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.

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.

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.

Clinical and therapeutic potential of protein kinase PKR in cancer and metabolism.

The protein kinase R (PKR, also called EIF2AK2) is an interferon-inducible double-stranded RNA protein kinase with multiple effects on cells that plays an active part in the cellular response to numerous types of stress. PKR has been extensively studied and documented for its relevance as an antiviral agent and a cell growth regulator. Recently, the role of PKR related to metabolism, inflammatory processes, cancer and neurodegenerative diseases has gained interest. In this review, we summarise and discuss the involvement of PKR in several cancer signalling pathways and the dual role that this kinase plays in cancer disease. We emphasise the importance of PKR as a molecular target for both conventional chemotherapeutics and emerging treatments based on novel drugs, and its potential as a biomarker and therapeutic target for several pathologies. Finally, we discuss the impact that the recent knowledge regarding PKR involvement in metabolism has in our understanding of the complex processes of cancer and metabolism pathologies, highlighting the translational research establishing the clinical and therapeutic potential of this pleiotropic kinase.

Biofunctional Ionic-Doped Calcium Phosphates: Silk Fibroin Composites for Bone Tissue Engineering Scaffolding.

The treatment and regeneration of bone defects caused by traumatism or diseases have not been completely addressed by current therapies. Lately, advanced tools and technologies have been successfully developed for bone tissue regeneration. Functional scaffolding materials such as biopolymers and bioresorbable fillers have gained particular attention, owing to their ability to promote cell adhesion, proliferation, and extracellular matrix production, which promote new bone growth. Here, we present novel biofunctional scaffolds for bone regeneration composed of silk fibroin (SF) and ß-tricalcium phosphate (ß-TCP) and incorporating Sr, Zn, and Mn, which were successfully developed using salt-leaching followed by a freeze-drying technique. The scaffolds presented a suitable pore size, porosity, and high interconnectivity, adequate for promoting cell attachment and proliferation. The degradation behavior and compressive mechanical strengths showed that SF/ionic-doped TCP scaffolds exhibit improved characteristics for bone tissue engineering when compared with SF scaffolds alone. The in vitro bioactivity assays using a simulated body fluid showed the growth of an apatite layer. Furthermore, in vitro assays using human adipose-derived stem cells presented different effects on cell proliferation/differentiation when varying the doping agents in the biofunctional scaffolds. The incorporation of Zn into the scaffolds led to improved proliferation, while the Sr- and Mn-doped scaffolds presented higher osteogenic potential as demonstrated by DNA quantification and alkaline phosphatase activity. The combination of Sr with Zn led to an influence on cell proliferation and osteogenesis when compared with single ions. Our results indicate that biofunctional ionic-doped composite scaffolds are good candidates for further in vivo studies on bone tissue regeneration.

Chromosome structure deficiencies in MCPH1 syndrome.

Mutations in the MCPH1 gene result in primary microcephaly in combination with a unique cellular phenotype of defective chromosome condensation. MCPH1 patient cells display premature chromosome condensation in G2 phase of the cell cycle and delayed decondensation in early G1 phase, observable as an increased proportion of cells with prophase-like appearance. MCPH1 deficiency thus appears to uncouple the chromosome cycle from the coordinated series of events that take place during mitosis such as some phases of the centrosome cycle and nuclear envelope breakdown. Here, we provide a further characterization of the effects of MCPH1 loss-of-function on chromosome morphology. In comparison to healthy controls, chromosomes of MCPH1 patients are shorter and display a pronounced coiling of their central chromatid axes. In addition, a substantial fraction of metaphase chromosomes shows apparently unresolved chromatids with twisted appearance. The patient chromosomes also showed signs of defective centromeric cohesion, which become more apparent and pronounced after harsh hypotonic conditions. Taking together, the observed alterations indicate additional so far unknown functions of MCPH1 during chromosome shaping and dynamics.

Epigenetic modifications in sex heterochromatin of vole rodents.

