Untersuchungen zum Einfluss von thrombozytären Wachstumsfaktoren auf den zellvermittelten Abbau eines nanopartikulären Knochenersatzstoffes auf Hydroxylapatitbasis : eine experimentelle Studie am Miniaturschwein

Ziel der vorliegenden tierexperimentellen Studie am Miniaturschwein war es, den Einfluss von plättchenreichem Plasma (PRP) auf den zellvermittelten Abbau eines nanopartikulären Hydroxylapatits (HA) in der Frühphase der Knochendefektheilung zu untersuchen. Hierzu wurden 26 männliche Miniaturschweine der Rasse Mini-Lewe in drei Versuchsgruppen eingeteilt und jeweils ein standardisierter Knochendefekt in der Intercondylarregion des rechten Femurs angelegt. Die Defekte wurden entweder mit dem Knochenersatzstoff (Gruppe I/PRP-,n = 11) oder dem Knochenersatzstoff kombiniert mit PRP (Gruppe II/PRP+, n = 11) befüllt. In einer Kontrollgruppe (n = 4) blieben die Defekte unbefüllt. Während der Implantationsoperation wurden bei sechs Tieren jeweils 250 ml Vollblut entnommen, aus dem anschließend durch fraktionierte Zentrifugation plättchenreiches Plasma gewonnen wurde. Die enthaltenen Thrombozyten wurden durch den Zusatz von Thrombin und Kalziumglukonat zur Degranulation angeregt, wodurch die enthaltenen Wachstumsfaktoren aus den alpha-Granula freigesetzt wurden. Zu diesen Wachstumsfaktoren gehören Platelet Derived Growth Factor AB und BB (PDGF AB, BB), Transforming Growth Factor ß 1 (TGF beta1), Vascular Endothelial Growth Factor (VEGF) und basic Fibroblast Growth Factor (bFGF). Die Konzentration der genannten Wachstumsfaktoren wurde mit Hilfe der ELISA-Technik bestimmt. Sie lagen zwischen Faktor 1,6 für TGF-beta1 und Faktor 24,4 für bFGF. 20 Tage post operationem fand die Explantation der operierten distalen Femura statt. Zur lichtmikroskopischen Untersuchung fanden die Knochen-Implantat-Proben Eingang in unterschiedliche Techniken der Einbettung (Paraffin-, Kunststoffeinbettungen), Präparation (Paraffinschnitte, Kunststoffschnitte und Schliffpräparationen), Färbung (Toluidinblau, Haematoxylin-Eosin, Safranin) und Histochemie (Enzym-, Immunhistochemie). Darüber hinaus wurden transmissionselektronenmikroskopische und computergestützte histomorphometrische Untersuchungen durchgeführt. Wie die Ergebnisse der Licht- und Transmissionselektronenmikroskopie aufgezeigt haben, erfolgt in den mit Knochenersatzmaterial behandelten Versuchsgruppen, unabhängig von der PRP-Applikation, die HA-Degradation hydrolytisch und Makrophagen-vermittelt. Die Makrophagen-Population wird durch Riesenzellen vom Langhans-Typ repräsentiert. Diese polarisierten Polykaryen adhärieren über ihre apikale Membrandomäne an den Implantatoberflächen. Das subplasmalemmale Zytoplasma ist immunhistochemisch durch Vimentin-Kondensationen gekennzeichnet. Nicht-adhärente, frei im Granulationsgewebe lokalisierte Polykaryen zeigen dagegen ein homogenes Vimentin-Verteilungsmuster im Zytoplasma. Der zelluläre Abbau des HA erfolgt mittels Phagozytose, indem die Polykaryen den "Fremdkörper" mit pseudopodienartigen Zytoplasmaausläufern umschließen und in ihr Zytoplasma inkorporieren. Diese Art der Degradation wird durch den post implantationem stattfindenden Zerfall des Knochenersatzmaterials in zahlreiche kleine Partikel unterstützt. Die hieraus resultierende Vergrößerung der Implantatoberfläche bietet einer Vielzahl von Zellen die Möglichkeit zur Haftung. Die festgestellten Expressionsmuster des CD44- Membranglykoproteins verweisen auf dessen funktionelle Rolle im Rahmen der Fusion mononukleärer Makrophagen zu multinukleären Riesenzellen. Die darüber hinaus beobachtete Umverteilung von CD44 von der apikalen zur basalen Membrandomäne bei Implantatassoziierten Polykaryen ist als transientes Geschehen im Zuge der Adhäsion zu interpretieren. Der hohe Aktivitätsstatus der adhärenten Polykaryen ist immunhistochemisch durch eine intensive Kathepsin K-Expression gekennzeichnet. Die vergleichende histomorphometrische Auswertung der mit HA aufgefüllten Defekte dokumentiert eine Verdopplung der Anzahl von Polykaryen in der Gruppe "Knochenersatzstoff mit PRP". Ein auf Basis der Messergebnisse durchgeführter Wilcoxon-Rangsummentest verweist auf den hochsignifikanten Einfluss (p < 0,01) des Faktors PRP auf die Ausdehnung Tartrat-resistenter saurer Phosphatase-positiver Areale in den Präparaten. Diese Effekte können sowohl auf den im PRP angereicherten Wachstumsfaktoren als auch auf dem homologen Charakter der PRP-Zubereitung