Development, characterization and sterilisation of Nanocellulose-alginate-(hyaluronic acid)- bioinks and 3D bioprinted scaffolds for tissue engineering.

3D-bioprinting is an emerging technology of high potential in tissue engineering (TE), since it shows effective control over scaffold fabrication and cell distribution. Biopolymers such as alginate (Alg), nanofibrillated cellulose (NC) and hyaluronic acid (HA) offer excellent characteristics for use as bioinks due to their excellent biocompatibility and rheological properties. Cell incorporation into the bioink requires sterilisation assurance, and autoclave, ß-radiation and γ-radiation are widely used sterilisation techniques in biomedicine; however, their use in 3D-bioprinting for bioinks sterilisation is still in their early stages. In this study, different sterilisation procedures were applied on NC-Alg and NC-Alg-HA bioinks and their effect on several parameters was evaluated. Results demonstrated that NC-Alg and NC-Alg-HA bioinks suffered relevant rheological and physicochemical modifications after sterilisation; yet, it can be concluded that the short cycle autoclave is the best option to sterilise both NC-Alg based cell-free bioinks, and that the incorporation of HA to the NC-Alg bioink improves its characteristics. Additionally, 3D scaffolds were bioprinted and specifically characterized as well as the D1 mesenchymal stromal cells (D1-MSCs) embedded for cell viability analysis. Notably, the addition of HA demonstrates better scaffold properties, together with higher biocompatibility and cell viability in comparison with the NC-Alg scaffolds. Thus, the use of MSCs containing NC-Alg based scaffolds may become a feasible tissue engineering approach for regenerative medicine.

Advances of hyaluronic acid in stem cell therapy and tissue engineering, including current clinical trials.

Hyaluronic acid (HA), as one of the main components of the extracellular matrix (ECM), plays a significant role in a multitude of biological processes involving cell migration, proliferation, differentiation, wound healing and inflammation. Thanks to its excellent biocompatibility, biodegradability and hygroscopic properties, HA has been used in its natural form for joint lubrication and ocular treatment. The chemical structure of HA can be easily modified by direct reaction with its carboxyl and hydroxyl groups. Recently, HA derivatives have been synthesised with the aim of developing HA-based materials with increased mechanical strength, improved cell interactions and reduced biodegradation and studied for regenerative medicine purposes, including cell therapy and tissue engineering. In this context, the present manuscript reviews HA applications from a basic point of view - including chemical modifications and cellular biology aspects related to clinical translation - and future perspectives of using biofabrication technologies for regenerative medicine. A detailed description of current clinical trials, testing advanced therapies based on combination of stem cells and HA formulations, is included. The final goal was to offer an integral portrait and a deeper comprehension of the current applications of HA from bench to bedside.

Volume-by-volume bioprinting of chondrocytes-alginate bioinks in high temperature thermoplastic scaffolds for cartilage regeneration.

IMPACT STATEMENT: 3D bioprinting represents a novel advance in the area of regenerative biomedicine and tissue engineering for the treatment of different pathologies, among which are those related to cartilage. Currently, the use of different thermoplastic polymers, such as PLA or PCL, for bioprinting processes presents an important limitation: the high temperatures that are required for extrusion affect the cell viability and the final characteristics of the construct. In this work, we present a novel bioprinting process called volume-by-volume (VbV) that allows us to preserve cell viability after bioprinting. This procedure allows cell injection at a safe thermoplastic temperature, and also allows the cells to be deposited in the desired areas of the construct, without the limitations caused by high temperatures. The VbV process could make it easier to bring 3D bioprinting into the clinic, allowing the generation of tissue constructs with polymers that are currently approved for clinical use.

Therapeutic strategies for skin regeneration based on biomedical substitutes.

Regenerative medicine and tissue engineering (TE) have experienced significant advances in the development of in vitro engineered skin substitutes, either for replacement of lost tissue in skin injuries or for the generation of in vitro human skin models to research. However, currently available skin substitutes present different limitations such as expensive costs, abnormal skin microstructure and engraftment failure. Given these limitations, new technologies, based on advanced therapies and regenerative medicine, have been applied to develop skin substitutes with several pharmaceutical applications that include injectable cell suspensions, cell-spray devices, sheets or 3Dscaffolds for skin tissue regeneration and others. Clinical practice for skin injuries has evolved to incorporate these innovative applications to facilitate wound healing, improve the barrier function of the skin, prevent infections, manage pain and even to ameliorate long-term aesthetic results. In this article, we review current commercially available skin substitutes for clinical use, as well as the latest advances in biomedical and pharmaceutical applications used to design advanced therapies and medical products for wound healing and skin regeneration. We highlight the current progress in clinical trials for wound healing as well as the new technologies that are being developed and hold the potential to generate skin substitutes such as 3D bioprinting-based strategies.

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Impact of TGF-ß family-related growth factors on chondrogenic differentiation of adipose-derived stem cells isolated from lipoaspirates and infrapatellar fat pads of osteoarthritic patients.

