Advancing the 3Rs: innovation, implementation, ethics and society.

The 3Rs principle of replacing, reducing and refining the use of animals in science has been gaining widespread support in the international research community and appears in transnational legislation such as the European Directive 2010/63/EU, a number of national legislative frameworks like in Switzerland and the UK, and other rules and guidance in place in countries around the world. At the same time, progress in technical and biomedical research, along with the changing status of animals in many societies, challenges the view of the 3Rs principle as a sufficient and effective approach to the moral challenges set by animal use in research. Given this growing awareness of our moral responsibilities to animals, the aim of this paper is to address the question: Can the 3Rs, as a policy instrument for science and research, still guide the morally acceptable use of animals for scientific purposes, and if so, how? The fact that the increased availability of alternatives to animal models has not correlated inversely with a decrease in the number of animals used in research has led to public and political calls for more radical action. However, a focus on the simple measure of total animal numbers distracts from the need for a more nuanced understanding of how the 3Rs principle can have a genuine influence as a guiding instrument in research and testing. Hence, we focus on three core dimensions of the 3Rs in contemporary research: (1) What scientific innovations are needed to advance the goals of the 3Rs? (2) What can be done to facilitate the implementation of existing and new 3R methods? (3) Do the 3Rs still offer an adequate ethical framework given the increasing social awareness of animal needs and human moral responsibilities? By answering these questions, we will identify core perspectives in the debate over the advancement of the 3Rs.

Osteopromotion with a plasmatransglutaminase on a beta-TCP ceramic.

We investigated the osteopromotive properties of plasmatransglutaminase (F XIII), bone marrow and venous blood on a resorbable beta-tricalcium phosphate (beta-TCP) scaffold. A baseline binding and release study of F XIII from the scaffold showed a continuous release of 18% of the total dose after 48 h. The main study consisted of 18 adult sheep with cylindrical defects in both tibiae. The defects were filled with a beta-TCP cylinder impregnated either with bone marrow, venous blood, F XIII or sheep were treated with 1250 IU F XIII intravenously over 14 days (n = 4 in each group). The defects were left open in two sheep. QCT and histology was performed after 6 and 12 weeks. The best bone ingrowth was seen after 6 weeks in the bone marrow group and after 12 weeks in the local F XIII group. The highest ingrowth on the inside of the cylinder proving the osteopromoting potential of F XIII was found in the local F XIII group. In our opinion F XIII is a good and readily available osteopromoting agent which can be used with beta-TCP in cases of bone deficit to promote bone regeneration.

Inhibition of vertebral endplate perfusion results in decreased intervertebral disc intranuclear diffusive transport.

Impaired nutrition of the intervertebral disc has been hypothesized to be one of the causes of disc degeneration. However, no causal relationship between decreased endplate perfusion and limited nutrient transport has been demonstrated to support this pathogenic mechanism. To determine the importance of endplate perfusion on solute diffusion into the nucleus pulposus and to show causality of endplate perfusion on intranuclear diffusion in large animal lumbar intervertebral discs, diffusive transport into ovine lumbar intervertebral discs was evaluated after inhibiting adjacent vertebral endplate perfusion. Partial perfusion blocks were created in vertebrae close and parallel to both endplates of lumbar discs of anaesthetized sheep. To assess diffusivity of small molecules through the endplate, N2O was introduced into the inhalation gas mixture and concentrations of intranuclear N2O were measured for 35 min thereafter. Post mortem, procion red was infused through the spinal vasculature and perfusion through the endplate was assessed by quantifying the density of dye-perfused endplate vascular buds in histology sections. Perfusion of the endplates overlying the nucleus pulposus was inhibited by almost 50% in the partially blocked discs relative to the control discs. There was also a nine-fold decreased transport rate of intranuclear N2O in partially blocked discs compared with control discs. The density of perfused endplate vascular buds correlated significantly to the amount of transported intranuclear N2O (r2 = 0.52, P = 0.008). The vertebral endplate was demonstrated to be the main route of intravascular solute transport into the nucleus pulposus of intervertebral discs, and inhibition of endplate perfusion can cause inhibited solute transport into the disc intranuclear tissue.

Mechanical and radiological assessment of the influence of rhTGFbeta-3 on bone regeneration in a segmental defect in the ovine tibia: pilot study.

Limitations in the use of autologous bone graft, which is the gold standard therapy in bone defect healing, drive the search for alternative treatments. In this study the influence of rhTGFbeta-3 on mechanical and radiological parameters of a healing bone defect in the sheep tibia was assessed. In the sheep, an 18-mm long osteoperiosteal defect in the tibia was treated by rhTGFbeta-3 seeded on a poly(L/DL-lactide) carrier (n = 4). In a second group (n = 4), the defect was treated by the carrier only, in a third group (n = 4) by autologous cancellous bone graft, and in a fourth group (n = 2) the defect remained blank. The healing process of the defect was assessed by weekly in vivo stiffness measurements and radiology as well as by quantitative computed tomographic assessment of bone mineral density (BMD) every 4 weeks. The duration of the experiment was 12 weeks under loading conditions. In the bone graft group, a marginally significant higher increase in stiffness was observed than in the PLA/rhTGFbeta-3 group (p = 0.06) and a significantly higher increase than in the PLA-only group (p = 0.03). The radiographic as well as the computed tomographic evaluation yielded significant differences between the groups (p = 0.03), indicating the bone graft treatment (bone/per area, 83%; BMD, 0.57 g/cm(3)) performing better than the PLA/rhTGFbeta-3 (38%; 0.23 g/cm(3)) and the PLA-only treatment (2.5%; 0.09 g/cm(3)), respectively. Regarding the mechanical and radiological parameters assessed in this study, we conclude that rhTGFbeta-3 has a promoting effect on bone regeneration. However, under the conditions of this study, this effect does not reach the potential of autologous cancellous bone graft transplantation.

Osteopromotion by a beta-tricalcium phosphate/bone marrow hybrid implant for use in spine surgery.

STUDY DESIGN: Experimental animal study. OBJECTIVE.: Evaluate osteopromotive properties of a beta-tricalcium phosphate (TCP) implant with different osteogenic substances in an animal study. SUMMARY OF BACKGROUND DATA: Current research in spine surgery is focusing on resorbable bone implants because of the high morbidity after iliac crest graft harvesting. Therefore, several osteoconductive scaffolds are combined with osteoinductive substances. METHODS: In 14 sheep, a critical size defect was performed on both tibiae. The sheep were randomized into 3 groups (4 sheep each) and a control group (2 sheep). In the treatment groups, the defects were filled with the beta-TCP scaffold impregnated with either venous blood, bone marrow from sternal aspiration, or a concentrated mononuclear cell suspension derived from sternal bone marrow aspiration. The sheep in each group were euthanized 6 and 12 weeks after the operation; the investigation included quantitative computerized tomography and histology. RESULTS: The best bone formation occurred in the bone marrow group after 6 and 12 weeks, whereas no difference was found between the cell and venous blood groups. Only the bone marrow group showed bone formation inside the scaffold after 6 weeks. We conclude that a beta-TCP scaffold filled with bone marrow aspiration creates a biologic resorbable bone substitute with high osteopromoting capacity. Surprisingly, no better bone formation occurred in the concentrated cells group, which may be a result of technical reasons and needs to be further evaluated. CONCLUSIONS: The combination of beta-TCP and bone marrow has superior osteopromotive properties to venous blood or concentrated mononuclear cells and can be used effectively as a substitute to iliac crest graft.