The genome of some vole rodents contains large blocks of heterochromatin coupled to the sex chromosomes. While the DNA content of these heterochromatic blocks has been extensively analyzed, little is known about the epigenetic modifications controlling their structure and dynamics. To better understand its organization and functions within the nucleus, we have compared the distribution pattern of several epigenetic marks in cells from two species, Microtus agrestis and Microtus cabrerae. We first could show that the heterochromatic blocks are identifiable within the nuclei due to their AT enrichment detectable by DAPI staining. By immunostaining analyses, we demonstrated that enrichment in H3K9me3 and HP1, depletion of DNA methylation as well as H4K8ac and H3K4me2, are major conserved epigenetic features of this heterochromatin in both sex chromosomes. Furthermore, we provide evidence of transcriptional activity for some repeated DNAs in cultivated cells. These transcripts are partially polyadenylated and their levels are not altered during mitotic arrest. In summary, we show here that enrichment in H3K9me3 and HP1, DNA demethylation, and transcriptional activity are major epigenetic features of sex heterochromatin in vole rodents.

Disparate molecular evolution of two types of repetitive DNAs in the genome of the grasshopper Eyprepocnemis plorans.

Wide arrays of repetitive DNA sequences form an important part of eukaryotic genomes. These repeats appear to evolve as coherent families, where repeats within a family are more similar to each other than to other orthologous representatives in related species. The continuous homogenization of repeats, through selective and non-selective processes, is termed concerted evolution. Ascertaining the level of variation between repeats is crucial to determining which evolutionary model best explains the homogenization observed for these sequences. Here, for the grasshopper Eyprepocnemis plorans, we present the analysis of intragenomic diversity for two repetitive DNA sequences (a satellite DNA (satDNA) and the 45S rDNA) resulting from the independent microdissection of several chromosomes. Our results show different homogenization patterns for these two kinds of paralogous DNA sequences, with a high between-chromosome structure for rDNA but no structure at all for the satDNA. This difference is puzzling, considering the adjacent localization of the two repetitive DNAs on paracentromeric regions in most chromosomes. The disparate homogenization patterns detected for these two repetitive DNA sequences suggest that several processes participate in the concerted evolution in E. plorans, and that these mechanisms might not work as genome-wide processes but rather as sequence-specific ones.

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.

Rapid, independent, and extensive amplification of telomeric repeats in pericentromeric regions in karyotypes of arvicoline rodents.

The distribution of telomeric repeats was analyzed by fluorescence in situ hybridization in 15 species of arvicoline rodents, included in three different genera: Chionomys, Arvicola, and Microtus. The results demonstrated that in most or the analyzed species, telomeric sequences are present, in addition to normal telomeres localization, as large blocks in pericentromeric regions. The number, localization, and degree of amplification of telomeric sequences blocks varied with the karyotype and the morphology of the chromosomes. Also, in some cases telomeric amplification at non-pericentromeric regions is described. The interstitial telomeric sequences are evolutionary modern and have rapidly colonized and spread in pericentromeric regions of chromosomes by different mechanisms and probably independently in each species. Additionally, we colocalized telomeric repeats and the satellite DNA Msat-160 (also located in pericentromeric regions) in three species and cloned telomeric repeats in one of them. Finally, we discuss about the possible origin and implication of telomeric repeats in the high rate of karyotypic evolution reported for this rodent group.

Comparative analysis by chromosome painting of the sex chromosomes in arvicolid rodents.

Sex chromosome evolution in mammals has been extensively investigated through chromosome-painting analyses. In some rodent species from the subfamily Arvicolinae the sex chromosomes contain remarkable features such as giant size, a consequence of heterochromatic enlargement, or asynaptic behaviour during male meiosis. Here, we have made a comparative study of the sex chromosomes in 6 arvicolid species using different probes from the X and Y chromosomes of 3 species, in order to gain knowledge about intra- or interspecific preservation of euchromatic regions. Our results clearly reveal poor conservation of the euchromatic region of the Y chromosome within these species, while the euchromatin on the X chromosome is extremely well preserved. Furthermore, we detected no clear correlation between the synaptic/asynaptic behaviour of the sex chromosomes, and the presence or absence of sequence homology within their euchromatic regions. Notably, our study has shown a new relationship between the giant sex chromosomes of 2 species, Microtus agrestis and Microtus cabrerae, that is, both X and Y share a novel region of common sequences in the euchromatin that is not present in the other species analysed. This interspecific euchromatic conservation, limited to the giant sex chromosomes, could point towards a common evolutionary origin for the heterochromatic enlargement process that has characterized the evolution of the sex chromosomes in some arvicolid species.

