A large animal model for standardized testing of bone regeneration strategies

Abstract Background The need for bone graft substitutes including those being developed to be applied together with new strategies of bone regeneration such as tissue engineering and cell-based approaches is growing. No large animal model of bone regeneration has been accepted as a standard testing model. Standardization may be the key to moving systematically towards better bone regeneration. This study aimed to establish a model of bone regeneration in the sheep that lends itself to strict standardization and in which a number of substances can be tested within the same animal. To this end the caudal border of the ovine scapula was used as a consistent bed of mineralized tissue that provided sufficient room for a serial alignment of multiple experimental drill holes. Results The findings show that for the sake of standardization, surgery should be restricted to the middle part of the caudal margin, an area at least 80 mm proximal from the Glenoid cavity, but not more than 140 mm away from it, in the adult female Land Merino sheep. A distance of 5 mm from the caudal margin should also be observed. Conclusions This standardized model with defined uniform defects and defect sites results in predictable and reproducible bone regeneration processes. Defects are placed unilaterally in only one limb of the animal, avoiding morbidity in multiple limbs. The fact that five defects per animal can be evaluated is conducive to intra-animal comparisons and reduces the number of animals that have to be subject to experimentation

Model of continuous infection in the absence of DIPs.

<p>(A) Schematic representation of the model for continuous influenza A virus infection in the absence of DIPs (see Eq. (2)). The continuous harvest of cells and viruses was omitted for illustrative reasons. (B, C) Simulated virus titers for a dilution rate of the virus reactor D which is (B) lower than the specific growth rate µ and (C) higher than the specific growth rate µ. Parameters were chosen according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072288#pone.0072288.s001" target="_blank">Table S1</a> except that the dilution rate in (B) was reduced to D = 10⁻⁸ 1/h.</p

Continuous propagation of influenza A virus.

<p>(A) Concentrations of AGE1.CR cells in the cell and virus bioreactor. (B) Virus titers determined by HA and TCID₅₀ assay. (C) MOI in the virus bioreactor based on the ratio of TCID₅₀ to cell count at each sampling time point. Results of two independent cultivations are shown. During the first cultivation additional trypsin (+T), seed virus (+V) or both were added to the virus bioreactor at indicated time points as an attempt to counteract decreasing virus titers.</p

Model of continuous infection in the presence of DIPs.

<p>(A) Schematic representation of the model for continuous influenza A virus infection in the presence of DIPs (see Eq. (1)). Dashed arrows indicate apoptosis or virus degradation. The continuous harvest of cells and viruses was omitted for illustrative reasons. (B) Simulated virus titers for the parameters used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072288#pone.0072288.s001" target="_blank">Table S1</a>. (C, D) Log₁₀ HA units/100 µL over process time for (C) various ratios of initial DIPs (V_d0) to STVs (V_s0) neglecting <i>de novo</i> DIP generation (f = 0) and (D) different rates of <i>de novo</i> DIP generation by STV-infected cells (f denoting the fraction of DIP to STV production) without DIPs being initially present (V_d0 = 0).</p

Overview of two-stage bioreactor setup for continuous virus propagation.

<p>AGE1.CR cells were cultivated in two bioreactors. At time of infection, the influenza strain A/Puerto Rico/8/34 was added to the virus bioreactor at a multiplicity of infection of 0.025. Subsequently, the cell concentration in the cell bioreactor was kept at approx. 4–5×10⁶ cells/mL and cells were constantly fed into the virus bioreactor (feeding rates are depicted). Trypsin was added either to the feed or directly into the virus bioreactor. All green components correspond to the cell bioreactor, all red components to the virus bioreactor. Both reactors are connected via the purple tubing.</p

Primers used for the segment-specific PCR.

<p>Primers used for the segment-specific PCR.</p