Filippo Causa

Osteoblast growth and function in porous poly ε-caprolactone matrices for bone repair: a preliminary study

Abstract Current methods for the replacement of skeletal tissue involve the use of autografts, allografts and, recently, synthetic substitutes, which provide a proper amount of material to repair large bone defects. Engineered bone seems a promising approach, but a number of variables have to be set prior to any clinical application. In this study, four different poly caprolactone-based polymers (PCL) were prepared and tested in vitro using osteoblast-like Saos-2 cells. Differences among three-dimensional polymers include porosity, addition of hydroxyapatite (HA) particles, and treatment with simulated body fluid. Biochemical parameters to assess cell/material interactions include viability, growth, alkaline phosphatase release, and mineralization of osteoblastic cells seeded onto three-dimensional samples, while their morphology was observed using light microscopy and SEM. Preliminary results show that the polymers, though degrading in the medium, have a positive interaction with cells, as they support cell growth and functions. In the short-term culture (3–7 days) of Saos-2 on polymers, little differences were found among PCL samples, with the presence of HA moderately improving the number of cells onto the surfaces. In the long term (3–4 weeks), it was found that the HA-added polymers obtained the best colonization by cells, and more mineral formation was observed after coating with SBF. It can be concluded that PCL is a promising material for three-dimensional scaffold for bone formation, and the presence of bone-like components improves osteoblast activity.

Bioactive scaffolds for bone and ligament tissue

Bone and ligament injuries present the greatest challenges in connective tissue regeneration. The design of materials for these applications lies at the forefront of material science and is the epitome of its current ambition. Indeed, its goal is to design and fabricate reproducible, bioactive and bioresorbable 3D scaffolds with tailored properties that are able to maintain their structure and integrity for predictable times, even under load-bearing conditions. Unfortunately, the mechanical properties of today’s available porous scaffolds fall short of those exhibited by complex human tissues, such as bone and ligament. The manipulation of structural parameters in the design of scaffolds and their bioactivation, through the incorporation of soluble and insoluble signals capable of promoting cell activities, are discussed as possible strategies to improve the formation of new tissues both in vitro and in vivo. This review focuses on the different approaches adopted to develop bioactive composite systems for use as temporary scaffolds for bone and anterior ligament regeneration.

Cells preferentially grow on rough substrates

Substrate nanotopography affects cell adhesion and proliferation and is fundamental to the rational design of bio-adhesives, to tissue engineering and to the development of assays for in-vitro screening. Cell behavior on rough substrates is still elusive, and the results presented in the open literature remain controversial. Here, the proliferation of cells on electrochemically etched silicon substrates with different roughness and nearly similar surface energy was studied over three days with confocal and atomic force microscopy. The surface profile of the substrates is a self-affine fractal with a roughness R(a) growing with the etching time from approximately 2 to 100 nm and a fractal dimension D ranging between about 2 (nominally flat surface) and 2.6. For four cell types, the number of adhering cells and their proliferation rates exhibited a maximum on moderately rough (R(a) approximately 10-45 nm) nearly Brownian (D approximately 2.5) substrates. The observed cell behavior was satisfactorily interpreted within the theory of adhesion to randomly rough solids. These findings demonstrated the importance of nanogeometry in cell stable adhesion and growth, suggesting that moderately rough substrates with large fractal dimension could selectively boost cell proliferation.

Polylactic acid fibre-reinforced polycaprolactone scaffolds for bone tissue engineering.

