Cryoreservation of alginate-fibrin beads involving bone marrow derived mesenchymal stromal cells by vitrification

Application of cell-–biomaterial systems in regenerative medicine can be facilitated by their successful low temperature preservation. Vitrification, which avoids ice crystal formation by amorphous solidification, is an emerging approach to cryopreservation. Developing vitrification strategy, effective cryopreservation of alginate–fibrin beads with porcine mesenchymal stromal cells has been achieved in this study. The cell–biomaterial constructs were pre-cultured for 20 days before cryopreservation, allowing for cell proliferation and construct stabilization. Ethylene glycol (EG) was employed as the basic cryoprotectant for two equilibration solutions. Successful cryopreservation of the constructs was achieved using vitrification solution composed of penetrating (EG MW 62 Da) and non-penetrating (sucrose MW 342 Da) cryoprotectants. Stepwise procedure of introduction to and removal of cryoprotectants was brief; direct plunging into liquid nitrogen was applied. Cell viability, evaluated by combining live/death staining and confocal laser microscopy, was similar for both control and vitrified cells in the beads. No detectable damage of microstructure of cryopreserved beads was found as shown by scanning electron microscopy. Both osteogenically induced control and vitrified cells in the constructs were equally capable of mineral production and deposition. There was no statistically significant difference in metabolic activity and proliferation between both groups during the entire culture period. Our study leads to the conclusion that the developed cryopreservation protocol allowed to maintain the integrity of the beads while preserving the ability of the pig bone marrow derived mesenchymal stromal cells to proliferate and subsequently differentiate; demonstrating that vitrification is a promising approach for cryopreser-vation of “ready-to-use” cell–biomaterial constructs.

Biomechanical aspects of bone microstructure in vertebrates : potential approach to paleontological investigations

The biomechanical or biophysical principles can be applied to study biological structures in their modern or fossil form. Bone is an important tissue in paleontological studies as it is a commonly preserved element in most fossil vertebrates, and can often allow its microstructures such as lacuna and canaliculi to be studied in detail. In this context, the principles of Fluid Mechanics and Scaling Laws have been previously applied to enhance the understanding of bone microarchitecture and their implications for the evolution of hydraulic structures to transport fluid. It has been shown that the microstructure of bone has evolved to maintain efficient transport between the nutrient supply and cells, the living components of the tissue. Application of the principle of minimal expenditure of energy to this analysis shows that the path distance comprising five or six lamellar regions represents an effective limit for fluid and solute transport between the nutrient supply and cells; beyond this threshold, hydraulic resistance in the network increases and additional energy expenditure is necessary for further transportation. This suggests an optimization of the size of bone’s building blocks (such as osteon or trabecular thickness) to meet the metabolic demand concomitant to minimal expenditure of energy. This biomechanical aspect of bone microstructure is corroborated from the ratio of osteon to Haversian canal diameters and scaling constants of several mammals considered in this study. This aspect of vertebrate bone microstructure and physiology may provide a basis of understanding of the form and function relationship in both extinct and extant taxa.

Influences of age and mechanical stability on volume, microstructure, and mineralization of the fracture callus during bone healing : Is osteoclast activity the key to age-related impaired healing?

