyte development.     

Cellular Immunity in COVID-19 Convalescents with PCR-Confirmed Infection but with Undetectable SARS-CoV-2–Specific IgG

We investigated immune responses against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) among a group of convalescent, potential blood donors in Germany who had PCR-confirmed SARS-CoV-2 infection. Sixty days after onset of symptoms, 13/78 (17%) study participants had borderline or negative results to an ELISA detecting IgG against the S1 protein of SARS-CoV-2. We analyzed participants with PCR-confirmed infection who had strong antibody responses (ratio >3) as positive controls and participants without symptoms of SARS-CoV-2 infection and without household contact with infected patients as negative controls. Using interferon-γ ELISpot, we observed that 78% of PCR-positive volunteers with undetectable antibodies showed T cell immunity against SARS-CoV-2. We observed a similar frequency (80%) of T-cell immunity in convalescent donors with strong antibody responses but did not detect immunity in negative controls. We concluded that, in convalescent patients with undetectable SARS-CoV-2 IgG, immunity may be mediated through T cells.     

Successful 18-h acellular extracorporeal perfusion and replantation of porcine limbs - Histology versus nerve stimulation

The current standard for composite tissue preservation is static cold storage (SCS) and is limited to 6 h until irreversible muscle damage occurs. Extracorporeal perfusion (ECP) is a promising technique for prolonged preservation, however, functional results have been scarcely researched. This article assessed neuromuscular function and compared results to histological alterations to predict muscle damage after ECP. Forelimbs of twelve Dutch landrace pigs were amputated and preserved by 4 h SCS at 4–6 °C (n = 6) or 18 h mid-thermic ECP with University of Wisconsin solution (n = 6). Limbs were replanted and observed for 12 h. Sham surgery was performed on contralateral forelimbs (n = 12). Histology analysis scored four subgroups representing different alterations (higher score equals more damage). Muscle contraction after median nerve stimulation was comparable between ECP, SCS, and sham limbs (P = 0.193). Histology scores were higher in ECP limbs compared to SCS limbs (4.8 vs. 1.5, P = 0.013). This was mainly based on more oedema in these limbs. In-vivo muscle contraction was well preserved after 18 h ECP compared to short SCS, although histology seemed inferior in this group. Histology, therefore, did not correlate to muscle function at 12 h after replantation. This leads to the question whether histology or neuromuscular function is the best predictor for transplant success.     

Significance of wet autoclave pretreatment in immunohistochemistry

Until recently the only way to rescue masked epitopes in routinely processed surgical pathological material was enzymatic digestion. The use of heat for antigen retrieval, first by microwave irradiation, represents an important breakthrough in immunohistochemistry. With the acceptance of microwave oven pretreatment, various modified techniques and alternative heating methods have also been proposed. Wet autoclave pretreatment for tissue proteolysis is a highly reliable alternative to the microwave antigen retrieval technique. It provides uniform heating of the slides, hence an even enhancement of staining intensity in a variety of formalin-sensitive antigens, and it also offers consistent interlaboratory results. The method has been introduced in routine diagnostic immunohistochemistry for the detection of estrogen-and progesterone receptors, L26-, Ki-67- and bcl-2 antigens and variable types of cytokeratins (1/5/10/11, 8, 13, 19). Experimentally, wet autoclaving can be used very successfully for the immunophenotyping of p53 and mdm2 expression, for the detection of adhesion molecules (CD44, integrins) and some anti-inflammatory molecules (annexins), among others. It has produced a substantial improvement in the visualisation of silver-stained nucleolar organizer regionsassociated proteins (AgNORs) in routine paraffin sections and along with modified silver staining and standardized AgNOR parameters assessed by image analysis. Wet autoclaving-based AgNOR staining has been proposed by a European multicentric study group as the standardized method for AgNOR analysis in archival material.     

Proliferative (mib1, mdm2) versus anti-proliferative (p53) markers in head and neck cancer. An Immunohistochemical study

Formalin fixed and paraffin embedded samples from 36 squamous cell carcinomas of the larynx and the oral cavity (pT₂N₀M₀, R₀) surrounded by non-tumorous mucosa were studied immunohistochemically using a panel of four different anti-p53 antibodies (CM1, PAbl801, D07, PAb240), a monoclonal anti-mdm2 antibody and MIB1, following wet autoclave antigen retrieval. P53 immunoreactivity was detected in 11/14 laryngeal and in 9/22 oral carcinomas. All p53 positive oral, and all but one laryngeal tumors revealed mdm2 positivity as well, whereas in p53 negative tumors 4/12 and 1/3 mdm2 immunopositive cases were demonstrated, respectively. MIB1 labeling indices of the tumors ranged between 18% – 64% in p53 positive cases, and 10% – 53% in p53 negative ones. The difference was not statistically significant. Close spatial coexpression of p53, mdm2 and MIB1 immunoreactivity was observed at the invasive front of the carcinomas and in the basal and suprabasal layers of the non-tumorous epithelium in all p53 positive cases. However, the MIB1 expression was similarly increased at the invasive margins in carcinomas lacking immunohistochemically detectable p53 alterations. Our results strongly suggest that p53 overexpression does not necessarily correspond to increased rate of proliferation, but rather to mdm2 overexpression and is largely dependent on the anatomical site in case of small and localized squamous cell carcinomas of the head and neck region.     