The employment of composite scaffolds with a well-organized architecture and multi-scale porosity certainly represents a valuable approach for achieving a tissue engineered construct to reproduce the middle and long-term behaviour of hierarchically complex tissues such as spongy bone. In this paper, fibre-reinforced composites scaffold for bone tissue engineering applications is described. These are composed of poly-L-lactide acid (PLLA) fibres embedded in a porous poly(epsilon-caprolactone) matrix, and were obtained by synergistic use of phase inversion/particulate leaching technique and filament winding technology. Porosity degree as high as 79.7% was achieved, the bimodal pore size distribution showing peaks at ca 10 and 200 microm diameter, respectively, accounting for 53.7% and 46.3% of the total porosity. In vitro degradation was carried out in PBS and SBF without significant degradation of the scaffold after 35 days, while in NaOH solution, a linear increase of weight lost was observed with preferential degradation of PLLA component. Subsequently, marrow stromal cells (MSC) and human osteoblasts (HOB) reached a plateau at 3 weeks, while at 5 weeks the number of cells was almost the same. Human marrow stromal cell and trabecular osteoblasts rapidly proliferate on the scaffold up to 3 weeks, promoting an oriented migration of bone cells along the fibre arrangement. Moreover, the role of seeded HOB and MSC on composite degradation mechanism was assessed by demonstrating a more relevant contribution to PLLA degradation of MSC when compared to HOB. The novel PCL/PLLA composite scaffolds thus showed promise whenever tuneable porosity, controlled degradability and guided cell-material interaction are simultaneously requested.

Determinants of cell -material crosstalk at the interface: towards engineering of cell instructive materials

The development of novel biomaterials able to control cell activities and direct their fate is warranted for engineering functional biological tissues, advanced cell culture systems, single-cell diagnosis as well as for cell sorting and differentiation. It is well established that crosstalk at the cell–material interface occurs and this has a profound influence on cell behaviour. However, the complete deciphering of the cell–material communication code is still far away. A variety of material surface properties have been reported to affect the strength and the nature of the cell–material interactions, including biological cues, topography and mechanical properties. Novel experimental evidence bears out the hypothesis that these three different signals participate in the same material–cytoskeleton crosstalk pathway via adhesion plaque formation dynamics. In this review, we present the relevant findings on material-induced cell response along with the description of cell behaviour when exposed to arrays of signals—biochemical, topographical and mechanical. Finally, with the aid of literature data, we attempt to draw unifying elements of the material–cytoskeleton–cell fate chain.

A unique three-dimensional SCID-polymeric scaffold (SCID-synth-hu) model for in vivo expansion of human primary multiple myeloma cells.

Multiple myeloma (MM) cells home to the bone marrow (BM) and adhere to extracellular matrix (ECM) proteins and to bone marrow stromal cells (BMSCs). The close cross talk between MM cells and cells of the non-tumor compartment within the BM has a key role in supporting tumor growth, survival and development of drug resistance. This biological scenario has led to a growing interest in novel drugs, targeting MM cells and/or interfering with their human BM milieu (HuBMM).1, 2 Based on this, appropriate in vivo models that recapitulate the complex interactions occurring between MM and its HuBMM are required for preclinical evaluation of new anti-MM agents. To date, the in vivo study of MM pathobiology and the validation of therapeutic anti-MM agents has been carried out using a variety of models of murine MM or human MM xenografts in immunocompromised mice.3 These models, however, do not replicate the HuBMM. The development of the SCID (severe combined immunodeficiency)-hu model has been an important advance, as it was the first model to recapitulate a HuBMM in mice.4, 5, 6 However, although the SCID-hu system remains a highly relevant model for preclinical investigation, it does have important limitations: (i) restricted availability of human fetal bone chips; (ii) the allogeneic nature of the fetal BM milieu versus MM cells; and (iii) the significant heterogeneity of implanted human bone chips, collected from different individuals at different gestational age, that may produce experimental variability.

Surface investigation on biomimetic materials to control cell adhesion: the case of RGD conjugation on PCL.

The cell recognition of bioactive ligands immobilized on polymeric surfaces is strongly dependent on ligand presentation at the cell/material interface. While small peptide sequences such as Arg-Gly-Asp (RGD) are being widely used to obtain biomimetic interfaces, surface characteristics after immobilization as well as presentation of such ligands to cell receptors deserve more detailed investigation. Here, we immobilized an RGD-based sequence on poly(epsilon-caprolactone) (PCL), a largely widespread polymeric material used in biomedical applications, after polymer aminolysis. The surface characteristics along with the efficacy of the functionalization was monitored by surface analysis (FTIR-ATR, contact angle measurements, surface free energy determination) and spectrophotometric assays specially adapted for the analytical quantification of functional groups and/or peptides at the interface. Particular attention was paid to the evaluation of a number, morphology, and penetration depth of immobilized functional groups and/or peptides engrafted on polymeric substrates. In particular, a typical morphology in peptide distribution was evidenced on the surface raised from polymer crystallites, while a significant penetration depth of the engrafted molecules was revealed. NIH3T3 fibroblast adhesion studies verified the correct presentation of the ligand with enhanced cell attachment after peptide conjugation. Such work proposes a morphological and analytical approach in surface characterization to study the surface treatment and the distribution of ligands immobilized on polymeric substrates.

