Enhancing osteoconduction of PLLA-based nanocomposite scaffolds for bone regeneration using different biomimetic signals to MSCs.

In bone engineering, the adhesion, proliferation and differentiation of mesenchymal stromal cells rely on signaling from chemico-physical structure of the substrate, therefore prompting the design of mimetic "extracellular matrix"-like scaffolds. In this study, three-dimensional porous poly-L-lactic acid (PLLA)-based scaffolds have been mixed with different components, including single walled carbon nanotubes (CNT), micro-hydroxyapatite particles (HA), and BMP2, and treated with plasma (PT), to obtain four different nanocomposites: PLLA + CNT, PLLA + CNTHA, PLLA + CNT + HA + BMP2 and PLLA + CNT + HA + PT. Adult bone marrow mesenchymal stromal cells (MSCs) were derived from the femur of orthopaedic patients, seeded on the scaffolds and cultured under osteogenic induction up to differentiation and mineralization. The release of specific metabolites and temporal gene expression profiles of marrow-derived osteoprogenitors were analyzed at definite time points, relevant to in vitro culture as well as in vivo differentiation. As a result, the role of the different biomimetic components added to the PLLA matrix was deciphered, with BMP2-added scaffolds showing the highest biomimetic activity on cells differentiating to mature osteoblasts. The modification of a polymeric scaffold with reinforcing components which also work as biomimetic cues for cells can effectively direct osteoprogenitor cells differentiation, so as to shorten the time required for mineralization.

Enhanced Adipogenic Differentiation of Human Dental Pulp Stem Cells in Enzymatically Decellularized Adipose Tissue Solid Foams.

Engineered 3D human adipose tissue models and the development of physiological human 3D in vitro models to test new therapeutic compounds and advance in the study of pathophysiological mechanisms of disease is still technically challenging and expensive. To reduce costs and develop new technologies to study human adipogenesis and stem cell differentiation in a controlled in vitro system, here we report the design, characterization, and validation of extracellular matrix (ECM)-based materials of decellularized human adipose tissue (hDAT) or bovine collagen-I (bCOL-I) for 3D adipogenic stem cell culture. We aimed at recapitulating the dynamics, composition, and structure of the native ECM to optimize the adipogenic differentiation of human mesenchymal stem cells. hDAT was obtained by a two-enzymatic step decellularization protocol and post-processed by freeze-drying to produce 3D solid foams. These solid foams were employed either as pure hDAT, or combined with bCOL-I in a 3:1 proportion, to recreate a microenvironment compatible with stem cell survival and differentiation. We sought to investigate the effect of the adipogenic inductive extracellular 3D-microenvironment on human multipotent dental pulp stem cells (hDPSCs). We found that solid foams supported hDPSC viability and proliferation. Incubation of hDPSCs with adipogenic medium in hDAT-based solid foams increased the expression of mature adipocyte LPL and c/EBP gene markers as determined by RT-qPCR, with respect to bCOL-I solid foams. Moreover, hDPSC capability to differentiate towards adipocytes was assessed by PPAR-γ immunostaining and Oil-red lipid droplet staining. We found out that both hDAT and mixed 3:1 hDAT-COL-I solid foams could support adipogenesis in 3D-hDPSC stem cell cultures significantly more efficiently than solid foams of bCOL-I, opening the possibility to obtain hDAT-based solid foams with customized properties. The combination of human-derived ECM biomaterials with synthetic proteins can, thus, be envisaged to reduce fabrication costs, thus facilitating the widespread use of autologous stem cells and biomaterials for personalized medicine.

Novel bioactive scaffolds incorporating nanogels as potential drug eluting devices.

Big advances are being achieved in the design of new implantable devices with enhanced properties. For example, synthetic porous three-dimensional structures can mimic the architecture of the tissues, and serve as templates for cell seeding. In addition, polymeric nanoparticles are able to provide a programmable and sustained local delivery of different types of biomolecules. In this study novel alternative scaffolds with controlled bioactive properties and architectures are presented. Two complementary approaches are described. Firstly, scaffolds with nanogels as active controlled release devices incorporated inside the three-dimensional structure are obtained using the thermally induced phase separation (TIPS) method. Secondly, a novel coating method using the spraying technique to load these nanometric crosslinked hydrogels on the surface of two-dimensional (2D) and three-dimensional (3D) biodegradable scaffolds is described. The scanning electron microscopy (SEM) images show the distribution of the nanogels on the surface of different substrates and also inside the porous structure of poly-alpha-hydroxy ester derivative foams. Both of them are compared in terms of manufacturability, dispersion and other processing variables.

