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"Publication Date (Web): February 20, 2015" ; In the last few years, great progress has been made to validate tissue engineering strategies in preclinical studies and clinical trials on the regeneration of osteochondral defects. In the preclinical studies, one of the dominant strategies comprises the development of biomimetic/bioactive scaffolds, which are used alone or incorporated with growth factors and/or stem cells. Many new trends are emerging for modulation of stem cell fate towards osteogenic and chondrogenic differentiations, but bone/cartilage interface regeneration and physical stimulus have been showing great promise. Besides the matrix-associated autologous chondrocyte implantation (MACI) procedure, the matrix-associated stem cells implantation (MASI) and layered scaffolds in acellular or cellular strategy are also applied in clinic. This review outlines the progresses at preclinical and clinical levels, and identifies the new challenges in osteochondral tissue engineering. Future perspectives are provided, e.g., the applications of extracellular matrix-like biomaterials, computer-aided design/manufacture of osteochondral implant and reprogrammed cells for osteochondral regeneration. ; The authors thank the Portuguese Foundation for Science and Technology (FCT) through the projects TISSUE2TISSUE (PTDC/CTM/105703/2008) and OsteoCart (PTDC/CTM-BPC/115977/2009). We also acknowledge European Union's Seventh Framework Programme (FP7/2007-2013) under grant agreement REGPOT-CT2012-316331-POLARIS. L-P.Y. acknowledges the PhD scholarship from FCT (SFRH/BD/64717/2009). The FCT distinction attributed to J.M.O. and A.L.O. under the Investigator FCT program (IF/00423/2012) and (IF/00411/2013) are also greatly ...

Macro/micro porous silk/nano-sized calcium phosphate scaffolds (SC16) with bioactive and superior physicochemical properties have been recently developed. In this study, we aim at evaluating the new bone formation ability of the SC 16 scaffolds in vivo, using silk fibroin scaffolds (S16) as control. The CaP distribution profile in the scaffolds was characterized by Micro-Computed Tomography. The in vitro mineralization behavior was examined by immersion in Simulated Body Fluid solution from 1 to 14 days. The long-term hydration degree and weight loss ratio of the scaffolds were evaluated by immersion in an Isotonic Saline Solution from 1 month to 1 year. In vivo osteogenesis properties of the scaffolds were screened by implantation into the rat femur defects for 3 weeks. The results showed that the CaP phase distributed homogeneously in the SC16 scaffolds. Mineralization was only observed in SC16 scaffolds, and both scaffolds gradually degraded with time. The staining of the explants showed that new bone formation with higher density was observed in the SC16 scaffolds as compared to S16 scaffolds, guiding the growth of new bone directly onto its surface. These results demonstrated that the SC16 hybrid scaffolds are osteoconductive and can be good candidates for bone tissue engineering as promoted superior de novo bone formation. ; This study was supported by the Portuguese Foundation for Science and Technology (FCT) projects OsteoCart (PTDC/CTM-BPC/115977/2009) and Tissue2Tissue (PTDC/CTM/105703/2008). Research leading to these results has received funding from the European Union's Seventh Framework Programme (FP7/2007-2013) under grant agreement no REGPOT-CT2012-316331-POLARIS. Le-Ping Yan is an FCT PhD scholarship holder ...