Earlier studies have shown that the influence of fixation stability on bone healing diminishes with advanced age. The goal of this study was to unravel the relationship between mechanical stimulus and age on callus competence at a tissue level. Using 3D in vitro micro-computed tomography derived metrics, 2D in vivo radiography, and histology, we investigated the influences of age and varying fixation stability on callus size, geometry, microstructure, composition, remodeling, and vascularity. Compared were four groups with a 1.5-mm osteotomy gap in the femora of Sprague–Dawley rats: Young rigid (YR), Young semirigid (YSR), Old rigid (OR), Old semirigid (OSR). Hypothesis was that calcified callus microstructure and composition is impaired due to the influence of advanced age, and these individuals would show a reduced response to fixation stabilities. Semirigid fixations resulted in a larger ΔCSA (Callus cross-sectional area) compared to rigid groups. In vitro μCT analysis at 6 weeks postmortem showed callus bridging scores in younger animals to be superior than their older counterparts (pb0.01). Younger animals showed (i) larger callus strut thickness (pb0.001), (ii) lower perforation in struts (pb0.01), and (iii) higher mineralization of callus struts (pb0.001). Callus mineralization was reduced in young animals with semirigid fracture fixation but remained unaffected in the aged group. While stability had an influence, age showed none on callus size and geometry of callus. With no differences observed in relative osteoid areas in the callus ROI, old as well as semirigid fixated animals showed a higher osteoclast count (pb0.05). Blood vessel density was reduced in animals with semirigid fixation (pb0.05). In conclusion, in vivo monitoring indicated delayed callus maturation in aged individuals. Callus bridging and callus competence (microstructure and mineralization) were impaired in individuals with an advanced age. This matched with increased bone resorption due to higher osteoclast numbers. Varying fixator configurations in older individuals did not alter the dominant effect of advanced age on callus tissue mineralization, unlike in their younger counterparts. Age-associated influences appeared independent from stability. This study illustrates the dominating role of osteoclastic activity in age-related impaired healing, while demonstrating the optimization of fixation parameters such as stiffness appeared to be less effective in influencing healing in aged individuals.

Influence of hydrocarbon contamination on clay soil microstructure

Microstructural (fabric, forces and composition) changes due to hydrocarbon contamination in a clay soil were studied using Scanning Electron Microscope (micro-fabric analysis), Atomic Force Microscope (forces measurement) and sedimentation bench test (particle size measurements). The non-polluted and polluted glacial till from north-eastern Poland (area of a fuel terminal) were used for the study. Electrostatic repelling forces for the polluted sample were much lower than for the non-polluted sample. In comparison to non-polluted sample, the polluted sample exhibited lower electric charge, attractive forces on approach and strong adhesion on retrieve. The results of the sedimentation tests indicate that clay particles form larger aggregates and settle out of the suspension rapidly in diesel oil. In non-polluted soil, the fabric is strongly aggregated – densely packed, dominate the face-to-face and edge-to-edge types of contacts, clay film tightly adheres to the surface of larger grains and interparticle pores are more common. In polluted soil, the clay matrix is less aggregated – loosely packed, dominate the edge-to-face types of contacts and inter-micro-aggregate pores are more frequent. Substantial differences were observed in the morphometric and geometrical parameters of pore space. The polluted soil micro-fabric proved to be more isotropic and less oriented than in non-polluted soil. The polluted soil, in which electrostatic forces were suppressed by hydrocarbon interaction, displays more open porosity and larger voids than non-polluted soil, which is characterized by occurrence of the strong electrostatic interaction between clay particles.

CaSiO3 microstructure modulating the in vitro and in vivo bioactivity of poly (lactide-co-glycolide) microspheres

Poly (lactide-co-glycolide) (PLGA) microspheres have been used for regenerative medicine due to their ability for drug delivery and generally good biocompatibility, but they lack adequate bioactivity for bone repair application. CaSiO3 (CS) has been proposed as a new class of material suitable for bone tissue repair due to its excellent bioactivity. In this study, we set out to incorporate CS into PLGA microspheres to investigate how the phase structure (amorphous and crystal) of CS influences the in vitro and in vivo bioactivity of the composite microspheres, with a view to the application for bone regeneration. X-ray diffraction (XRD), N2 adsorption-desorption analysis and scanning electron microscopy (SEM) were used to analyze the phase structure, surface area/pore volume, and microstructure of amorphous CS (aCS) and crystal CS (cCS), as well as their composite microspheres. The in vitro bioactivity of aCS and cCS – PLGA microspheres was evaluated by investigating their apatite-mineralization ability in simulated body fluids (SBF) and the viability of human bone mesenchymal stem cells (BMSCs). The in vivo bioactivity was investigated by measuring their de novo bone-formation ability. The results showed that the incorporation of both aCS and cCS enhanced the in vitro and in vivo bioactivity of PLGA microspheres. cCS/PLGA microspheres improved better in vitro BMSC viability and de novo bone-formation ability in vivo, compared to aCS/PLGA microspheres. Our study indicates that controlling the phase structure of CS is a promising method to modulate the bioactivity of polymer microsphere system for potential bone tissue regeneration.