Treatment of Osteochondral Defects in the Rabbit's Knee Joint by Implantation of Allogeneic Mesenchymal Stem Cells in Fibrin Clots

The treatment of osteochondral articular defects has been challenging physicians for many years. The better understanding of interactions of articular cartilage and subchondral bone in recent years led to increased attention to restoration of the entire osteochondral unit. In comparison to chondral lesions the regeneration of osteochondral defects is much more complex and a far greater surgical and therapeutic challenge. The damaged tissue does not only include the superficial cartilage layer but also the subchondral bone. For deep, osteochondral damage, as it occurs for example with osteochondrosis dissecans, the full thickness of the defect needs to be replaced to restore the joint surface ¹. Eligible therapeutic procedures have to consider these two different tissues with their different intrinsic healing potential ². In the last decades, several surgical treatment options have emerged and have already been clinically established ^3-6.Autologous or allogeneic osteochondral transplants consist of articular cartilage and subchondral bone and allow the replacement of the entire osteochondral unit. The defects are filled with cylindrical osteochondral grafts that aim to provide a congruent hyaline cartilage covered surface ^3,7,8. Disadvantages are the limited amount of available grafts, donor site morbidity (for autologous transplants) and the incongruence of the surface; thereby the application of this method is especially limited for large defects.New approaches in the field of tissue engineering opened up promising possibilities for regenerative osteochondral therapy. The implantation of autologous chondrocytes marked the first cell based biological approach for the treatment of full-thickness cartilage lesions and is now worldwide established with good clinical results even 10 to 20 years after implantation ^9,10. However, to date, this technique is not suitable for the treatment of all types of lesions such as deep defects involving the subchondral bone ¹¹.The sandwich-technique combines bone grafting with current approaches in Tissue Engineering ^5,6. This combination seems to be able to overcome the limitations seen in osteochondral grafts alone. After autologous bone grafting to the subchondral defect area, a membrane seeded with autologous chondrocytes is sutured above and facilitates to match the topology of the graft with the injured site. Of course, the previous bone reconstruction needs additional surgical time and often even an additional surgery. Moreover, to date, long-term data is missing ¹².Tissue Engineering without additional bone grafting aims to restore the complex structure and properties of native articular cartilage by chondrogenic and osteogenic potential of the transplanted cells. However, again, it is usually only the cartilage tissue that is more or less regenerated. Additional osteochondral damage needs a specific further treatment. In order to achieve a regeneration of the multilayered structure of osteochondral defects, three-dimensional tissue engineered products seeded with autologous/allogeneic cells might provide a good regeneration capacity ¹¹.Beside autologous chondrocytes, mesenchymal stem cells (MSC) seem to be an attractive alternative for the development of a full-thickness cartilage tissue. In numerous preclinical in vitro and in vivo studies, mesenchymal stem cells have displayed excellent tissue regeneration potential ^13,14. The important advantage of mesenchymal stem cells especially for the treatment of osteochondral defects is that they have the capacity to differentiate in osteocytes as well as chondrocytes. Therefore, they potentially allow a multilayered regeneration of the defect.In recent years, several scaffolds with osteochondral regenerative potential have therefore been developed and evaluated with promising preliminary results ^1,15-18. Furthermore, fibrin glue as a cell carrier became one of the preferred techniques in experimental cartilage repair and has already successfully been used in several animal studies ^19-21 and even first human trials ²².The following protocol will demonstrate an experimental technique for isolating mesenchymal stem cells from a rabbit''s bone marrow, for subsequent proliferation in cell culture and for preparing a standardized in vitro-model for fibrin-cell-clots. Finally, a technique for the implantation of pre-established fibrin-cell-clots into artificial osteochondral defects of the rabbit''s knee joint will be described.     

