Regeneration of bone defects in elderly patients is limited due to the decreased function of bone forming cells and compromised tissue physiology. Previous studies suggested that the regenerative activity of stem cells from aged tissues can be enhanced by exposure to young systemic and tissue microenvironments. The aim of our project was to investigate whether extracellular matrix (ECM) engineered from human induced pluripotent stem cells (hiPSCs) can enhance the bone regeneration potential of aged human bone marrow stromal cells (hBMSCs). ECM was engineered from hiPSC-derived mesenchymal-like progenitors (hiPSC-MPs), as well as young (<30 years) and aged (>70 years) hBMSCs. ECM structure and composition were characterized before and after decellularization using immunofluorescence and biochemical assays. Three hBMSCs of different ages were cultured on engineered ECMs. Growth and differentiation responses were compared to tissue culture plastic, as well as to collagen and fibronectin coated plates. Decellularized ECMs contained collagens type I and IV, fibronectin, laminin and < 5% residual DNA, suggesting efficient cell elimination. Cultivation of young and aged hBMSCs on the hiPSC-ECM in osteogenic medium significantly increased hBMSC growth and markers of osteogenesis, including collagen deposition, alkaline phosphatase activity, bone sialoprotein expression and matrix mineralization compared to plastic controls and single protein substrates. In aged BMSCs, matrix mineralization was only detected in ECM cultures in osteogenic medium. Comparison of ECMs engineered from hiPSC-MPs and hBMSCs of different ages suggested similar structure, composition and potential to enhance osteogenic responses in aged BMSCs. Engineered ECM induced a higher osteogenic response compared to specific matrix components. Our studies suggest that aged BMSCs osteogenic activity can be enhanced by culture on engineered ECM. hiPSCs represent a scalable cell source, and tissue engineering strategies employing engineered ECM materials could potentially enhance bone regeneration in elderly patients

Introduction and Objective. Guided Bone Regeneration (GBR) uses biodegradable collagen membranes of animal origin tissues (dermis and pericardium). Their barrier effect prevents soft tissues to interfere with the regeneration of alveolar bone. However, their xenogeneic origin involves heavy chemical treatments which impact their bioactivity. Wharton's Jelly (WJ) from the umbilical cord is a recoverable surgery waste. WJ is mostly made from collagen fibers, proteoglycans, hyaluronic acid, and growth factors. WJ with immunologically privileged status and bioactive properties lends credence to its use as an allograft. Nevertheless, low mechanical properties limit its use in bone regenerative strategies. Herein, our objective is to develop a crosslinked WJ-based membrane to improve its strength and thus its potential use as a GBR membrane. Materials and Methods. The umbilical cords are collected after delivery and then stored at −20°C until use. The WJ membranes (1 × 5 × 12 mm) were obtained after the removal of blood vessels and amniotic tissue, washed, lyophilized, and stored at −20°C. WJ membranes were incubated in genipin solutions in decreasing concentrations (0.3 g / 100 mL − 0.03 g / 100 mL) for 24 hours at 37°C. The crosslinking degree was estimated by ninhydrin and confirmed by FTIR (Fourier-transform infrared spectroscopy) assays. The swelling rate was obtained after the rehydration of dry crosslinked WJ-membrane for 10 min in D-PBS. The mechanical properties were assessed in hydrated conditions on a tensile bench. The resistance to the degradation was evaluated by collagenase digestion (1 mg/mL for 60 hours) assay. The cytotoxicity of crosslinked WJ-membrane was evaluated in accordance with the standard ISO.10993-5 (i.e. Mitochondrial activity and Lactate Dehydrogenase release) against Mesenchymal Stem Cells (MSCs). Finally, the MSCs colonization and proliferation were followed after 21 days of culture on crosslinked WJ-membranes. Results. The increase of crosslinking rates from 30% to 90% of the WJ membrane was demonstrated by the ninhydrin assay. FTIR analysis showed a prominent peak at 1732 cm. -1. , confirming the incorporation of genipin in the WJ. The swelling rate of crosslinked WJ-membrane decreased with an increase of the crosslinking rate. An increase in elastic modulus and an increase in the resistance to the collagenase degradation were observed along with an increase in the crosslinking degree. Cytotoxicity investigations did not elicit a harmful effect of the genipin, however, a poor MSCs adhesion on the crosslinked membrane was observed. Conclusions. Our results show that a membrane can be developed from Wharton's jelly. The mechanical and degradation properties can be improved by crosslinking with genipin without inducing any cytotoxicity effect. However, the percentage of crosslinking has an influence on the adhesion of the cells to the membranes. The crosslinked WJ-membrane bioactivity and the osteo-regenerative potential in vitro/in vivo will be evaluate