The role of hydroxyapatite as solid signal on performance of PCL porous scaffolds for bone tissue regeneration.

Highly porous composites made up of biodegradable poly-epsilon-caprolactone (PCL) and stoichiometric hydroxyapatite (HA) particles have been developed as substrate for bone-tissue regeneration. The processing technique consists of phase inversion and particulate (salt crystals) leaching. Three different HA contents (13, 20 and 26 vol %) in PCL-based composite were considered in this study. Pore microstructure with fully interconnected network and pore sizes ranging around a few hundred of mum (macroporosity) was obtained as a result of salt particles removal by leaching process. Several microns (microporosity) porosity was also created through phase inversion of polymer solution. Total porosity up to 95% was achieved. Human marrow stromal cells (MSC) were seeded onto porous PCL-based composites for 1-5 weeks and cultured in osteogenic medium. MSC were able to adhere and grow on PCL-based substrates with a plateau at 3-4 weeks. However, the small effect of bioactive signals on the biological response evaluated in MSC cell culture suggests a prior role of topography on the biological response. Importantly, the presence of HA as a bioactive solid signal determines an increase of mechanical properties. On the overall, the results indicated that porous PCL-based composites are potential candidate for bone substitution with beneficial influence on structural characteristics by solid signal addition.

Rheological Characterization of Hyaluronic Acid Derivatives as Injectable Materials Toward Nucleus Pulposus Regeneration

Nucleus pulposus (NP) is the soft center of the intervertebral disc (IVD), able to resist compressive loads, while the annulus fibrosus withstands tension and gives mechanical strength. NP function may be altered as consequence of several pathologies or injury and when a damaged IVD does not properly play its role. In the past years, a great effort has been devoted to the design of injectable systems as NP substitutes. The different synthetic- and natural hydrogel-based materials proposed, present many drawbacks and, in particular, they do not seem to mimic the required behavior. In the search for natural-based systems a dodecylamide of hyaluronic acid (HA), HYADD3®, has been proved as bioactive and suitable vehicle to carry cells for NP tissue engineering, while a crosslinked HA ester, HYAFF120® showed interesting results if used as injectable acellular material. Even though these derivatives showed appropriate biological behavior up to now, data on mechanical behavior of these derivatives are still missing. In this frame, the aim of this study was to provide a rheological characterization of these HA derivatives to asses their biomechanical compatibility with the NP tissue. To this, the rheological properties of these derivatives were studied through dynamic shear tests before and after injection through needles used in the current surgical procedure. Both HA derivatives showed a ‘gel-like’ rheological behavior similar to the native NP tissue and this behavior was not altered by injection.

Fluorescence Lifetimes and Quantum Yields of Rhodamine Derivatives: New Insights from Theory and Experiment

Although lifetimes and quantum yields of widely used fluorophores are often largely characterized, a systematic approach providing a rationale of their photophysical behavior on a quantitative basis is still a challenging goal. Here we combine methods rooted in the time-dependent density functional theory and fluorescence lifetime imaging microscopy to accurately determine and analyze fluorescence signatures (lifetime, quantum yield, and band peaks) of several commonly used rhodamine and pyronin dyes. We show that the radiative lifetime of rhodamines can be correlated to the charge transfer from the phenyl toward the xanthene moiety occurring upon the S(0) ← S(1) de-excitation, and to the xanthene/phenyl relative orientation assumed in the S(1) minimum structure, which in turn is variable upon the amino and the phenyl substituents. These findings encourage the synergy of experiment and theory as unique tool to design finely tuned fluorescent probes, such those conceived for modern optical sensors.