Cyclo-saligenyl-5-fluoro-2′-deoxyuridinemonophosphate (cycloSal-FdUMP) — A New Prodrug Approach for FdUMP

Abstract

The synthesis of cycloSal-FdUMP 3a-d as a new prodrug approach for FdU 1 is described. Phosphotriesters 3 release the FdUMP 2 selectively by a controlled, chemically induced tandem reaction in hydrolysis studies. The biological activity (IC₅₀) of cycloSal-phosphotriesters 3 was evaluated in FM3A/O cells and FM3A/TK^? cells.     

Chemical Diversity of the Natural Populations of Dalmatian Pyrethrum (Tanacetum cinerariifolium (Trevir.) Sch.Bip.) in Croatia

Dalmatian pyrethrum (Tanacetum cinerariifolium (Trevir. ) Sch.Bip. ) is a plant species endemic to the east Adriatic coast. The bioactive substance of Dalmatian pyrethrum is a natural insecticide, pyrethrin, a mixture of six active components (pyrethrins I and II, cinerins I and II, and jasmolins I and II). The insecticidal potential of pyrethrin was recognized decades ago, and dried and ground flowers have traditionally been used in Croatian agriculture and households. A total of 25 Dalmatian pyrethrum populations from Croatia were studied to determine the pyrethrin content and composition, and to identify distinct chemotypes. The total pyrethrin content ranged from 0.36 to 1.30% (dry flower weight; DW) and the pyrethrin I/pyrethrin II ratio ranged from 0.64 to 3.33%. The statistical analyses revealed that the correlations between the percentage of pyrethrin I and of all the other components were significant and negative. The total pyrethrin content was positively correlated with the percentage of pyrethrin I and negatively correlated with cinerin II. The multivariate analysis of the chemical variability enabled the identification of five chemotypes among 25 Dalmatian pyrethrum populations. The chemical characterization of indigenous Dalmatian pyrethrum populations may serve as a good background for future breeding and agricultural exploitation.     

Production of human platelet lysate by use of ultrasound for ex vivo expansion of human bone marrow–derived mesenchymal stromal cells

Background aimsA medium supplemented with fetal bovine serum (FBS) is of common use for the expansion of human mesenchymal stromal cells (MSCs). However, its use is discouraged by regulatory authorities because of the risk of zoonoses and immune reactions. Human platelet lysate (PL) obtained by freezing/thawing disruption of platelets has been proposed as a possible substitute of FBS. The process is time-consuming and not well standardized. A new method for obtaining PL that is based on the use of ultrasound is proposed.MethodsPlatelet sonication was performed by submerging platelet-containing plastic bags in an ultrasonic bath. To evaluate platelet lysis we measured platelet-derived growth factor-AB release. PL efficiency was tested by expanding bone marrow (BM)-MSCs, measuring population doubling time, differentiation capacity and immunogenic properties. Safety was evaluated by karyotyping expanded cells.ResultsAfter 30 minutes of sonication, 74% of platelet derived growth factor-AB was released. PL enhanced BM-MSC proliferation rate compared with FBS. The mean cumulative population doubling (cPD) of cells growth in PL at 10%, 7.5% and 5% was better compared with cPD obtained with 10% FBS. PD time (hours) of MSCs with PL obtained by sonication was shorter than for cPD with PL obtained by freezing/thawing (18.9 versus 17.4, P < 0.01). BM mononucleated cells expressed MSC markers and were able to differentiate into adipogenic, osteogenic and chondrogenic lineages. When BM-MSCs and T cells were co-cultured in close contact, immunosuppressive activity of BM-MSCs was maintained. Cell karyotype showed no genetic alterations.ConclusionsThe proposed method for the production of PL by sonication could be a safe, efficient and fast substitute of FBS, without the potential risks of FBS.     

Differences in acute stress responses between wild-caught and captive-bred birds: a physiological mechanism contributing to current avian invasions?