Regeneration of bone defects in elderly patients is limited due to the decreased function of bone forming cells and compromised tissue physiology. Previous studies suggested that the regenerative activity of stem cells from aged tissues can be enhanced by exposure to young systemic and tissue microenvironments. The aim of our project was to investigate whether extracellular matrix (ECM) engineered from human induced pluripotent stem cells (hiPSCs) can enhance the bone regeneration potential of aged human bone marrow stromal cells (hBMSCs). ECM was engineered from hiPSC-derived mesenchymal-like progenitors (hiPSC-MPs), as well as young (70 years) hBMSCs. ECM structure and composition were characterized before and after decellularization using immunofluorescence and biochemical assays. Three hBMSCs of different ages were cultured on engineered ECMs. Growth and differentiation responses were compared to tissue culture plastic controls. Decellularized ECMs contained collagens type I and IV, fibronectin, laminin and < 5% residual DNA. Cultivation of young and aged hBMSCs on the hiPSC-ECM in osteogenic medium significantly increased hBMSC growth and markers of osteogenesis, including collagen deposition, alkaline phosphatase activity, bone sialoprotein expression and matrix mineralization compared to plastic controls. In aged BMSCs, matrix mineralization was only detected in ECM cultures in osteogenic medium. Comparison of ECMs engineered from hiPSC-MPs and hBMSCs of different ages suggested similar structure, composition and potential to enhance osteogenic responses in aged BMSCs. Our studies suggest that aged BMSCs regenerative activity can be enhanced by culture on hiPSC-engineered ECM

In the treatment of bone non-unions an alternative to bone autografts is the use of bone morphogenetic proteins (BMP-2, BMP-7) with powerful osteoinductive and osteogenic properties. In clinical settings, BMPs are applied using absorbable collagen sponges. Supraphysiological doses are needed and major side effects may occur as induce ectopic bone formation, chronic inflammation and excessive bone resorption. In order to increase the efficiency of the delivered for BMPs we designed cryostructured collagen scaffolds functionalized with hydroxyapatite, mimicking the structure of cortical bone (aligned porosity, anisotropic, ANI) or trabecular bone (random distributed porosity, isotropic, ISO). We hypothesize that anisotropic structure would enhance osteoconductive properties of the scaffolds increasing rhBMP-2 regenerative properties. In vitro, both scaffolds presented similar mechanical properties, rhBMP-2 retention and delivery capacity. For in vivo testing, a rat femoral critical size defect model was created. Four groups were assessed depending on the implant applied to the bone defect: ISO, unloaded isotropic sponge; ISO-BMP, isotropic sponge loaded with 5 μg of hrBMP-2; ANI, unloaded anisotropic sponge; and ANI-BMP, anisotropic sponge loaded with 5 μg of hrBMP-2. Regeneration was allowed for 10 weeks. X-ray, μCT, biomechanical testing and histology were used to evaluate repair. Independently of their structure, sponges loaded with rhBMP-2 demonstrate increased bone volume, and biomechanical properties than their controls (p<0.01 and p<0.05 respectively). Globally, ANI-BMP group demonstrated better bone regeneration outputs with increased defect bridging (p<0.05 when compared ANI-BMP vs ISO-BMP groups). In conclusion, anisotropic cryostructured collagen scaffolds improve the efficiency of rhBMP-2 in bone regeneration

Amniotic membrane (AM) and amnion/chorion foetal membranes (ACM) are mainly composed of collagen & laminin layers and constitute relatively new materials to the dental market. They have proven effective for periodontal treatments such as Guided Tissue Regeneration (GTR) [1–3]. Based on our expertise in the field of lyophilisation & securisation of human bone allograft (Phoenix® process), we aimed to develop our own process applied to ACM and to control its in vivoefficacy in GTR indication. Human placentas were donated under informed consent. ACM were separated from placenta and processed with a proprietary AMTRIX (TBF) Process. Resulting product was called ACMTRIX. The effectiveness of ACMTRIX in GTR was evaluated using an in vivorat calvaria defect model as followed:. Empty defect (2 animals),. ACMTRIX apposed onto the defect (4 animals),. 3 Bone substitutes (allogenic – mineralized cortical bone powder (Phoenix®); demineralized cancellous bone powder mixed with hydroxyapatite and demineralized bone matrix (DBM) cancellous block) filled in the defect and covered by ACMTRIX (4 animals). One animal per study group was sacrificed after 8 weeks, all others after 8 weeks. Evaluations were performed by: macroscopic observations, X Ray micro-CT, and histological analysis. For all groups using ACMTRIX, no major sign of inflammation were observed macroscopically and histologically. Moreover, bone tissue was already mature from 8 weeks and bone filling was slight to moderate. The higher mean rate of mineralization was obtained for the group associating DBM cancellous block + ACMTRIX. Although a xenogenic material, ACMTRIX was very well integrated without significant inflammatory reaction compared to empty defect and fully integrated in subcutaneous area. The mineralization was superior with DBM cancellous block probably thanks to the stabilization of the material in the defect. Used alone, ACMTRIX has no osteogenic potential. In conclusion, ACMTRIX has the potential to function as barrier for GTR and the unique properties associated with this material can augment its potential as a matrix for periodontal regeneration

Stem cell therapy for the intervertebral disc (IVD) is highly debated but holds great promises. From previous studies, it is known that notochordal cells are highly regenerative and may stimulate other differentiated cells to produce more matrix. Lately, a particular tissue-specific progenitor cell population has been identified in the centre of the intervertebral disc (IVD. The current hope is that these nucleus pulposus progenitor cells (NPPC) could play a particular role in IVD regeneration.

