Joaquim M. Oliveira

Natural-based nanocomposites for bone tissue engineering and regenerative medicine : a review

Tissue engineering and regenerative medicine has been providing exciting technologies for the development of functional substitutes aimed to repair and regenerate damaged tissues and organs. Inspired by the hierarchical nature of bone, nanostructured biomaterials are gaining a singular attention for tissue engineering, owing their ability to promote cell adhesion and proliferation, and hence new bone growth, compared with conventional microsized materials. Of particular interest are nanocomposites involving biopolymeric matrices and bioactive nanosized fillers. Biodegradability, high mechanical strength, and osteointegration and formation of ligamentous tissue are properties required for such materials. Biopolymers are advantageous due to their similarities with extracellular matrices, specific degradation rates, and good biological performance. By its turn, calcium phosphates possess favorable osteoconductivity, resorbability, and biocompatibility. Herein, an overview on the available natural polymer/calcium phosphate nanocomposite materials, their design, and properties is presented. Scaffolds, hydrogels, and fibers as biomimetic strategies for tissue engineering, and processing methodologies are described. The specific biological properties of the nanocomposites, as well as their interaction with cells, including the use of bioactive molecules, are highlighted. Nanocomposites in vivo studies using animal models are also reviewed and discussed.

The osteogenic differentiation of rat bone marrow stromal cells cultured with dexamethasone-loaded carboxymethylchitosan/poly(amidoamine) dendrimer nanoparticles

There is an increasing interest in developing novel macromolecular vehicles for the intracellular and controlled delivery of bioactive molecules, since they can allow modulation of the cellular functions in a more effective manner ex vivo, and maintain the cellular phenotype in vivo upon re-implantation. The present study was designed to investigate the effect of combining novel dexamethasone-loaded carboxymethylchitosan/poly(amidoamine) dendrimer (Dex-loaded CMCht/PAMAM) nanoparticles and, both HA and SPCL scaffolds (3D system) on the proliferation and osteogenic differentiation of rat bone marrow stromal cells (RBMSCs) in vitro. A luminescent cell viability assay using RBMSCs was performed for screening cytotoxicity of the developed HA and SPCL scaffolds. Results corroborated previous ones which have demonstrated in vitro, the superior performance of the HA and SPCL scaffolds on supporting cells adhesion and proliferation. Furthermore, this work showed that RBMSCs seeded onto the surface of both HA and SPCL scaffolds differentiate into osteoblasts when cultured in the presence of 0.01 mg ml(-1) Dex-loaded CMCht/PAMAM dendrimer nanoparticles. In addition, results demonstrated that Dex-loaded CMCht/PAMAM dendrimer nanoparticles combined with the HA enhance osteogenesis by increasing ALP activity and mineralization of the extra-cellular matrix. The pre-incubation of stem cells with these kinds of nanoparticles allows the delivery of Dex inside the cells and directly influences their cellular fate, being a promising new tool to be used in cells and tissue engineering strategies.

Hydrogels in acellular and cellular strategies for intervertebral disc regeneration.

Low back pain is an extremely common illness syndrome that causes patient suffering and disability and requires urgent solutions to improve the quality of life of these patients. Treatment options aimed to regenerate the intervertebral disc (IVD) are still under development. The cellular complexity of IVD, and consequently its fine regulatory system, makes it a challenge to the scientific community. Biomaterials‐based therapies are the most interesting solutions to date, whereby tissue engineering and regenerative medicine (TE&RM) strategies are included. By using such strategies, i.e., combining biomaterials, cells, and biomolecules, the ultimate goal of reaching a complete integration between native and neo‐tissue can be achieved. Hydrogels are promising materials for restoring IVD, mainly nucleus pulposus (NP). This study presents an overview of the use of hydrogels in acellular and cellular strategies for intervertebral disc regeneration. To better understand IVD and its functioning, this study will focus on several aspects: anatomy, pathophysiology, cellular and biomolecular performance, intrinsic healing processes, and current therapies. In addition, the application of hydrogels as NP substitutes will be addressed due to their similarities to NP mechanical properties and extracellular matrix. These hydrogels can be used in cellular strategies when combined with cells from different sources, or in acellular strategies by performing the functionalization of the hydrogels with biomolecules. In addition, a brief summary of therapies based on simple injection for primary biological repair will be examined. Finally, special emphasis will focus on reviewing original studies reporting on the use of autologous cells and biomolecules such as platelet‐rich plasma and their potential clinical applications. Copyright

Ex vivo culturing of stromal cells with dexamethasone-loaded carboxymethylchitosan/poly(amidoamine) dendrimer nanoparticles promotes ectopic bone formation.