Bioactivity of dexamethasone-releasing coatings on polymer/magnesium composites.

We developed biodegradable polymeric coatings loaded with increasing amounts of dexamethasone on composites based on polylactic acid and Mg particles for bone repair. Incorporation of Mg particles into the polymeric matrix improves the compressive behaviour of the polymer. Mg-containing composites release Mg2+ ions into the culture medium and improve mesenchymal stem cell (MSC) viability, enhance their osteogenic potential and promote the release of angiogenic factors. Dexamethasone-loaded coatings deposited on composites delay Mg2+ ion dissolution while releasing controlled amounts of the drug, which are highly dependent on initial payload. Release kinetic of dexamethasone from the coatings exhibits a fast initial release of the drug followed by a slower secondary release. Bioactivity of the released dexamethasone was explored by monitoring dose-dependent responses of MSCs and macrophages. Biological effects exerted by the released drug are similar to those observed in cells treated with solutions of the glucocorticoid, indicating that the method employed for inclusion of dexamethasone into the coatings does not impair its bioactive behaviour. Culturing MSCs on dexamethasone-releasing coatings enhances extracellular matrix production and initial induction to osteogenic commitment as a function of drug payload. Dexamethasone incorporated into the coatings presents anti-inflammatory activity, as shown by the decrease in the production of cytokines and angiogenic factors by macrophages and MSCs. Deposition of dexamethasone-releasing coatings on polymer/Mg composites appears to be a promising approach to delay composite degradation at the early stage of implantation and may be useful to attenuate inflammation and adverse foreign body reactions.

Effect of nanotubes and apatite on growth factor release from PLLA scaffolds.

There is an evident clinical need for artificial bone restorative materials. In this respect, novel composites based on poly(L-lactic acid) (PLLA) have been described. The bone response of such polymer-based composites is usually improved by the addition of bone morphogenetic protein-2 (BMP-2). However, released BMP-2 is cleared almost immediately from the site of implantation by diffusion, whereas a prolonged retention of BMP-2 onto the scaffold has been suggested to be more favourable. Besides the ability to improve the mechanical strength and osteoconductivity of polymeric scaffolds, both carbon nanotubes (CNTs) and microhydroxyapatite (µHA) have been described to facilitate such retention of BMP-2 when incorporated into a composite scaffold. Therefore, in the current study, radiolabelled BMP-2 was loaded onto plain PLLA and composite PLLA-CNT-µHA scaffolds. Subsequently, the scaffolds were implanted subcutaneously for 5 weeks in rats and BMP-2 release was measured. Release started with an initial phase of quick release, followed by a gradual release of BMP-2. Both scaffold types comprised the same in vivo release properties for BMP-2. The bioactivity of the BMP-2 remained unaltered. It can be concluded that incorporated CNTs and µHA did not affect BMP-2 release from composite scaffold materials.

Genetic profiling of osteoblast-like cells cultured on a novel bone reconstructive material, consisting of poly-L-lactide, carbon nanotubes and microhydroxyapatite, in the presence of bone morphogenetic protein-2.

In bone tissue engineering composite materials have been introduced, combining a degradable polymer matrix with, for instance, carbon nanotubes (CNTs) to improve mechanical properties or with microhydroxyapatite (µHA) to improve osteoconduction. The addition of bone morphogenetic protein-2 (BMP-2) can further improve the biological response to the material. However, the influence of such an elaborate composite formation on osteoprogenitor cells is unknown. To examine this, rat bone marrow (RBM) cells were cultured on porous poly-L-lactic acid and composite scaffolds, with or without added BMP-2. Cell proliferation and differentiation were studied using DNA, alkaline phosphatase and scanning electron microscopic analysis. Further, genetic profiles were examined by microarray investigation. Results showed that the composite scaffold had no significant effect on the proliferation of RBM cells, but indicated a negative effect on cell differentiation. The addition of BMP-2 also had no significant effect on the proliferation of RBM cells, but differentiation towards the osteogenic lineage was confirmed. In the arrays results, the addition of BMP-2 alone led to the expression of genes involved in (minor) inflammation. The composite scaffold, and even more distinctly the combination of the composite scaffold with BMP-2, led to the expression of genes, based on gene ontology, connected to tumorigenesis. Therefore, CNT- and µHA-containing composite materials are not recommended as a bone restorative material.