Recent studies suggest that bone marrow stromal cells are a potential source of osteoblasts and chondrocytes and can be used to regenerate damaged tissues using a tissue-engineering (TE) approach. However, these strategies require the use of an appropriate scaffold architecture that can support the formation de novo of either bone and cartilage tissue, or both, as in the case of osteochondral defects. The later has been attracting a great deal of attention since it is considered a difficult goal to achieve. This work consisted on developing novel hydroxyapatite/chitosan (HA/CS) bilayered scaffold by combining a sintering and a freeze-drying technique, and aims to show the potential of such type of scaffolds for being used in TE of osteochondral defects. The developed HA/CS bilayered scaffolds were characterized by Fourier transform infra-red spectroscopy, X-ray diffraction analysis, micro-computed tomography, and scanning electron microscopy (SEM). Additionally, the mechanical properties of HA/CS bilayered scaffolds were assessed under compression. In vitro tests were also carried out, in order to study the water-uptake and weight loss profile of the HA/CS bilayered scaffolds. This was done by means of soaking the scaffolds into a phosphate buffered saline for 1 up to 30 days. The intrinsic cytotoxicity of the HA scaffolds and HA/CS bilayered scaffolds extract fluids was investigated by carrying out a cellular viability assay (MTS test) using Mouse fibroblastic-like cells. Results have shown that materials do not exert any cytotoxic effect. Complementarily, in vitro (phase I) cell culture studies were carried out to evaluate the capacity of HA and CS layers to separately, support the growth and differentiation of goat marrow stromal cells (GBMCs) into osteoblasts and chondrocytes, respectively. Cell adhesion and morphology were analysed by SEM while the cell viability and proliferation were assessed by MTS test and DNA quantification. The chondrogenic differentiation of GBMCs was evaluated measuring the glucosaminoglycans synthesis. Data showed that GBMCs were able to adhere, proliferate and osteogenic differentiation was evaluated by alkaline phosphatase activity and immunocytochemistry assays after 14 days in osteogenic medium and into chondrocytes after 21 days in culture with chondrogenic medium. The obtained results concerning the physicochemical and biological properties of the developed HA/CS bilayered scaffolds, show that these constructs exhibit great potential for their use in TE strategies leading to the formation of adequate tissue substitutes for the regeneration of osteochondral defects.
The positive interaction of materials with tissues is an important step in regenerative medicine strategies. Hydrogels that are obtained from polysaccharides and proteins are expected to mimic the natural cartilage environment and thus provide an optimum milleu for tissue growth and regeneration. In this work, novel hydrogels composed of blends of chitosan and Bombyx mori silk fibroin were cross-linked with genipin (G) and were freeze dried to obtain chitosan/silk (CSG) sponges. CSG sponges possess stable and ordered structures because of protein conformational changes from alpha-helix/random-coil to beta-sheet structure, distinct surface morphologies, and pH/swelling dependence at pH 3, 7.4, and 9. We investigated the cytotoxicity of CSG sponge extracts by using L929 fibroblast-like cells. Furthermore, we cultured ATDC5 cells onto the sponges to evaluate the CSG sponges' potential in cartilage repair strategies. These novel sponges promoted adhesion, proliferation, and matrix production of chondrocyte-like cells. Sponges' intrinsic properties and biological results suggest that CSG sponges may be potential candidates for cartilage tissue engineering (TE) strategies.
Musculoskeletal diseases are one of the leading causes of disability worldwide. Among them, tendon and ligament injuries represent an important aspect to consider in both athletes and active working people. Tendon and ligament damage is an important cause of joint instability, and progresses into early onset of osteoarthritis, pain, disability and eventually the need for joint replacement surgery. The social and economical burden associated with these medical conditions presents a compelling argument for greater understanding and expanding research on this issue. The particular physiology of tendons and ligaments (avascular, hypocellular and overall structural mechanical features) makes it difficult for currently available treatments to reach a complete and long-term functional repair of the damaged tissue, especially when complete tear occurs. Despite the effort, the treatment modalities for tendon and ligament are suboptimal, which have led to the development of alternative therapies, such as the delivery of growth factors, development of engineered scaffolds or the application of stem cells, which have been approached in this review.