Recently, our group has proposed a combinatorial strategy in tissue engineering principles employing carboxymethylchitosan/poly(amidoamine) dendrimer nanoparticles (CMCht/PAMAM) towards the intracellular release and regimented supply of dexamethasone (Dex) aimed at controlling stem cell osteogenic differentiation in the absence of typical osteogenic inducers, in vivo. In this work, we have investigated if the Dex-loaded CMCht/PAMAM dendrimer nanoparticles could play a crucial role in the regulation of osteogenesis, in vivo. Macroporous hydroxyapatite (HA) scaffolds were seeded with rat bone marrow stromal cells (RBMSCs), whose cells were expanded in MEM medium supplemented with 0.01 mg ml(-1) Dex-loaded CMCht/PAMAM dendrimer nanoparticles and implanted subcutaneously on the back of rats for 2 and 4 weeks. HA porous ceramics without RBMSCs and RBMSCs/HA scaffold constructs seeded with cells expanded in the presence and absence of 10(-8) M Dex were used as controls. The effect of initial cell number seeded in the HA scaffolds on the bone-forming ability of the constructs was also investigated. Qualitative and quantitative new bone formation was evaluated in a non-destructive manner using micro-computed tomography analyses of the explants. Haematoxylin and Eosin stained implant sections were also used for the histomorphometrical analysis. Toluidine blue staining was carried out to investigate the synthesis of proteoglycan extracellular matrix. In addition, alkaline phosphatase and osteocalcin levels in the explants were also quantified, since these markers denote osteogenic differentiation. At 4 weeks post-implantation results have shown that the novel Dex-loaded carboxymethylchitosan/poly(amidoamine) dendrimer nanoparticles may be beneficial as an intracellular nanocarrier, supplying Dex in a regimented manner and promoting superior ectopic de novo bone formation.

Novel poly(L-lactic acid)/hyaluronic acid macroporous hybrid scaffolds: characterization and assessment of cytotoxicity.

Poly(L-lactic acid), PLLA, a synthetic biodegradable polyester, is widely accepted in tissue engineering. Hyaluronic acid (HA), a natural polymer, exhibits an excellent biocompatibility, influences cell signaling, proliferation, and differentiation. In this study, HA crosslinking was performed by immersion of the polysaccharide in water-acetone mixtures containing glutaraldehyde (GA). The objective of this work is to produce PLLA scaffolds with the pores coated with HA, that could be beneficial for bone tissue engineering applications. PLLA tridimensional scaffolds were prepared by compression molding followed by salt leaching. After the scaffolds impregnation with soluble HA solutions of distinct concentration, a GA-crosslinking reaction followed by inactivation of the unreacted GA with glycine was carried out. An increase on surface roughness is shown by scanning electron microscopy (SEM) with the addition of HA. Toluidine blue staining indicates the present of stable crosslinked HA. An estimation of the HA original weight in the hybrid scaffolds was performed using thermal gravimetric analyses. FTIR-ATR and XPS confirmed the crosslinking reaction. Preliminary in vitro cell culture studies were carried out using a mouse lung fibroblast cell line (L929). SEM micrographs of L929 showed that cells adhered well, spread actively throughout all scaffolds, and grew favorably. A MTS test indicated that cells were viable when cultured onto the surface of all scaffolds, suggesting that the introduction of crosslinked HA did not increase the cytotoxicity of the hybrid scaffolds.