Since the pioneering work dealing with the synthesis and physicochemical aspects of dendrimers, a predictable and tunable set of compositions for therapeutic, scaffolding and imaging systems has been reported. These are well documented, butmanyhot issues should be examined and reviewed. Herein, a review is given on dendritic nanopolymers and their applications that show promise in the field of regenerative medicine. This review begins with a brief overview on research merging nanotechnology and regenerative medicine. Fundamentals of the synthesis and macromolecular structure of dendritic polymers are provided. Dendrimers fulfill the requirements as carriers for gene, nucleic acids, bioactive molecules and peptide/protein delivery aimed at modulate the cells functions, in vitro and in vivo. However, to make use of this potential, toxicological, drug-loading capacity, surface engineering and host–guest chemistries in dendrimers must be addressed and thus are also discussed. We focus on recent work involving dendrimers with applications in tissue engineering and the central nervous system. Due to their innovative character, applications beyond drug delivery systems became possible, namely as scaffolding and chemoattractants for tissue regeneration, and implantable biodegradable nanomaterialbased medical devices integrated with drug delivery functions (theranostics). Finally, we highlight promising areas for further research and comment on how and why dendrimer and dendron technology should be viewed as the next generation of biomaterials for the 21st century. ; The authors wish to acknowledge the funding provided by the Portuguese Foundation for Science and Technology (FCT) through the POCTI and FEDER programs, and FCT project (SMARTCARBO). This work was carried out under the scope of the European NoE EXPERTISSUES (NMP3-CT-2004-500283) and European Union HIPPOCRATES STREP Project (NMP3-CT-2003-505758). Canon Foundation in Europe is also gratefully ...

An alternative, green method was used to develop chitin-based biocomposite (ChHA) materials by an integrated strategy using ionic liquids, supercritical fluid drying, and salt leaching. ChHA matrices were produced by dissolving chitin in 1-butyl-methylimidazolium acetate along with salt and/or hydroxyapatite particles and then subsequent drying. The ChHA composite formed had a heterogeneous porous microstructure with 65%–85% porosity and pore sizes in the range of 100–300 µm. The hydroxyapatite was found to be well distributed within the composite structures and had a positive effect in the viability and proliferation of osteoblast-like cells, in vitro. Our findings indicate that these ChHA matrices have potential applications in bone tissue engineering. ; Simone S. Silva and Miguel Oliveira received financial support from the Portuguese Foundation for Science and Technology-FCT (Grant SFRH/BPD/45307/2008 and SFRH/BPD/21786/2009, respectively), "Fundo Social Europeu"-FSE, and "Programa Diferencial de Potencial Humano-POPH." This work was partially supported by the FEDER through POCTEP 0330_IBEROMARE_1_P and European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement no. KBBE-2010-266033 (project ...

A developmental composite scaffold for bone tissue engineering applications composed of hydroxyapatite (HA) and carboxymethylchitosan (CMC) was obtained using a coprecipitation method, which is based on the ''autocatalytic'' electroless deposition route. The results revealed that the pores of the scaffold were regular, interconnected, and possess a size in the range of 20–500 lm. Furthermore, the Fourier transform infra-red spectrum of the composite scaffolds exhibited all the characteristic peaks of apatite, and the appearance of typical bands from CMC, thus showing that coprecipitation of both organic and inorganic phases was effective. The X-ray diffraction pattern of composite scaffolds demonstrated that calciumphosphates consisted of crystalline HA. From microcomputed tomography analysis, it was possible to determine that composite scaffolds possess a 58.9% 6 6% of porosity. The 2D morphometric analysis demonstrated that on average the scaffolds consisted of 24% HA and 76% CMC. The mechanical properties were assessed using compressive tests, both in dry and wet states. Additionally, in vitro tests were carried out to evaluate the wateruptake capability, weight loss, and bioactive behavior of the composite scaffolds. The novel hydroxyapatite/ carboxymethylchitosan composite scaffolds showed promise whenever degradability and bioactivity are simultaneously desired, as in the case of bone tissue-engineering scaffolding applications. ; Contract grant sponsor: European Union (STREP Project HIPPOCRATES); contract grant number: NMP3-CT-2003-505758