Current avian invasions are often the result of exotic birds accidentally escaping from cages. It has been hypothesised that the higher invasiveness of wild-caught cage-birds compared to captive-bred ones could be related to the loss of ability in captive-bred birds to cope with new environments. The acute stress response plays an important role in how animals cope with challenges because elevated corticosterone (CORT) levels can mediate learning and memory consolidation and help to increase their survival prospects. We experimentally tested whether exotic wild-caught and captive-bred cage-birds differ in their responses to acute stress using a representative sample of parrots. Wild-caught individuals showed longer CORT responses to acute stress than captive-bred ones, both at inter- and intra-specific levels when comparing wild-birds to the first generation born in captivity. Captive-bred birds may have attenuated their CORT responses due to acclimation, while high mortality rates during the international trade of wild-caught birds could have selected those individuals that are better able to cope with stress. We suggest that the longer acute response found in wild-caught parrots could help them to escape from cages and survive when facing challenges in new wild environments, possibly contributing to their higher invasiveness.     

Adapt or die—Response of large herbivores to environmental changes in Europe during the Holocene

Climate warming and human landscape transformation during the Holocene resulted in environmental changes for wild animals. The last remnants of the European Pleistocene megafauna that survived into the Holocene were particularly vulnerable to changes in habitat. To track the response of habitat use and foraging of large herbivores to natural and anthropogenic changes in environmental conditions during the Holocene, we investigated carbon (δ¹³C) and nitrogen (δ¹⁵N) stable isotope composition in bone collagen of moose (Alces alces), European bison (Bison bonasus) and aurochs (Bos primigenius) in Central and Eastern Europe. We found strong variations in isotope compositions in the studied species throughout the Holocene and diverse responses to changing environmental conditions. All three species showed significant changes in their δ¹³C values reflecting a shift of foraging habitats from more open in the Early and pre‐Neolithic Holocene to more forest during the Neolithic and Late Holocene. This shift was strongest in European bison, suggesting higher plasticity, more limited in moose, and the least in aurochs. Significant increases of δ¹⁵N values in European bison and moose are evidence of a diet change towards more grazing, but may also reflect increased nitrogen in soils following deglaciation and global temperature increases. Among the factors explaining the observed isotope variations were time (age of samples), longitude and elevation in European bison, and time, longitude and forest cover in aurochs. None of the analysed factors explained isotope variations in moose. Our results demonstrate the strong influence of natural (forest expansion) and anthropogenic (deforestation and human pressure) changes on the foraging ecology of large herbivores, with forests playing a major role as a refugial habitat since the Neolithic, particularly for European bison and aurochs. We propose that high flexibility in foraging strategy was the key for survival of large herbivores in the changing environmental conditions of the Holocene.     

Divergent responses of Atlantic cod to ocean acidification and food limitation

In order to understand the effect of global change on marine fishes, it is imperative to quantify the effects on fundamental parameters such as survival and growth. Larval survival and recruitment of the Atlantic cod (Gadus morhua) were found to be heavily impaired by end‐of‐century levels of ocean acidification. Here, we analysed larval growth among 35–36 days old surviving larvae, along with organ development and ossification of the skeleton. We combined CO₂ treatments (ambient: 503 µatm, elevated: 1,179 µatm) with food availability in order to evaluate the effect of energy limitation in addition to the ocean acidification stressor. As expected, larval size (as a proxy for growth) and skeletogenesis were positively affected by high food availability. We found significant interactions between acidification and food availability. Larvae fed ad libitum showed little difference in growth and skeletogenesis due to the CO₂ treatment. Larvae under energy limitation were significantly larger and had further developed skeletal structures in the elevated CO₂ treatment compared to the ambient CO₂ treatment. However, the elevated CO₂ group revealed impairments in critically important organs, such as the liver, and had comparatively smaller functional gills indicating a mismatch between size and function. It is therefore likely that individual larvae that had survived acidification treatments will suffer from impairments later during ontogeny. Our study highlights important allocation trade‐off between growth and organ development, which is critically important to interpret acidification effects on early life stages of fish.