Ana I. Gonçalves

Understanding the role of growth factors in modulating stem cell tenogenesis

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.

Urinary estrogen metabolites and self-reported infertility in women infected with Schistosoma haematobium

Background Schistosomiasis is a neglected tropical disease, endemic in 76 countries, that afflicts more than 240 million people. The impact of schistosomiasis on infertility may be underestimated according to recent literature. Extracts of Schistosoma haematobium include estrogen-like metabolites termed catechol-estrogens that down regulate estrogen receptors alpha and beta in estrogen responsive cells. In addition, schistosome derived catechol-estrogens induce genotoxicity that result in estrogen-DNA adducts. These catechol estrogens and the catechol-estrogen-DNA adducts can be isolated from sera of people infected with S. haematobium. The aim of this study was to study infertility in females infected with S. haematobium and its association with the presence of schistosome-derived catechol-estrogens. Methodology/Principal Findings A cross-sectional study was undertaken of female residents of a region in Bengo province, Angola, endemic for schistosomiasis haematobia. Ninety-three women and girls, aged from two (parents interviewed) to 94 years were interviewed on present and previous urinary, urogenital and gynecological symptoms and complaints. Urine was collected from the participants for egg-based parasitological assessment of schistosome infection, and for liquid chromatography diode array detection electron spray ionization mass spectrometry (LC/UV-DAD/ESI-MSn) to investigate estrogen metabolites in the urine. Novel estrogen-like metabolites, potentially of schistosome origin, were detected in the urine of participants who were positive for eggs of S. haematobium, but not detected in urines negative for S. haematobium eggs. The catechol-estrogens/ DNA adducts were significantly associated with schistosomiasis (OR 3.35; 95% CI 2.32–4.84; P≤0.001). In addition, presence of these metabolites was positively associated with infertility (OR 4.33; 95% CI 1.13–16.70; P≤0.05). Conclusions/Significance Estrogen metabolites occur widely in diverse metabolic pathways. In view of the statistically significant association between catechol-estrogens/ DNA adducts and self-reported infertility, we propose that an estrogen-DNA adduct mediated pathway in S. haematobium-induced ovarian hormonal deregulation could be involved. In addition, the catechol-estrogens/ DNA adducts described here represent potential biomarkers for schistosomiasis haematobia.

Exploring the Potential of Starch/Polycaprolactone Aligned Magnetic Responsive Scaffolds for Tendon Regeneration

The application of magnetic nanoparticles (MNPs) in tissue engineering (TE) approaches opens several new research possibilities in this field, enabling a new generation of multifunctional constructs for tissue regeneration. This study describes the development of sophisticated magnetic polymer scaffolds with aligned structural features aimed at applications in tendon tissue engineering (TTE). Tissue engineering magnetic scaffolds are prepared by incorporating iron oxide MNPs into a 3D structure of aligned SPCL (starch and polycaprolactone) fibers fabricated by rapid prototyping (RP) technology. The 3D architecture, composition, and magnetic properties are characterized. Furthermore, the effect of an externally applied magnetic field is investigated on the tenogenic differentiation of adipose stem cells (ASCs) cultured onto the developed magnetic scaffolds, demonstrating that ASCs undergo tenogenic differentiation synthesizing a Tenascin C and Collagen type I rich matrix under magneto-stimulation conditions. Finally, the developed magnetic scaffolds were implanted in an ectopic rat model, evidencing good biocompatibility and integration within the surrounding tissues. Together, these results suggest that the effect of the magnetic aligned scaffolds structure combined with magnetic stimulation has a significant potential to impact the field of tendon tissue engineering toward the development of more efficient regeneration therapies.

In vitro and in vivo assessment of magnetically actuated biomaterials and prospects in tendon healing

AIM To expand our understanding on the effect of magnetically actuated biomaterials in stem cells, inflammation and fibrous tissue growth. MATERIALS & METHODS Magnetic biomaterials were obtained by doping iron oxide particles into starch poly-ϵ-caprolactone (SPCL) to create two formulations, magSPCL-1.8 and 3.6. Stem cell behavior was assessed in vitro and the inflammatory response, subcutaneously in Wistar rats. RESULTS Metabolic activity and proliferation increased significantly overtime in SPCL and magSPCL-1.8. Electromagnetic fields attenuated the presence of mast cells and macrophages in tissues surrounding SPCL and magSPCL-1.8, between weeks 1 and 9. Macrophage reduction was more pronounced for magSPCL-1.8, which could explain why this material prevented growth of fibrous tissue overtime. CONCLUSION Magnetically actuated biomaterials have potential to modulate inflammation and the growth of fibrous tissue.

New massive parallel sequencing approach improves the genetic characterization of congenital myopathies

Congenital myopathies (CMs) are a heterogeneous group of muscle diseases characterized by hypotonia, delayed motor skills and muscle weakness with onset during the first years of life. The diagnostic workup of CM is highly dependent on the interpretation of the muscle histology, where typical pathognomonic findings are suggestive of a CM but are not necessarily gene specific. Over 20 loci have been linked to these myopathies, including three exceptionally large genes (TTN, NEB and RYR1), which are a challenge for molecular diagnosis. We developed a new approach using massive parallel sequencing (MPS) technology to simultaneously analyze 20 genes linked to CMs. Assay design was based on the Ion AmpliSeq strategy and sequencing runs were performed on an Ion PGM system. A total of 12 patients were analyzed in this study. Among the 2534 variants detected, 14 pathogenic mutations were successfully identified in the DNM2, NEB, RYR1, SEPN1 and TTN genes. Most of these had not been documented and/or fully characterized, hereby contributing to expand the CM mutational spectrum. The utility of this approach was demonstrated by the identification of mutations in 70% of the patients included in this study, which is relevant for CMs especially considering its wide phenotypic and genetic heterogeneity.