In this study, the stability and biocompatibility of methacrylated gellan gum hydrogels, obtained either by ionic- (iGG-MA) or photo-crosslinking (phGGMA), were evaluated in vitro and in vivo. Size exclusion chromatography analysis of the methacrylated gellan gum (GG-MA) powder revealed that molecular weight is lower as compared to the non-modifi ed material, i.e., low acyl gellan gum. The water uptake and swelling of iGG-MA and phGGMA hydrogels were investigated in phosphate-buffered saline solution (pH 7.4). The biocompatibility of the hydrogels was fi rstly evaluated by producing cell-laden hydrogels. The in vitro cells encapsulation study showed that lung fi broblast cells (L929 cells) and human intervertebral disc (hIVD) cells are viable when cultured within both hydrogels, up to 21 days of culturing. The iGG-MA and phGG-MA hydrogels were also subcutaneously implanted in Lewis rats for 10 and 18 days. Tissue response to the hydrogels implantation was determined by histological analysis (haematoxylin-eosin staining). A thin fi brous capsule was observed around the implanted hydrogels. No necrosis, calcifi cation, and acute infl ammatory reaction were observed. The results presented in this study demonstrate that iGG-MA and phGG-MA hydrogels are stable in vitro and in vivo, support L929 and hIVD cells' encapsulation and viability, and were found to be well-tolerated and non-toxic in vivo. ; The authors thank the funds provided by Portuguese Foundation for Science and Technology (FCT) through POCTI and FEDER programmes. This work was also supported by the European Union funded Collaborative Project Disc Regeneration ...

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. ; Contract grant sponsor: Portuguese Foundation for Science and Technology (FCT) through POCTIContract grant sponsor: FEDER programs including project ProteoLight; contract grant number: PTDC/FIS/68517/2006Contract grant sponsor: European Union funded STREP Project HIPPOCRATES; contract grant number: NMP3-CT-2003-505758Contract grant sponsor: European NoE EXPERTISSUES; contract grant number: NMP3-CT-2004-500283Contract grant sponsor: Spanish ...

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 Dexloaded 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. ; The authors thank the funds provided by Portuguese Foundation for Science and Technology (FCT) through POCTI and FEDER programmes including project ProteoLight (PTDC/FIS/68517/2006). This work was also carried out with the support of the European Union funded STREP Project HIPPOCRATES (NMP3-CF-2003-505758) and European NOE EXPERTISSUES (NMP3-CT-2004-500283). The funding provided by Canon Foundation in Europe is gratefully ...

First published online: 2012 ; 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. ; The authors thank the Portuguese Foundation for Science and Technology (FCT) for financial support through POCTI and FEDER programmes. This work was also conducted with the support of the European Union funded Collaborative Project Disc Regeneration (NMP3-LA-2008-213904), MBC(A04003) and the World Class University project ...

Novel porous bilayered scaffolds, fully integrating a silk fibroin (SF) layer and a silk-nano calcium phosphate (Silk-NanoCaP) layer for osteochondral defect (OCD) regeneration were developed. Homogeneous porosity distribution was achieved in the scaffolds, with calcium phosphate phase only retained in the Silk-NanoCaP layer. The scaffold presented compressive moduli of 0.4 MPa in wet state. Rabbit bone marrow mesenchymal stromal cells (RBMSCs) were cultured on the scaffolds, and good adhesion and proliferation were observed. The Silk-NanoCaP layer showed a higher alkaline phosphatase level than the silk layer in osteogenic conditions. Subcutaneous implantation in rabbits demonstrated weak inflammation. In a rabbit knee critical size OCD model, the scaffolds firmly integrated into the host tissue. The histological analysis and immunohistochemistry analysis showed that collagen II positive cartilage and glycosaminoglycan regeneration presented in the silk layer, and de novo bone ingrowths and vessel formation were observed in the Silk-NanoCaP layer. These bilayered scaffolds can therefore be promising candidates for OCD regeneration. ; This study was funded by the Portuguese Foundation for Science and Technology (FCT) projects Tissue2Tissue (PTDC/CTM/105703/2008) and OsteoCart (PTDC/CTM-BPC/115977/2009), as well as the European Union's FP7 Programme under grant agreement no REGPOT-CT2012-316331-POLARIS. The authors are grateful to Viviana P. Ribeiro and Teresa Oliveira for the assistance with the immunohistochemical staining. L-P.Y. was awarded a FCT PhD scholarship (SFRH/BD/64717/2009). The FCT distinction attributed to J.M.O. under the Investigator FCT program (IF/00423/2012) is also greatly ...