Toward a mechanically biocompatible intervertebral disc: Engineering of combined biomimetic annulus fibrosus and nucleus pulposus analogs.

Intervertebral disc (IVD) degeneration and accompanying lower back pain impose global medical and societal challenges, affecting over 600 million people worldwide. The IVD complex fibrocartilaginous structure is responsible for the spine biomechanical function. The nucleus pulposus (NP), composed of swellable glycosaminoglycan (GAG), transfers compressive loads to the surrounding fiber-reinforced annulus fibrosus (AF) lamellae, which stretches under tension. Together, these substructures allow the IVD to withstand extremely high and complex loads. Key to mimic the complete disc must consider the properties of its substructures. This study presents three novel substructures-a biomimetic silk-reinforced composite lamella for the AF, a GAG analog for the NP, and a novel biomimetic combined AF-NP construct. The biomimetic AF demonstrates nonlinear, hyperelastic, and anisotropic behavior similar to the native human AF, while the NP analog demonstrates mechanical behavior similar to the human NP. The synergized biomimetic AF-NP demonstrates similar behavior to the unconfined NP, with significantly increased deformations indicating improved performance. Validation of the AF-NP construct mechanics using a finite element model yields results compatible with native human IVD under various physiological loadings. The ability of our AF-NP construct to mimic the native IVD offers a revolutionary concept for the potential development of a fully functional IVD.

Encapsulation of Human-Bone-Marrow-Derived Mesenchymal Stem Cells in Small Alginate Beads Using One-Step Emulsification by Internal Gelation: In Vitro, and In Vivo Evaluation in Degenerate Intervertebral Disc Model.

Cell microencapsulation in gel beads contributes to many biomedical processes and pharmaceutical applications. Small beads (<300 µm) offer distinct advantages, mainly due to improved mass transfer and mechanical strength. Here, we describe, for the first time, the encapsulation of human-bone-marrow-derived mesenchymal stem cells (hBM-MSCs) in small-sized microspheres, using one-step emulsification by internal gelation. Small (127−257 µm) high-mannuronic-alginate microspheres were prepared at high agitation rates (800−1000 rpm), enabling control over the bead size and shape. The average viability of encapsulated hBM-MSCs after 2 weeks was 81 ± 4.3% for the higher agitation rates. hBM-MSC-loaded microspheres seeded within a glycosaminoglycan (GAG) analogue, which was previously proposed as a mechanically equivalent implant for degenerate discs, kept their viability, sphericity, and integrity for at least 6 weeks. A preliminary in vivo study of hBM-MSC-loaded microspheres implanted (via a GAG-analogue hydrogel) in a rat injured intervertebral disc model demonstrated long-lasting viability and biocompatibility for at least 8 weeks post-implantation. The proposed method offers an effective and reproducible way to maintain long-lasting viability in vitro and in vivo. This approach not only utilizes the benefits of a simple, mild, and scalable method, but also allows for the easy control of the bead size and shape by the agitation rate, which, overall, makes it a very attractive platform for regenerative-medicine applications.

Copper oxide loaded PLGA nanospheres: towards a multifunctional nanoscale platform for ultrasound-based imaging and therapy.

Copper oxide nanoparticles (CuO-NPs) are increasingly becoming the subject of investigation exploring their potential use for diagnostic and therapeutic purposes. Recent work has demonstrated their anticancer potential, as well as contrast agent capabilities for magnetic resonance imaging (MRI) and through-transmission ultrasound. However, no capability of CuO-NPs has been demonstrated using conventional ultrasound systems, which, unlike the former, are widely deployed in the clinic. Furthermore, in spite of their potential as multifunctional nano-based materials for diagnosis and therapy, CuO-NPs have been delayed from further clinical application due to their inherent toxicity. Herein, we present the synthesis of a novel nanoscale system, composed of CuO-loaded PLGA nanospheres (CuO-PLGA-NS), and demonstrate its imaging detectability and augmented heating effect by therapeutic ultrasound. The CuO-PLGA-NS were prepared by a double emulsion (W/O/W) method with subsequent solvent evaporation. They were characterized as sphere-shaped, with size approximately 200 nm. Preliminary results showed that the viability of PANC-1, human pancreatic adenocarcinoma cells was not affected after 72 h exposure to CuO-PLGA-NS, implying that PLGA masks the toxic effects of CuO-NPs. A systematic ultrasound imaging evaluation of CuO-PLGA-NS, using a conventional system, was performed in vitro and ex vivo using poultry heart and liver, and also in vivo using mice, all yielding a significant contrast enhancement. In contrast to CuO-PLGA-NS, neither bare CuO-NPs nor blank PLGA-NS possess these unique advantageous ultrasonic properties. Furthermore, CuO-PLGA-NS accelerated ultrasound-induced temperature elevation by more than 4 °C within 2 min. The heating efficiency (cumulative equivalent minutes at 43 °C) was increased approximately six-fold, demonstrating the potential for improved ultrasound ablation. In conclusion, CuO-PLGA-NS constitute a versatile platform, potentially useful for combined imaging and therapeutic ultrasound-based procedures.