beruhen. Die beobachteten Polykaryen – sogenannte "Fremdkörperriesenzellen" – sind auch immer Indikatoren einer stattfindenden Entzündungsreaktion. Die histomorphometrisch dargestellte, deutlich verstärkte Fremdkörperreaktion in Gruppe II/PRP+ kann auf die PRP-Applikation zurückgeführt werden. Im weiteren Heilungsverlauf kann dies zu einer Verzögerung der knöchernen Konsolidierung der Defekte führen.Aim of the current experimental study in Minipigs was to examine the effects of homologous platelet-rich plasma (PRP) on the cell-mediated degradation of a nanoparticulate hydroxyapatite (HA) during the early phase of bone defect healing. Twenty-six male "Lewe" minipigs were divided into three groups. Standardized bone defects were created in the intercondylar region of the right femur of each pig and were filled with HA (Group I/PRP-, n = 11) or HA + PRP (Group II/PRP+, n = 11). The defects of the control group (n = 4) were left empty. During the implantation procedure blood was drawn from six minipigs (250 ml each). PRP was isolated from these blood samples after several centrifugation steps. After the addition of thrombin and calcium gluconate growth factors were released from the alpha-granules of the thrombocytes which were enriched within the PRP. Some of these growth factors are Platelet Derived Growth Factor AB and BB (PDGF AB, BB), Transforming Growth Factor ß 1 (TGF beta1), Vascular Endothelial Growth Factor (VEGF) and basic Fibroblast Growth Factor (bFGF). The level of enrichment of these growth factors was controlled by the ELISA technique. Growth factor enrichment within the PRP ranged from 1.6 fold (TGF-beta1) to 24.4 fold (bFGF). After 20 days the treated distal femura were explanted. For light microscopical examination different tissue embedding methods (paraffine, plastic, resin), sectioning techniques (paraffine sections, plastic and resin sections, sawing and grinding sections), staining procedures (toluidine blue, hematoxylin eosin, safranin) and histochemical methods (enzyme- and immunohistochemistry) were performed. Additionally transmission electron microscopy and computer-assisted histomorphometry were used. The results of light microscopy and transmission electron microscopy showed that regardless of the addition of PRP, the HA is degraded by hydrolysis and macrophages. The population of macrophages consists of Langhans-type giant cells. The adhesion of the polarized polykaryons at the surfaces of the implant is mediated by the apical domain of the plasmamembranes. Vimentin condensations of the cytoplasm are attached to the apical plasmalemma. In contrast, non-adherent polykaryons of the granulation tissue reveal a homogeneous Vimentin distribution pattern within their cytoplasma. As shown ultrastructurally, the implant is degraded by means of phagocytosis. The implant particles are encircled by pseudopodia of the polykaryons and become incorporated into the cytoplasma. The degradation process is supported by disintegration of the bone substitute into numerous small particles after implantation. This disintegration causes enlargement of the implant surface and increases the probability of phagocyte adhesion. The pattern of CD44 expression points towards a functional role of the molecule during fusion of mononucleated macrophages into multinucleated giant cells. Implant-associated polykaryons show CD44 immunoreactivity only along the basal domains of the cytomembrane. This pattern can be interpreted as a temporal event during adhesion. Adherent polykaryons are further characterized by strong cathepsin K expression. The histomorphometric examination demonstrates twice as much foreign body giant cells in "Group II/PRP+" as in "Group I/PRP-". Based on these results, a Wilcoxon-signed-rank test was performed and a highly significant effect (p < 0.01) of PRP on the expansion of tartrate resistent acid phophatase (TRAP)-positive areas within bone defects could be demonstrated. This effect could be a result of the substution of PRP or of its homologous character. The polykaryons descibed in this work - so-called Foreign Body Giant Cells - are also indicators of inflammation. The enhanced cellular reaction observed in Group II/PRP+ must be interpreted as a strong foreign body reaction, triggered by the addition of PRP. It cannot be excluded that the strong inflammation reaction will lead to delayed bone formation in the course of healing