Comparative cytogenetic study of two sister species of Iberian ground voles, Microtus (Terricola) duodecimcostatus and M. (T.) lusitanicus (rodentia, cricetidae).

The two Iberian species of pine voles, Microtus (Terricola) duodecimcostatus and M. (T.) lusitanicus of the subfamily Arvicolinae (Cricetidae, Rodentia), were compared after G- and C-banding and chromosomal mapping of ribosomal RNA genes (rDNA), telomeric repeats, and satellite DNA Msat-160. Notwithstanding their close relationship (one sister group in phylogenetic analyses) and sharing of the diploid and fundamental chromosome numbers, the 2 species show notable differences in the sex chromosome morphology, the number and distribution of rDNA sites, constitutive heterochromatin and satDNA patterns. The only telomeric repeats showed normal, all-telomeric, distribution in karyotypes of both species. The data are discussed with regard to interspecific and intrageneric variation of the analyzed characters and the chromosomal evolution in the genus Microtus.

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.

Pore geometry influences growth and cell adhesion of infrapatellar mesenchymal stem cells in biofabricated 3D thermoplastic scaffolds useful for cartilage tissue engineering.

The most pressing need in cartilage tissue engineering (CTE) is the creation of a biomaterial capable to tailor the complex extracellular matrix of the tissue. Despite the standardized used of polycaprolactone (PCL) for osteochondral scaffolds, the pronounced stiffness mismatch between PCL scaffold and the tissue it replaces remarks the biomechanical incompatibility as main limitation. To overcome it, the present work was focused in the design and analysis of several geometries and pore sizes and how they affect cell adhesion and proliferation of infrapatellar fat pad-derived mesenchymal stem cells (IPFP-MSCs) loaded in biofabricated 3D thermoplastic scaffolds. A novel biomaterial for CTE, the 1,4-butanediol thermoplastic polyurethane (b-TPUe) together PCL were studied to compare their mechanical properties. Three different geometrical patterns were included: hexagonal (H), square (S), and, triangular (T); each one was printed with three different pore sizes (PS): 1, 1.5 and 2 mm. Results showed differences in cell adhesion, cell proliferation and mechanical properties depending on the geometry, porosity and type of biomaterial used. Finally, the microstructure of the two optimal geometries (T1.5 and T2) was deeply analyzed using multiaxial mechanical tests, with and without perimeters, µCT for microstructure analysis, DNA quantification and degradation assays. In conclusion, our results evidenced that IPFP-MSCs-loaded b-TPUe scaffolds had higher similarity with cartilage mechanics and T1.5 was the best adapted morphology for CTE.

A new pericentromeric repeated DNA sequence in Microtus thomasi.

Several karyotypic forms have been previously described in populations of the vole species Microtus thomasi from Greece. In particular, the karyomorphs Microtus thomasi 'thomasi' and 'atticus' differ in X chromosome morphology, being acrocentric and subtelocentric, respectively. Furthermore, remarkable heterochromatin content variability has been described in sex chromosomes of both karyomorphs. Genomic DNA digestion with AluI allowed us to clone an 884 bp long repeated DNA sequence (Mth-Alu900) from the karyomorph M. thomasi 'atticus'. This repeated DNA is AT rich and seems to be organized mainly as a dimer of the 884-bp unit, which presents three simple repeats (CAAAT, CAGAT and CAGAC) that constitute 80% of the total unit length. This repeated DNA is exclusive to M. thomasi, since it is absent from the genome of other studied Arvicolinae species. The chromosomal location of Mth-Alu900 was analyzed on M. thomasi 'thomasi' and M. thomasi 'atticus' karyomorphs, with different sex chromosome constitution. It was mainly located on the pericentromeric heterochromatin of most autosomes and X chromosomes on both karyomorphs. Results are also discussed in relation to karyotypic and sex chromosome variations in M. thomasi. To our knowledge, Mth-Alu900 constitutes a new - the third discovered so far - pericentromeric repeated DNA sequence described in Microtus species.

Retroelements (LINEs and SINEs) in vole genomes: differential distribution in the constitutive heterochromatin.