Tendon Stem Cell Niche

Tendon stem cells constitute a heterogeneous population of stem and progenitor cells that is involved in tendon formation and healing. The basic understanding of the interactions between tendon cells and their surroundings can enable the development of improved regenerative therapies. In this chapter, the main characteristics of their microenvironment—niche —are reviewed. In particular, the importance of signaling molecules and extracellular matrix (ECM) are highlighted, focusing on their potential role in tendon regeneration.

Human adipose tissue-derived tenomodulin positive subpopulation of stem cells: a promising source of tendon progenitor cells

Cell‐based therapies are of particular interest for tendon and ligament regeneration given the low regenerative potential of these tissues. Adipose tissue is an abundant source of stem cells, which may be employed for the healing of tendon lesions. However, human adult multipotent adipose‐derived stem cells (hASCs) isolated from the stromal vascular fraction of adipose tissue originate highly heterogeneous cell populations that hinder their use in specific tissue‐oriented applications. In this study, distinct subpopulations of hASCs were immunomagnetic separated and their tenogenic differentiation capacity evaluated in the presence of several growth factors (GFs), namely endothelial GF, basic‐fibroblast GF, transforming GF‐β1 and platelet‐derived GF‐BB, which are well‐known regulators of tendon development, growth and healing. Among the screened hASCs subpopulations, tenomodulin‐positive cells were shown to be more promising for tenogenic applications and therefore this subpopulation was further studied, assessing tendon‐related markers (scleraxis, tenomodulin, tenascin C and decorin) both at gene and protein level. Additionally, the ability for depositing collagen type I and III forming extracellular matrix structures were weekly assessed up to 28 days. The results obtained indicated that tenomodulin‐positive cells exhibit phenotypical features of tendon progenitor cells and can be biochemically induced towards tenogenic lineage, demonstrating that this subset of hASCs can provide a reliable source of progenitor cells for therapies targeting tendon regeneration.

Tissue-engineered magnetic cell sheet patches for advanced strategies in tendon regeneration

Tendons are powerful 3D biomechanically structures combining a few cells in an intrincated and highly hierarchical niche environment. When tendon homeostasis is compromised, restoration of functionality upon injury is limited and requires alternatives to current augmentation or replacement strategies. Cell sheet technologies are a powerful tool for the fabrication of living extracellular-rich patches towards regeneration of tenotopic defects. Thus, we originally propose the development of magnetically responsive tenogenic patches through magnetic cell sheet (magCSs) technology that enable the remote control upon implantation of the tendon-mimicking constructs. A Tenomodulin positive (TNMD+) subpopulation of cells sorted from a crude population of human adipose stem cells (hASCs) previously identified as being prone to tenogenesis was selected for the magCSs patch construction. We investigated the stability, the cellular co-location of the iron oxide nanoparticles (MNPs), as well as the morphology and mechanical properties of the developed magCSs. Moreover, the expression of tendon markers and collagenous tendon-like matrix were further assessed under the actuation of an external magnetic field. Overall, this study confirms the potential to bioengineer tendon patches using a magnetic cell sheet construction with magnetic responsiveness, good mechanoelastic properties and a tenogenic prone stem cell population envisioning cell-based functional therapies towards tendon regeneration. STATEMENT OF SIGNIFICANCE The concept of magnetic force-based tissue engineering may assist the development of innovative solutions to treat tendon (or other tissues) disorders upon remote control of biological processes as cell migration or differentiation. Herein, we originally fabricated magnetic responsive cell sheets (magCSs) with a Tenomodulin positive subpopulation of adipose tissue derived stem cells identified to commit to the tenogenic lineage. To the best of authors knowledge, this is the first time a tendon oriented strategy resorting on magCSsis reported. Moreover, the promising role of tenogenic living constructs fabricated as magnetically responsive ECM-rich patches is highlighted, envisioning the stimulation of endogenous regenerative mechanisms. Altogether, these findings contribute to future stem cell studies and their translation toward tendon therapies.

Fabrication of hierarchical and biomimetic fibrous structures to support the regeneration of tendon tissues

Abstract Tendons are connective tissues mainly composed of hierarchically organized extracellular matrix (ECM) whose function is transmitting forces between muscles and bones. Natural healing of tendons is highly ineffective and current repair strategies present numerous limitations. Thus, tissue engineering (TE) may provide alternative therapies for the treatment of tendon injuries. For this purpose, multiple strategies are being developed to fabricate biomimetic materials that recapitulate native tissue microenvironment, especially the tendons hierarchical architecture and biomechanical behavior. Given the fibrous nature of tendon ECM, fibrous biomaterials have been obvious candidates as scaffolds for tendon TE. In this chapter, several fiber-based approaches for tendon scaffolding are reviewed, as well as their assembly into higher hierarchical structures using textile techniques. In particular, the usefulness and particularities of the referred methods are discussed, focusing on their potential for the fabrication of hierarchical and biomimetic materials for tendon regeneration.

Cell-based approaches for tendon regeneration

Abstract Cell-based therapies have been proposed as potential alternative solutions toward efficient regenerative strategies for damaged tendon tissues. These approaches traditionally envision the delivery of cells, mostly stem cells or tendon cells, often combined with carriers and growth factors to provide the behavioral instructions similar to the ones found in native tissues. The purpose of cell-based therapies is to apply living cells to guide and promote enhanced tissue repair and functional regeneration of an injured or diseased tissue. This chapter will focus on the potential of several cell sources, including stem cells from different origins, in tendon regeneration, illustrated by their therapeutic role in animal models of tendinopathies. Furthermore, it will be also addressed experimental cellular therapies currently ongoing in clinical trials that hold the promise for innovative clinical outcomes in tendon regeneration.