Effect of Trp53 gene deficiency on brain injury after neonatal hypoxia-ischemia

Hypoxia-ischemia (HI) can result in permanent life-long injuries such as motor and cognitive deficits. In response to cellular stressors such as hypoxia, tumor suppressor protein p53 is activated, potently initiating apoptosis and promoting Bax-dependent mitochondrial outer membrane permeabilization. The aim of this study was to investigate the effect of Trp53 genetic inhibition on injury development in the immature brain following HI. HI (50 min or 60 min) was induced at postnatal day 9 (PND9) in Trp53 heterozygote (het) and wild type (WT) mice. Utilizing Cre-LoxP technology, CaMK2α-Cre mice were bred with Trp53-Lox mice, resulting in knockdown of Trp53 in CaMK2α neurons. HI was induced at PND12 (50 min) and PND28 (40 min). Extent of brain injury was assessed 7 days following HI. Following 50 min HI at PND9, Trp53 het mice showed protection in the posterior hippocampus and thalamus. No difference was seen between WT or Trp53 het mice following a severe, 60 min HI. Cre-Lox mice that were subjected to HI at PND12 showed no difference in injury, however we determined that neuronal specific CaMK2α-Cre recombinase activity was strongly expressed by PND28. Concomitantly, Trp53 was reduced at 6 weeks of age in KO-Lox Trp53 mice. Cre-Lox mice subjected to HI at PND28 showed no significant difference in brain injury. These data suggest that p53 has a limited contribution to the development of injury in the immature/juvenile brain following HI. Further studies are required to determine the effect of p53 on downstream targets

Induction of Mitochondrial Fragmentation and Mitophagy after Neonatal Hypoxia–Ischemia

Hypoxia–ischemia (HI) leads to immature brain injury mediated by mitochondrial stress. If damaged mitochondria cannot be repaired, mitochondrial permeabilization ensues, leading to cell death. Non-optimal turnover of mitochondria is critical as it affects short and long term structural and functional recovery and brain development. Therefore, disposal of deficient mitochondria via mitophagy and their replacement through biogenesis is needed. We utilized mt-Keima reporter mice to quantify mitochondrial morphology (fission, fusion) and mitophagy and their mechanisms in primary neurons after Oxygen Glucose Deprivation (OGD) and in brain sections after neonatal HI. Molecular mechanisms of PARK2-dependent and -independent pathways of mitophagy were investigated in vivo by PCR and Western blotting. Mitochondrial morphology and mitophagy were investigated using live cell microscopy. In primary neurons, we found a primary fission wave immediately after OGD with a significant increase in mitophagy followed by a secondary phase of fission at 24 h following recovery. Following HI, mitophagy was upregulated immediately after HI followed by a second wave at 7 days. Western blotting suggests that both PINK1/Parkin-dependent and -independent mechanisms, including NIX and FUNDC1, were upregulated immediately after HI, whereas a PINK1/Parkin mechanism predominated 7 days after HI. We hypothesize that excessive mitophagy in the early phase is a pathologic response which may contribute to secondary energy depletion, whereas secondary mitophagy may be involved in post-HI regeneration and repair

Induction of Mitochondrial Fragmentation and Mitophagy after Neonatal Hypoxia&ndash;Ischemia