Prolyl hydroxylation in elastin is not random.

BACKGROUND: This study aimed to investigate the prolyl and lysine hydroxylation in elastin from different species and tissues. METHODS: Enzymatic digests of elastin samples from human, cattle, pig and chicken were analyzed using mass spectrometry and bioinformatics tools. RESULTS: It was confirmed at the protein level that elastin does not contain hydroxylated lysine residues regardless of the species. In contrast, prolyl hydroxylation sites were identified in all elastin samples. Moreover, the analysis of the residues adjacent to prolines allowed the determination of the substrate site preferences of prolyl 4-hydroxylase. It was found that elastins from all analyzed species contain hydroxyproline and that at least 20%-24% of all proline residues were partially hydroxylated. Determination of the hydroxylation degrees of specific proline residues revealed that prolyl hydroxylation depends on both the species and the tissue, however, is independent of age. The fact that the highest hydroxylation degrees of proline residues were found for elastin from the intervertebral disc and knowledge of elastin arrangement in this tissue suggest that hydroxylation plays a biomechanical role. Interestingly, a proline-rich domain of tropoelastin (domain 24), which contains several repeats of bioactive motifs, does not show any hydroxyproline residues in the mammals studied. CONCLUSIONS: The results show that prolyl hydroxylation is not a coincidental feature and may contribute to the adaptation of the properties of elastin to meet the functional requirements of different tissues. GENERAL SIGNIFICANCE: The study for the first time shows that prolyl hydroxylation is highly regulated in elastin.

Factors regulating viable cell density in the intervertebral disc: blood supply in relation to disc height.

The intervertebral disc is an avascular tissue, maintained by a small population of cells that obtain nutrients mainly by diffusion from capillaries at the disc-vertebral body interface. Loss of this nutrient supply is thought to lead to disc degeneration, but how nutrient supply influences viable cell density is unclear. We investigated two factors that influence nutrient delivery to disc cells and hence cell viability: disc height and blood supply. We used bovine caudal discs as our model as these show a gradation in disc height. We found that although disc height varied twofold from the largest to the smallest disc studied, it had no significant effect on cell density, unlike the situation found in articular cartilage. The density of blood vessels supplying the discs was markedly greater for the largest disc than the smallest disc, as was the density of pores allowing capillary penetration through the bony endplate. Results indicate that changes in blood vessels in the vertebral bodies supplying the disc, as well as changes in endplate architecture appear to influence density of cells in intervertebral discs.

Aggrecan turnover in human intervertebral disc as determined by the racemization of aspartic acid.

We have used the racemization of aspartic acid as a marker for the "molecular age" of aggrecan components of the human intervertebral disc matrix (aggregating and non-aggregating proteoglycans as well as the different buoyant density fractions of aggrecan). By measuring the D/L(Asp) ratio of the various aggrecan species as a function of age and using the values of the racemization constant, k(i), found earlier for aggrecan in articular cartilage, we were able to establish directly the relative residence time of these molecules in human intervertebral disc matrix. For A1 preparations taken from normal tissue, turnover rates of 0.059 +/- 0.01 and 0.063 +/- 0.01/year correspond to half-life values of 12 +/- 2.0 and 11.23 +/- 1.9 years for nucleus pulposus and annulus fibrosus, respectively; the turnover rates of 0.084 +/- 0.022 and 0.092 +/- 0.034/year for degenerate tissue correspond to half-life values of 8.77 +/- 2.2 and 8.41 +/- 2.8 years, suggesting increased rate of removal of small aggrecan fragments. For the large monomer, fraction A1D1, turnover is 0.13 +/- 0.04/year, corresponding to a half-life of 5.56 +/- 1.58 years, similar to 3.4 years in human articular cartilage. For the binding region (A1D6), turnover is 0.033 +/- 0.0012/year, corresponding to a half-life of 21.53 +/- 0.6 years, similar to 23.5 years in articular cartilage. A1 preparations from nucleus pulposus contain a lower proportion of aggregating proteoglycans as compared with annulus fibrosus, suggesting increased proteolytic modification in the nucleus pulposus. D/L(Asp) values in aggregating and non-aggregating proteoglycans of a 24-year-old individual show similar results, suggesting that the non-aggregating molecules are synthesized initially as aggregating proteoglycans, which thereafter undergo cleavage and detachment from hyaluronan.