Assessment of left ventricular function: comparison between radionuclide angiography and semiquantitative two-dimensional echocardiographic analysis

Measurement of global left ventricular function is important in the follow-up of cardiac patients and is a good prognostic indicator in acute cardiac situations. We compared quantitative measurements of global left ventricular function made with radionuclide angiography (RNA) and contrast cardiac ventriculography (CVG) to visual semiquantitative estimates from two-dimensional echocardiographic images (2D-echo). Three hundred and thirty-nine consecutive patients who underwent RNA were assessed with 2D-echo within 3 months. In addition, 92 of these patients also underwent CVG (correlation of ejection fraction between CVG and RNA:r=0.82;P<0.0001). The RNA mean ejection fractions in the four 2D-echo groups (0=normal, 1=slightly, 2=moderate, or 3=severe reduced left ventricular function) differed markedly (P<0.0001); however, there was overlapping among the groups (2D-echo score/RNA ejection fraction: 0=57.3%±12.8%; 1=46.0%±12.9%; 2=29.6%±12.2%; and 3=24.6%±11.5%) and the difference between 2D-echo scores 2 and 3 was not significant. 2D-echo showed a good concordance in RNA classes (0=505; 1=35%–49%; 2=21%–34%; and 3=520% ejection fraction) 0 (133/166; 80%) and 3 (18/30; 60%) but low concordance in classes 1 (27/82; 33%) and 2 (21/61; 34%). For accurate assessment of global left ventricular ejection fraction, visual semiquantitative judgement of a 2D echocardiographic image is limited in comparison to CVG or RNA, especially in patients with a slight or moderate reduction in left ventricular ejection fraction.     

Die Veränderungen der physikalischen Eigenschaften von Polyacetalharz (Hostaform,C®), Polyester (Hostadur®), Polyäthylen (Hostalen®) und Teflon (Hostaflon®) nach tierischer Implantation und mehrfachem Autoklavieren

Zusammenfassung Nach 4- und 12wöchiger Implantation in der Rückenmuskulatur von 6 Monate alten gleichgewichtigen männlichen Kaninchen sowie nach ein- bis viermaligem Autoklavieren erfolgte die Untersuchung von standardisierten Probekörpern aus Polyacetalharz (Polyoxymethylen-Copolymer), Polyester (Polyäthylenterephthalat), Polyäthylen und Teflon (Polytetrafluoräthylen) im Rahmen eines Zugversuches. Dabei zeigte es sich, daß Polyester durch Implantation und Autoklavieren eine starke Verminderung der viscoelastischen Eigenschaften erfährt. Während Polyäthylen und Teflon nach beiden Behandlungen im wesentlichen unveränderte Eigenschaften aufweisen, läßt sich bei Polyacetalharz nach 12wöchiger Implantation und viermaligem Autoklavieren nur eine leichte Verminderung der viscoelastischen Eigenschaften im Sinne einer Versprödung von 10 bis 15% nachweisen.Die histologisch-qualitative Beurteilung des umliegenden Gewebes ergibt eine überragende Gewebsverträglichkeit von Polyäthylen, während bei den drei übrigen Kunststoffen Polyacetalharz, Polyester und Teflon eine vergleichbare Fremdkörperreaktion nach 4 Wochen erkennbar ist, die nach 12 Wochen jedoch deutlich abgenommen hat.

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The change of physical properties of plastics (polyoxymethylenecopolymer, polyethyleneterephthalate, polyethylene, polytetrafluorethylene) after animal implantation and autoclavation

Summary The change of physical properties of plastics (polyoxymethylene-copolymer, polyethyleneterephthalate, polyethylene, polytetrafluorethylene) and the bio-compatibility of these materials were examined by implantation in the backmuscle of 6-month-old male rabbits for 4 and 12 weeks and after autoclavation.We have found out, that after implantation and autoclavation polyethyleneterephthalate demonstrates a strong diminution of the visco-elastic qualities. Polyethylene and polytetrafluorethylene were not changed by these treatments. After an implantation of 12 weeks and an autoclavation of four times the visco-elastic properties of polyoxymethylene-copolymer were only slightly diminished by 10 to 15%.The histological investigation of the surrounding tissue demonstrated a very good bio-compatibility of polyethylene. After an implantation of 4 weeks polyoxymethylene-copolymer, polyethyleneterephthalate and polytetrafluorethylene produced a comparable foreign body reaction, which, however, was evidently diminished after an implantation of 12 weeks.

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Mit Unterstützung der DFG (Schwerpunkt Biopolymere und Biomechanik von Bindegewebssystemen).

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Für Beratung und Unterstützung danken wir Herrn Prof. Dr. H. Ueberberg, Biberach, Abteilung für Experimentelle Pathologie der Firma Dr. K. Thomae.