Hypoxia&ndash;ischemia (HI) leads to immature brain injury mediated by mitochondrial stress. If damaged mitochondria cannot be repaired, mitochondrial permeabilization ensues, leading to cell death. Non-optimal turnover of mitochondria is critical as it affects short and long term structural and functional recovery and brain development. Therefore, disposal of deficient mitochondria via mitophagy and their replacement through biogenesis is needed. We utilized mt-Keima reporter mice to quantify mitochondrial morphology (fission, fusion) and mitophagy and their mechanisms in primary neurons after Oxygen Glucose Deprivation (OGD) and in brain sections after neonatal HI. Molecular mechanisms of PARK2-dependent and -independent pathways of mitophagy were investigated in vivo by PCR and Western blotting. Mitochondrial morphology and mitophagy were investigated using live cell microscopy. In primary neurons, we found a primary fission wave immediately after OGD with a significant increase in mitophagy followed by a secondary phase of fission at 24 h following recovery. Following HI, mitophagy was upregulated immediately after HI followed by a second wave at 7 days. Western blotting suggests that both PINK1/Parkin-dependent and -independent mechanisms, including NIX and FUNDC1, were upregulated immediately after HI, whereas a PINK1/Parkin mechanism predominated 7 days after HI. We hypothesize that excessive mitophagy in the early phase is a pathologic response which may contribute to secondary energy depletion, whereas secondary mitophagy may be involved in post-HI regeneration and repair

Single sustained inflation followed by ventilation leads to rapid cardiorespiratory recovery but causes cerebral vascular leakage in asphyxiated near-term lambs

Background A sustained inflation (SI) rapidly restores cardiac function in asphyxic, bradycardic newborns but its effects on cerebral haemodynamics and brain injury are unknown. We determined the effect of different SI strategies on carotid blood flow (CaBF) and cerebral vascular integrity in asphyxiated near-term lambs. Methods Lambs were instrumented and delivered at 139 ± 2 d gestation and asphyxia was induced by delaying ventilation onset. Lambs were randomised to receive 5 consecutive 3 s SI (multiple SI; n = 6), a single 30 s SI (single SI; n = 6) or conventional ventilation (no SI; n = 6). Ventilation continued for 30 min in all lambs while CaBF and respiratory function parameters were recorded. Brains were assessed for gross histopathology and vascular leakage. Results CaBF increased more rapidly and to a greater extent during a single SI (p = 0.01), which then decreased below both other groups by 10 min, due to a higher cerebral oxygen delivery (p = 0.01). Blood brain barrier disruption was increased in single SI lambs as indicated by increased numbers of blood vessel profiles with plasma protein extravasation (p = 0.001) in the cerebral cortex. There were no differences in CaBF or cerebral oxygen delivery between the multiple SI and no SI lambs. Conclusions Ventilation with an initial single 30 s SI improves circulatory recovery, but is associated with greater disruption of blood brain barrier function, which may exacerbate brain injury suffered by asphyxiated newborns. This injury may occur as a direct result of the initial SI or to the higher tidal volumes delivered during subsequent ventilation

End diastolic pressure (BP_ED) expressed as a percentage change from before cord clamping measured in <i>in utero</i> (•) or <i>ex utero</i> (○) lambs before and after umbilical cord occlusion (designated as time 0).

<p>Data are mean ± SEM. * p&lt;0.05 <i>in utero</i> vs <i>ex utero</i>.</p

Max dP/dt expressed as a percentage change from before cord clamping measured in <i>in utero</i> (•) or <i>ex utero</i> (○) lambs before and after umbilical cord occlusion (designated as time 0).

<p>Data are mean ± SEM. * p&lt;0.05 <i>in utero</i> vs <i>ex utero</i>.</p

Lamb arterial blood gases and physiological parameters at the end of asphyxia.

<p>BP, arterial blood pressure; PaCO₂, partial pressure of carbon dioxide; PaO₂, partial pressure of oxygen; SaO₂, oxygen saturation; BE, base excess. Data are mean ± SD. * p&lt;0.05 <i>in utero</i> vs <i>ex utero</i>.</p><p>Lamb arterial blood gases and physiological parameters at the end of asphyxia.</p