The use of polymeric nanoparticles, especially those composed of natural polymers, has become a very interesting approach in drug delivery, mainly because of the advantages offered by their small dimensions. The aim of this work was to develop a novel formulation of nanoparticles comprised of two natural marine-derived polymers, namely chitosan and carrageenan, and to evaluate their potential for the association and controlled release of macromolecules. Nanoparticles were obtained in a hydrophilic environment, under very mild conditions, avoiding the use of organic solvents or other aggressive technologies for their preparation. The developed nanocarriers presented sizes within 350-650 nm and positive zeta potentials of 50-60 mV. Polymeric interactions between nanoparticles' components were evaluated by Fourier transform infrared spectroscopy. Using ovalbumin as model protein, nanoparticles evidenced loading capacity varying from 4% to 17% and demonstrated excellent capacity to provide a controlled release for up to 3 weeks. Furthermore, nanoparticles have demonstrated to exhibit a noncytotoxic behavior in biological in vitro tests performed using L929 fibroblasts, which is critical regarding the biocompatibility of those carriers. In summary, the developed chitosan-carrageenan nanoparticles have shown promising properties to be used as carriers of therapeutic macromolecules, with potential application not only strictly in drug delivery, but also in broader areas, such as tissue engineering and regenerative medicine.
Current treatments for tendon injuries often fail to fully restore joint biomechanics leading to the recurrence of symptoms, and thus resulting in a significant health problem with a relevant social impact worldwide. Cell-based approaches involving the use of stem cells might enable tailoring a successful tendon regeneration outcome. As growth factors (GFs) powerfully regulate the cell biological response, their exogenous addition can further stimulate stem cells into the tenogenic lineage, which might eventually depend on stem cells source. In the present study we investigate the tenogenic differentiation potential of human- amniotic fluid stem cells (hAFSCs) and adipose-derived stem cells (hASCs) with several GFs associated to tendon development and healing; namely, EGF, bFGF, PDGF-BB and TGF-β1. Stem cells response to biochemical stimuli was studied by screening of tendon-related genes (collagen type I, III, decorin, tenascin C and scleraxis) and proteins found in tendon extracellular matrix (ECM) (Collagen I, III, and Tenascin C). Despite the fact that GFs did not seem to influence the synthesis of tendon ECM proteins, EGF and bFGF influenced the expression of tendon-related genes in hAFSCs, while EGF and PDGF-BB stimulated the genetic expression in hASCs. Overall results on cellular alignment morphology, immunolocalization and PCR analysis indicated that both stem cell source can be biochemically induced towards tenogenic commitment, validating the potential of hASCs and hAFSCs for tendon regeneration strategies.
Tissue engineering (TE) is continuously evolving assimilating inputs from adjacent scientific areas and their technological advances, including nanotechnology developments that have been spawning the range of available options for the precise manipulation and control of cells and cellular environments. Simultaneously, with the maturation of the field, TE has a growing and marked impact in other fields, such as cancer and other diseases research, enabling tri-dimensional (3D) tumor/tissue models of increased complexity that more closely resemble living tissue dynamics, playing a decisive role in the development of new and improved therapies. Nevertheless, TE is still struggling with translational issues. On this matter, the advent of personalized and precision medicine has opened new perspectives, particularly with the striking evolutions enabled by 3D bioprinting technologies. Based on a modified methodology grounded in the past years' approach, we have identified and reviewed some of the most high-impact publications on the topics that are revolutionizing TE and helping to define the future directions of the field, namely: (1) New trends in TE: Personalized/precision regenerative medicine and 3D bioprinting, (2) Contributions of TE to other fields: microfabricated tissueengineered 3D models for cancer and other diseases research, and (3) Diagnostic and theranostic tools: monitoring and real-time control of TE systems.Keywords: 3D bioprinting, 3D disease models, nanotechnology, precision regenerative medicine, theranostic toolsThe Aim, Scope, and Methods of This Review S ince its origin, tissue engineering (TE) holds the promise of revolutionizing healthcare by providing artificially developed tissues and organs substitutes on demand. More recently, TE has expanded into different biomedical areas to feedback research and clinical needs, widening the range not only of possible successful therapies but also of diagnostic/screening tools. Considering the growing number of publications related with TE and approaching different scientific domains, such as cancer science or pharmaceutics research, the potential complementary role of the field in a multitude of clinical areas and therapies in the years to come is evident.Our review attempts to cover some of the most exciting research contributing to both regenerative medicine and in vitro research applications of engineered tissues, along with the novel technological approaches that are advancing the field. This is the fifth review of the kind in TE. The previous four articles [1][2][3][4] helped to establish the methodology adopted for selecting the areas of focus and the contributions to highlight. As in previous years, we started by searching the ISI Web of Knowledge database for articles in ''tissue engineering'' and ''regenerative medicine'' published during the period of September 2015 through December 2016, thus using a 3-month overlap with the previous review to not miss impactful articles in areas not addressed earlier.Similar to previous years, we also consid...
