Evaluation of Strontium-Containing PCL-PDIPF Scaffolds for Bone Tissue Engineering: In Vitro and In Vivo Studies.

Bone tissue engineering (BTE) has the general objective of restoring and improving damaged bone. A very interesting strategy for BTE is to combine an adequate polymeric scaffold with an osteoinductive compound. Strontium is a divalent cation that can substitute calcium in hydroxyapatite and induce both anabolic and anti-catabolic effects in bone. On the other hand, systemic increases in Sr2+ levels can provoke adverse cardiovascular effects. In the present study we have developed a compatibilized blend of poly-ε-caprolactone (PCL) and polydiisopropyl fumarate (PDIPF) enriched with 1% or 5% Sr2+ and evaluated the applicability of these biomaterials for BTE, both in vitro and in vivo. In vitro, whereas Blend + 5% Sr2+ was pro-inflammatory and anti-osteogenic, Blend + 1% Sr2+ released very low quantities of the cation; was not cytotoxic for cultured macrophages; and showed improved osteocompatibility when used as a substratum for primary cultures of bone marrow stromal cells. In vivo, implants with Blend + 1% Sr2+ significantly increased bone tissue regeneration and improved fibrous bridging (vs. Blend alone), while neither inducing a local inflammatory response nor increased serum levels of Sr2+. These results indicate that our compatibilized blend of PCL-PDIPF enriched with 1% Sr2+ could be useful for BTE.

Multi-Scale Approach for the Evaluation of Bone Mineralization in Strontium Ranelate-Treated Diabetic Rats.

Long-term diabetes mellitus can induce osteopenia and osteoporosis, an increase in the incidence of low-stress fractures, and/or delayed fracture healing. Strontium ranelate (SrR) is a dual-action anti-osteoporotic agent whose use in individuals with diabetic osteopathy has not been adequately evaluated. In this study, we studied the effects of an oral treatment with SrR and/or experimental diabetes on bone composition and biomechanics. Young male Wistar rats (half non-diabetic, half with streptozotocin/nicotinamide-induced diabetes) were either untreated or orally administered 625 mg/kg/day of SrR for 6 weeks. After sacrifice, femora from all animals were evaluated by a multi-scale approach (X-ray diffraction, Fourier transform infrared spectroscopy, inductively coupled plasma optical-emission spectrometry, static histomorphometry, pQCT, and mechanical testing) to determine chemical, crystalline, and biomechanical properties. Untreated diabetic animals (versus untreated non-diabetic) showed a decrease in femoral mineral carbonate content, in cortical thickness and BMC, in trabecular osteocyte density, in maximum load supported at rupture and at yield point, and in overall toughness at mid-shaft. Treatment of diabetic animals with SrR further affected several parameters of bone (some already impaired by diabetes): crystallinity index (indicating less mature apatite crystals); trabecular area, BMC, and vBMD; maximum load at yield point; and structural elastic rigidity. However, SrR was also able to prevent the diabetes-induced decreases in trabecular osteocyte density (completely) and in bone ultimate strength at rupture (partially). Our results indicate that SrR treatment can partially but significantly prevent some bone structural mechanical properties as previously affected by diabetes, but not others (which may even be worsened).

Efectos in vivo del ranelato de estroncio sobre células progenitoras de médula ósea de ratas diabéticas / In vivo effects of strontium ranelate on bone marrow progenitor cells of diabetic rats