Management of knee osteoarthritis: current status and future trends

Osteoarthritis (OA) affects a large number of the population, and its incidence is showing a growing trend with the increasing life span. OA is the most prevalent joint condition worldwide, and currently, there is no functional cure for it. This review seeks to briefly overview the management of knee OA concerning standardized pharmaceutical and clinical approaches, as well as the new biotechnological horizons of OA treatment. The potential of biomaterials and state of the art of advanced therapeutic approaches, such as cell and gene therapy focused primarily on cartilage regeneration are the main subjects of this review. Biotechnol. Bioeng. 2017;114: 717–739.

In vivo biodistribution of carboxymethylchitosan/poly(amidoamine) dendrimer nanoparticles in rats

Carboxymethylchitosan/poly(amidoamine) (CMCht/PAMAM) dendrimer nanoparticles, comprised of a PAMAM dendrimer core grafted with chains of CMCht, have recently been proposed for intracellular drug delivery. In previous reports, these nanoparticles had lower levels of cytotoxicity when compared with traditional dendrimers. In this study, the short-term in vivo biodistribution of fluorescein isothiocyanate (FITC)-labeled CMCht/PAMAM dendrimer nanoparticles after intravenous (IV) injections in Wistar Han rats was determined. The brain, liver, kidney, and lung were collected at 24, 48, and 72 h after injection and stained with phalloidin–tetramethylrhodamine isothiocyanate (TRITC, red) and 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI, blue) to trace the nanoparticles within these tissues. The liver, kidney, and lung were also stained for hematoxylin and eosin to assess any morphological alterations of these organs. CMCht/PAMAM dendrimer nanoparticles were observed within the vascular space and parenchyma of liver, kidney, and lung and in the choroid plexus, after each injection period. No particles were observed in the brain parenchyma, nor any apparent deleterious histological changes were observed within these organs. The CMCht/PAMAM dendrimer nanoparticles were stable in circulation for a period of up to 72 h, targeting the main organs/systems through internalization by the cells present in their parenchyma. These results provide positive indicators to their potential use in the future as intracellular drug delivery systems.

Biological evaluation of intervertebral disc cells in different formulations of gellan gum-based hydrogels

Gellan gum (GG)‐based hydrogels are advantageous in tissue engineering not only due to their ability to retain large quantities of water and provide a similar environment to that of natural extracellular matrix (ECM), but also because they can gelify in situ in seconds. Their mechanical properties can be fine‐tuned to mimic natural tissues such as the nucleus pulposus (NP). This study produced different formulations of GG hydrogels by mixing varying amounts of methacrylated (GG‐MA) and high‐acyl gellan gums (HA‐GG) for applications as acellular and cellular NP substitutes. The hydrogels were physicochemically characterized by dynamic mechanical analysis. Degradation and swelling abilities were assessed by soaking in a phosphate buffered saline solution for up to 170 h. Results showed that as HA‐GG content increased, the modulus of the hydrogels decreased. Moreover, increases in HA‐GG content induced greater weight loss in the GG‐MA/HA‐GG formulation compared to GG‐MA hydrogel. Potential cytotoxicity of the hydrogel was assessed by culturing rabbit NP cells up to 7 days. An MTS assay was performed by seeding rabbit NP cells onto the surface of 3D hydrogel disc formulations. Viability of rabbit NP cells encapsulated within the different hydrogel formulations was also evaluated by Calcein‐AM and ATP assays. Results showed that tunable GG‐MA/HA‐GG hydrogels were non‐cytotoxic and supported viability of rabbit NP cells. Copyright

The Meniscus: Basic Science

The menisci are semilunar discs of fibrocartilaginous tissue which play critical roles in knee joint biomechanics [1]. Despite, it has been described in the past as nearly useless with perhaps some minor roles on joint nutrition and stabilization [2]. These complex structures are primarily composed of an interlacing network of collagen fibers (predominantly type I collagen) interposed with cells, and an extracellular matrix (ECM) of proteoglycans and glycoproteins.

Meniscal Repair: Indications, Techniques, and Outcome

The current trend when dealing with meniscus tears is “preservation whenever possible.” This way, meniscal repair techniques have significantly developed in recent years. There have been advances in both surgical techniques (e.g., techniques for root tear repair) and related devices. However, anatomical knowledge is crucial, and learning curve time for any technique should be considered. Classification of tears, preoperative planning, and surgical training (including cadaver courses) are extremely important.