The chromosomal distribution of mobile genetic elements is scarcely known in Arvicolinae species, but could be of relevance to understand the origin and complex evolution of the sex chromosome heterochromatin. In this work we cloned two retrotransposon sequences, L1 and SINE-B1, from the genome of Chionomys nivalis and investigated their chromosomal distribution on several arvicoline species. Our results demonstrate first that both retroelements are the most abundant repeated DNA sequences in the genome of these species. L1 elements, in most species, are highly accumulated in the sex chromosomes compared to the autosomes. This favoured L1 insertion could have played an important role in the origin of the enlarged heterochromatic blocks existing in the sex chromosomes of some Microtus species. Also, we propose that L1 accumulation on the X heterochromatin could have been the consequence of different, independent and rapid amplification processes acting in each species. SINE elements, however, were completely lacking from the constitutive heterochromatin, either in autosomes or in the heterochromatic blocks of sex chromosomes. These data could indicate that some SINE elements are incompatible with the formation of heterochromatic complexes and hence are necessarily missing from the constitutive heterochromatin.

Cartilage biomechanics: A key factor for osteoarthritis regenerative medicine.

Osteoarthritis (OA) is a joint disorder that is highly extended in the global population. Several researches and therapeutic strategies have been probed on OA but without satisfactory long-term results in joint replacement. Recent evidences show how the cartilage biomechanics plays a crucial role in tissue development. This review describes how physics alters cartilage and its extracellular matrix (ECM); and its role in OA development. The ECM of the articular cartilage (AC) is widely involved in cartilage turnover processes being crucial in regeneration and joint diseases. We also review the importance of physicochemical pathways following the external forces in AC. Moreover, new techniques probed in cartilage tissue engineering for biomechanical stimulation are reviewed. The final objective of these novel approaches is to create a cellular implant that maintains all the biochemical and biomechanical properties of the original tissue for long-term replacements in patients with OA.

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.

Tumour malignancy loss and cell differentiation are associated with induction of gef gene in human melanoma cells.

BACKGROUND: Gene therapy is a new method used to induce cancer cell differentiation. Our group previously showed that transfection of the gef gene from Escherichia coli, related to cell-killing functions, may be a novel candidate for cancer gene therapy. Its expression leads to cell cycle arrest unrelated to the triggering of apoptosis in MS-36 melanoma cells. OBJECTIVES: To determine the basis of the antiproliferative effect of the gef gene in this cell line. METHODS: Transmission electron microscopy, apoptosis analysis by confocal microscopy, flow cytometry and immunocytochemical analysis were used. RESULTS: Ultrastructural analysis showed a strikingly different morphology after treatment with dexamethasone and expression of the gef gene, with large accumulations of pigment throughout the cell cytoplasm and presence of melanosomes in different stages of development. High mitochondrial turnover and myeloid bodies, characteristics of neurone cells, were also observed. In addition, both immunocytochemical and indirect immunofluorescence analysis demonstrated a significant decrease in HMB-45, Ki-67 and CD44 antigen expression and an increase in S100 and p53 expression in gef gene-transfected MS-36 melanoma cells that were correlated with the duration of dexamethasone treatment. In the present work, we report that gef gene not only reduces cell proliferation in transfected melanoma MS-36TG cell line but also induces morphological changes clearly indicative of melanoma cell differentiation and a reduction in tumour malignancy. CONCLUSIONS: These findings support the hypothesis that the gef gene offers a new approach to differentiation therapy in melanoma.

Albumin-covered lipid nanocapsules exhibit enhanced uptake performance by breast-tumor cells.

Liquid lipid nanocapsules (LLN) represent a promising new generation of drug-delivery systems. They can carry hydrophobic drugs in their oily core, but the composition and structure of the surrounding protective shell determine their capacity to survive in the circulatory system and to achieve their goal: penetrate tumor cells. Here, we present a study of LLN covered by the protein human serum albumin (HSA) and loaded with curcumin as a hydrophobic model drug. A cross-linking procedure was performed to further strengthen the protective protein layer. Physicochemical properties and release kinetics of the nanocapsules were investigated, and cellular uptake and killing capacity were evaluated on the human breast-cancer line MCF-7. The nanocapsules exhibited a half maximal inhibitory concentration (IC50) capacity similar to that of free curcumin, but avoiding problems associated with excipients, and displayed an outstanding uptake performance, entering cells massively in less than 1 min.