La diabetes mellitus (DM) crónica se asocia con reducción en el contenido mineral óseo (osteopenia y osteoporosis). El objetivo de este trabajo fue evaluar la acción del ranelato de estroncio (RaSr) administrado por vía oral a animales control y diabéticos, sobre el potencial osteogénico de células progenitoras de médula ósea (CPMO). Dieciséis ratas Wistar macho jóvenes se dividieron en dos grupos: controles (C) y diabéticas (D) con destrucción parcial de células b-pancreáticas mediante inyecciones intraperitoneales consecutivas de nicotinamida y estreptozotocina. Siete días después de la inyección, cada grupo se subdividió: sin tratamiento, o tratadas oralmente con RaSr (625 mg/kg/día) durante seis semanas, luego de lo cual los animales fueron sacrificados. Las CPMO se obtuvieron de ratas de los cuatro grupos, por lavados del canal diafisario medular (húmero o fémur o ambos) y cultivo hasta confluencia en DMEM-10% FBS. La proliferación celular se evaluó mediante el ensayo de MTT. Luego las CPMO se replaquearon e incubaron en un medio osteogénico durante 14 días (fosfatasa alcalina [FAL] y colágeno tipo 1) o 21 días (mineralización). Las CPMO del grupo C+RaSr mostraron un aumento significativo versus control en la proliferación (133%) y en la diferenciación osteogénica (colágeno 143%, FAL 168%, mineralización 117%). La DM (grupo D) disminuyó significativamente la proliferación y diferenciación osteoblástica de las CPMO. El tratamiento con RaSr (grupo D+RaSr) previno completamente estos efectos antiosteogénicos de la DM. Así, en nuestro modelo experimental in vivo, la DM disminuye el potencial osteogénico de CPMO, efecto que puede ser prevenido por un tratamiento oral con RaSr. (AU)

Chronic diabetes mellitus (DM) is associated with a reduction in bone mineral content (osteopenia and osteoporosis). The object of this study was to evaluate the in vivo effect of he anti-osteoporotic drug strontium ranelate (SrRa) administered orally to control and diabetic animals, on the osteogenic potential of bone marrow progenitor cells (BMPC). Sixteen young male Wistar rats were divided into two groups: control (C) and diabetic with partial beta-cell destruction via consecutive intra-peritoneal injections of nicotinamide and streptozotocin (D). Seven days postinjection, each group was sub-divided: without treatment, or oral treatment with SrRa (625 mg/kg/day) for six weeks, after which the animals were euthanised (groups C, C+SrRa, D, D+SrRa). BMPC were obtained from rats of all four groups by flushing of the diaphysary canal (humerus and/or femur). Adherent cells were then cultured until confluence in DMEM10% FBS. Cell proliferation was evaluated with the MTT mitogenic bioassay. BMPC were replated and incubated in an osteogenic medium for 14 days (determination of alkaline phosphatase [ALP] and type-1 collagen) or 21 days (evaluation of mineralisation). BMPC from C+SrRa rats showed a significant increase versus control in proliferation (133%) and in osteogenic differentiation (collagen 143%, ALP 168%, mineralisation 117%). Induction of diabetes (group D) significantly decreased the proliferation and osteoblastic differentiation of BMPC. Treatment of diabetic animals with SrRa (group D+SrRa) completely prevented these anti-osteogenic effects of Diabetes. Thus, in our experimental in vivo model, Diabetes decreases the osteogenic potential of BMPC, an effect that can be prevented by oral treatment with strontium ranelate. (AU)

Characterization of phenoloxidase activity from spider Polybetes pythagoricus hemocyanin.

Hemocyanin of the spider Polybetes pythagoricus, in addition to its typical role as an oxygen transporter, also exhibits a phenoloxidase activity induced by micellar concentrations of SDS. In the present work, we found the kinetic parameters Km and Vmax of Polybetes pythagoricus hemocyanin (PpHc) PO activity to be 0.407 mM and 0.081 µmolmin(-1) mg protein(-1) , respectively. Dopamine was used as the substrate with SDS at a final concentration of 10 mM and a 30-min incubation at 25°C. Conformational changes in Hc associated with the SDS treatment were analyzed using far-UV circular dichroism, intrinsic fluorescence and absorption spectroscopy. The secondary and tertiary structural changes of PpHc induced by SDS led to increases in α-helical content and tryptophan fluorescence intensity. A reduction in the absorption spectrum at 340 nm in the presence of SDS was also observed. These results suggest that the SDS-induced PO activity of PpHc can be ascribed to conformational changes in the local environment of the typer-3 copper active site.