The success of cell-based approaches for the treatment of cartilage defects requires an optimal autologous cell source with chondrogenic differentiation ability that maintains its differentiated properties and stability following implantation. The objective of this study was to compare the chondrogenic capacity of mesenchymal stem cells (MSCs) isolated from lipoaspirates (ASCs) and the infrapatellar fat pad (IFPSCs) of osteoarthritic patients and treated with transforming growth factor (TGF)-ß family-related growth factors. Cells were cultured for 6 weeks in a 3D pellet culture system with the chimeric activin A/bone morphogenic protein (BMP)-2 ligand (AB235), the chimeric nodal/BMP-2 ligand (NB260) or BMP-2. To investigate the stability of the new cartilage, ASCs-treated pellets were transplanted subcutaneously into severe combined immunodeficiency (SCID) mice. Histological and immunohistochemical assessment confirmed that the growth factors induced cartilage differentiation in both isolated cell types. However, reverse transcription-quantitative PCR results showed that ASCs presented a higher chondrogenic potential than IFPSCs. In vivo results revealed that AB235-treated ASCs pellets were larger in size and could form stable cartilage-like tissue as compared to NB260-treated pellets, while BMP-2-treated pellets underwent calcification. The chondrogenic induction of ASCs by AB235 treatment was mediated by SMAD2/3 activation, as proved by immunofluorescence analysis. The results of this study indicated that the combination of ASCs and AB235 might lead to a cell-based cartilage regeneration treatment.

Activin A/BMP2 chimera AB235 drives efficient redifferentiation of long term cultured autologous chondrocytes.

Autologous chondrocyte implantation (ACI) depends on the quality and quantity of implanted cells and is hindered by the fact that chondrocytes cultured for long periods of time undergo dedifferentiation. Here we have developed a reproducible and efficient chondrogenic protocol to redifferentiate chondrocytes isolated from osteoarthritis (OA) patients. We used morphological, histological and immunological analysis together with a RT-PCR detection of collagen I and collagen II gene expression to show that chondrocytes isolated from articular cartilage biopsies of patients and subjected to long-term culture undergo dedifferentiation and that these cells can be redifferentiated following treatment with the chimeric Activin A/BMP2 ligand AB235. Examination of AB235-treated cell pellets in both in vitro and in vivo experiments revealed that redifferentiated chondrocytes synthesized a cartilage-specific extracellular matrix (ECM), primarily consisting of vertically-orientated collagen fibres and cartilage-specific proteoglycans. AB235-treated cell pellets also integrated into the surrounding subcutaneous tissue following transplantation in mice as demonstrated by their dramatic increase in size while non-treated control pellets disintegrated upon transplantation. Thus, our findings describe an effective protocol for the promotion of redifferentiation of autologous chondrocytes obtained from OA patients and the formation of a cartilage-like ECM that can integrate into the surrounding tissue in vivo.

Chondrocytes extract from patients with osteoarthritis induces chondrogenesis in infrapatellar fat pad-derived stem cells.

OBJECTIVE: Infrapatellar fat pad of patients with osteoarthritis (OA) contains multipotent and highly clonogenic adipose-derived stem cells that can be isolated by low invasive methods. Moreover, nuclear and cytoplasmic cellular extracts have been showed to be effective in induction of cell differentiation and reprogramming. The aim of this study was to induce chondrogenic differentiation of autologous mesenchymal stem cells (MSCs) obtained from infrapatellar fat pad (IFPSCs) of patients with OA using cellular extracts-based transdifferentiation method. DESIGN: IFPSCs and chondrocytes were isolated and characterized by flow cytometry. IFPSCs were permeabilized with Streptolysin O and then exposed to a cell extract obtained from chondrocytes. Then, IFPSCs were cultured for 2 weeks and chondrogenesis was evaluated by morphologic and ultrastructural observations, immunologic detection, gene expression analysis and growth on 3-D poly (dl-lactic-co-glycolic acid) (PLGA) scaffolds. RESULTS: After isolation, both chondrocytes and IFPSCs displayed similar expression of MSCs surface makers. Collagen II was highly expressed in chondrocytes and showed a basal expression in IFPSCs. Cells exposed to chondrocyte extracts acquired a characteristic morphological and ultrastructural chondrocyte phenotype that was confirmed by the increased proteoglycan formation and enhanced collagen II immunostaining. Moreover, chondrocyte extracts induced an increase in mRNA expression of chondrogenic genes such as Sox9, L-Sox5, Sox6 and Col2a1. Interestingly, chondrocytes, IFPSCs and transdifferentiated IFPSCs were able to grow, expand and produce extracellular matrix (ECM) on 3D PLGA scaffolds. CONCLUSIONS: We demonstrate for the first time that extracts obtained from chondrocytes of osteoarthritic knees promote chondrogenic differentiation of autologous IFPSCs. Moreover, combination of transdifferentiated IFPSCs with biodegradable PLGA 3D scaffolds can serve as an efficient system for the maintenance and maturation of cartilage tissue. These findings suggest its usefulness to repair articular surface in OA.