Bone tissue engineering success strongly depends on our ability to develop new materials combining osteoconductive, osteoinductive and osteogenic properties. Recent studies suggest that biomaterials incorporating silanol (Si-OH) groups promote and maintain osteogenesis. The purpose of the present research work was to provide evidence that using wet-spinning technologies and a calcium silicate solution as a coagulation bath, it was possible to develop an in situ functionalization methodology to obtain 3D wet-spun fibre meshes with Si-OH groups, through a simple, economic and reliable process. SPCL (blend of starch with polycaprolactone) fibre meshes were produced by wet-spinning, using a calcium silicate solution as a non-solvent and functionalized in situ with Si-OH groups. In vitro tests, using goat bone marrow stromal cells (GBMSCs), showed that SPCL-Si scaffolds sustained cell viability and proliferation. Furthermore, high ALP activity and matrix production indicated that Si-OH groups improve cellular functionality towards the osteoblastic phenotype. Using this methodology, and assembling several wet-spun fibre meshes, 3D meshes can be developed, aiming at designing osteoconductive/osteoinductive 3D structures capable of stimulating bone ingrowth in vivo.
In this work, a new methodology is reported for developing hydroxyapatite (HA) scaffolds using an organic sacrifice template. The novelty of work consists of possibility of obtaining porous and highly interconnected scaffolds mimicking the sacrificial component. Our purpose consisted of evaluating the physicochemical properties of the HA scaffolds by means of Fourier transform infra-red spectroscopy, X-ray diffraction analysis, and scanning electron microscopy (SEM) attached with an X-ray detector. The HA scaffolds obtained possess a porosity of approximately 70%, and macropores diameter in the range of 50-600 microm. In contrast, results regarding the microcomputed tomography analysis have demonstrated both high pore uniformity and interconnectivity across the scaffolds. The compressive strength of the HA scaffolds was found to be 30.2 +/- 6.0 MPa. Bioactivity of the HA scaffolds was assessed by immersion into a simulated body fluid solution, in vitro. SEM observations have showed a deposition of apatite on the surface of the HA scaffolds, with a "cauliflower-like" morphology after 1 day, and tend to be more pronounced with the immersion time. The changes in calcium and phosphorus concentration were monitored by inductively-coupled plasma optical emission spectrometry. Cytotoxicity of the HA scaffolds was preliminarily investigated by carrying direct observation of mouse fibroblasts cells (L929 cell-line) death in the inverted microscope, and then cell viability was determined by means of carrying out a MTS assay. Complementarily, a luminescent cell viability assay based on the quantification of adenosine triphosphate was performed using rat bone marrow stromal cells (RBMSCs). A LIVE/DEAD assay and SEM analysis allowed the visualization of the RBMSCs adhesion and proliferation on the surface of the HA scaffolds. According to the results obtained from 3D architecture, mechanical properties, biocompatibility, and adhesion tests, it is suggested that HA scaffolds has potential to find applications in bone tissue engineering scaffolding.
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