New pulmonary hypertension model in conscious dogs to investigate pulmonary-selectivity of acute pharmacological interventions.

PURPOSE: Testing of investigational drugs in animal models is a critical step in drug development. Current models of pulmonary hypertension (PH) have limitations. The most relevant outcome parameters such as pulmonary artery pressure (PAP) are measured invasively which requires anesthesia of the animal. We developed a new canine PH model in which pulmonary vasodilators can be characterized in conscious dogs and lung selectivity can be assessed non-invasively. METHODS: Telemetry devices were implanted to measure relevant hemodynamic parameters in conscious dogs. A hypoxic chamber was constructed in which the animals were placed in a conscious state. By reducing the inspired oxygen fraction (FiO2) to 10%, a hypoxic pulmonary vasoconstriction was induced leading to PH. The PDE-5 inhibitor sildenafil, the current standard of care was compared to atrial natriuretic peptide (ANP). RESULTS: The new hypoxic chamber provided a stable hypoxic atmosphere during all experiments. The mean PAP under normoxic conditions was 15.8 ± 1.8 mmHg. Hypoxia caused a reliable increase in mean PAP (+ 12.2 ± 3.2 mmHg, p < 0.0001). Both, sildenafil (- 6.8 ± 4.4 mmHg) and ANP (- 6.4 ± 3.8 mmHg) significantly (p < 0.05) decreased PAP. Furthermore sildenafil and ANP showed similar effects on systemic hemodynamics. In subsequent studies, the in vitro effects and gene expression pattern of the two pathways were exemplified. CONCLUSIONS: By combining the hypoxic environment with the telemetric approach, we could successfully establish a new acute PH model. Sildenafil and ANP demonstrated equal effects regarding pulmonary selectivity. This non-invasive model could help to rapidly screen pulmonary vasodilators with decreased animal burden.

Effect of platelet-rich plasma on the in vitro proliferation and osteogenic differentiation of human mesenchymal stem cells on distinct calcium phosphate scaffolds: the specific surface area makes a difference.

The in vitro effect of platelet-rich plasma (PRP) on cell loading, proliferation, and osteogenic differentiation of human mesenchymal stem cells (MSC) is assessed on distinct resorbable and synthetic calcium phosphate scaffolds. A high specific surface area scaffold composed of calcium-deficient hydroxyapatite (CDHA; 48m2/g) is compared with one made out of beta-tricalcium phosphate (beta-TCP; surface area <0.5 m2/g). Fivefold concentrated fresh PRP is applied to scaffolds loaded with 2 x 10(5) MSC (n = 5). These constructs are kept in a medium with osteogenic supplements for 3 weeks. The addition of PRP leads to a higher cell loading efficiency of MSC on CDHA (p = 0.0001), that reaches the values of beta-TCP. Proliferation over 21 days is improved by PRP both on CDHA (p = 0.0001) and beta-TCP (p = 0.014) compared to MSC/calcium phosphate composites. Without the addition of PRP, CDHA has a lower cell loading efficiency (p= 0.0001) and proliferation (p= 0.001) than beta-TCP. The ALP activity is higher in the MSC/ceramics groups than in the monolayer controls (p<0.05). The addition of PRP does not significantly affect ALP activity. However, ALP activity varies considerably within the cell donors and different PRP-pools (p = 0.001), while the cell numbers do not vary within these two parameters. PRP generates a positive effect on the loading efficiency of MSC on the high specific surface scaffold CDHA that thereby reaches the loading efficiency of beta-TCP. PRP improved proliferation, but its osteogenic properties on both calcium phosphate scaffolds are weak.

Platelet-rich plasma improves expansion of human mesenchymal stem cells and retains differentiation capacity and in vivo bone formation in calcium phosphate ceramics.

INTRODUCTION: Mesenchymal stem cells (MSC) applied to bone substitution materials can improve bone healing. Bone formation in biocomposites is highly dependent on the kind of biomaterial, its pre-treatment and the applied cells. Potentially immunogenic or infectious supplements such as fetal calf serum (FCS) should be avoided in cell expansion media. Therefore, we developed an expansion protocol free of xenogenic supplements. Cells expanded with two different media were tested on distinct biomaterials for their bone formation capacity after ectopic implantation in vivo, as well as for their growth rate and differentiation capacity in vitro. METHODS: MSC of six donors were expanded with cell expansion medium containing FCS (2%) or platelet-rich plasma (PRP, 3%). Their growth rate and osteogenic, adipogenic and chondrogenic differentiation capacity were compared in vitro. For the in vivo bone formation assay, expanded cells (2 x 105 or 2 x 106) were seeded on calcium-deficient hydroxyapatite (CDHA; n = 12) and on beta-tricalcium phosphate (beta-TCP; n = 12) blocks, which had been coated with either fibronectin or human serum. They were then implanted subcutaneously in severe combined immunodeficient mice (SCID), harvested after 8 weeks and analysed by histology. Bone formation was assessed by a semi-quantitative bone score, after toluidine blue and alizarin red staining. Human cells were detected by an in situ hybridisation for human-specific alu sequences. RESULTS: PRP-supplemented expansion medium yielded two-fold higher cell numbers compared to medium with FCS (P = 0.046) after 3 weeks (four passages) and retained a similar capacity to differentiate towards the osteogenic, chondrogenic and adipogenic lineage. In vivo bone formation was equal for cells expanded with PRP and FCS and depended on the specific surface area of the carrier. CDHA (specific surface area (SSA) 48 m2/g) showed a significantly better bone formation in deep layers (P = 0.005) than beta-TCP (SSA 0.5 m2/g). Fibronectin-coating of the ceramics was slightly superior to coating with human serum (P = 0.045). CONCLUSIONS: The replacement of FCS by PRP eliminated risks connected with the use of xenogeneic supplements. It improved expansion of MSC and retained their differentiation and in vivo bone formation capacity in a setting adaptable to autogenous use.

Ectopic bone formation associated with mesenchymal stem cells in a resorbable calcium deficient hydroxyapatite carrier.

Bone substitute materials can induce bone formation in combination with mesenchymal stem cells (MSC). The aim of the current study was to examine ectopic in vivo bone formation with and without MSC on a new resorbable ceramic, called calcium deficient hydroxyapatite (CDHA). Ceramic blocks characterized by a large surface (48 m2/g) were compared with beta-tricalcium phosphate (beta-TCP), hydroxyapatite (HA) ceramics (both ca. 0.5 m2/g surface) and demineralized bone matrix (DBM). Before implantation in the back of SCID mice carriers were freshly loaded with 2x10(5) expanded human MSC or loaded with cells and kept under osteogenic conditions for two weeks in vitro. Culture conditions were kept free of xenogenic supplements. Deposits of osteoid at the margins of ceramic pores occurred independent of osteogenic pre-induction, contained human cells, and appeared in 416 MSC/CDHA composites compared to 216 MSC/beta-TCP composites. ALP activity was significantly higher in samples with MSC versus empty controls (p<0.001). Furthermore, ALP was significantly (p<0.05) higher for all ceramics when compared to the DBM matrix. Compared to previous studies, overall bone formation appeared to be reduced possibly due to the strict human protocol. Ectopic bone formation in the novel biomaterial CDHA varied considerably with the cell pool and was at least equal to beta-TCP blocks.