Current evidence confirms the presence of these cells in murine, canine, bovine and in the human fetal/surgical samples. Noteworthy, one of the main markers to identify these cells, i.e., Tie2, is a typical marker for endothelial cells. Thus, it is not very clear what their origin and their role might be in the context of developmental biology. In human surgical specimens, their presence is, even more, obscured depending on the donor's age and the condition of the IVD and other yet unknown factors.

Here, I revisit the recent literature on regenerative cells identified for the IVD in the past decades. Current evidence how these NPPC can be isolated and detected in various species and tissues will be recapitulated. Future directions will be provided on how these progenitor cells could be used for regenerative medicine and tissue engineering.

Despite the major advances in osteosynthesis after trauma, there remains a small proportion of patients (<10%) who exhibit delayed healing and/or eventual progression to non-union. While known risk factors exist, e.g. advanced age or diabetes, the exact molecular mechanism underlying the impaired healing is largely unknown and identifying which specific patient will develop healing complications is still not possible in clinical practice. The talk will cover our novel multimodal approaches in small animals, which have the potential to precisely capture and understand biological changes during fracture healing on an individual basis. Via combining emerging omics technologies with our recently developed femur defect loading equipment in mice, we provide a platform to precisely link mechanical and molecular analyses during fracture healing.

Bone regeneration is an area of acute medical need, but its clinical success is hampered by the need to ensure rapid vascularization of osteogenic grafts. Vascular Endothelial Growth Factor (VEGF) is the master regulator of vascular growth and during bone development angiogenesis and osteogenesis are physiologically coupled through so-called angiocrine factors produced by blood vessels. However, how to exploit this process for therapeutic bone regeneration remains a challenge (1).

Here we will describe recent work aiming at understanding the cross-talk between vascular growth and osteogenesis under conditions relevant for therapeutic bone regeneration. To this end we take advantage of a unique platform to generate controlled signalling microenvironments, by the covalent decoration of fibrin matrices with tunable doses and combinations of engineered growth factors. The combination of human osteoprogenitors and hydroxyapatite in these engineered fibrin matrices provides a controlled model to investigate how specific molecular signals regulate vascular invasion and bone formation in vivo. In particular, we found that:

In conclusion, vascularization of osteogenic grafts is not simply necessary in order to enable progenitor survival. Rather, blood vessels can actively stimulate bone regeneration in engineered grafts through specific molecular signals that can be harnessed for therapeutic purposes.

Acknowledgements: This work was supported in part by the European Union Horizon 2020 Program (Grant agreement 874790 – cmRNAbone).

For any figures and tables, please contact the authors directly.

For chondral damage in younger patients, surgical best practice is microfracture, which involves drilling into the bone to liberate the bone marrow. This leads to a mechanically inferior fibrocartilage formed over the defect as opposed to the desired hyaline cartilage that properly withstands joint loading. While some devices have been developed to aid microfracture and enable its use in larger defects, fibrocartilage is still produced and there is no clear clinical improvement over microfracture alone in the long term. Our goal is to develop 3D printed devices, which surgeons can implant with a minimally invasive technique. The scaffolds should match the functional properties of cartilage and expose endogenous marrow cells to suitable mechanobiological stimuli in-situ, in order to promote healing of articular cartilage lesions before they progress to osteoarthritis, and rapidly restore joint health and mobility. Importantly, scaffolds should direct a physiological host reaction, instead of a foreign body reaction, associated with chronic inflammation and fibrous capsule formation, negatively influencing the regenerative outcome.

Our novel silica/polytetrahydrofuran/polycaprolactone hybrids were prepared by sol-gel synthesis and scaffolds were 3D printed by direct ink writing. 3D printed hybrid scaffolds with pore channels of ~250 µm mimic the compressive behaviour of cartilage. Our results show that these scaffolds support human bone marrow stem/stromal cell (hMSC) differentiation towards chondrogenesis in vitro under hypoxic conditions to produce markers integral to articular cartilage-like matrix evaluated by immunostaining and gene expression analysis. Macroscopic and microscopic evaluation of subcutaneously implanted scaffolds in mice showed that scaffolds caused a minimal resolving inflammatory response. Our findings show that 3D printed hybrid scaffolds have the potential to support cartilage regeneration.

Acknowledgements: Authors acknowledge funding provided by EPSRC grant EP/N025059/1.

Bone tissue is known to possess an intrinsic regeneration potential. However, in cases of major injury, trauma, and disease, bone loss is present, and the regeneration potential of the tissue is often impaired. The process of bone regeneration relies on a complex interaction of molecules. MicroRNAs (miRNA) are small, non-coding RNAs that inhibit messenger RNAs (mRNA). One miRNA can inhibit several mRNAs and one mRNA can be inhibited by several miRNAs. Functionally, miRNAs regulate the entire proteome via the local inhibition of translation. In fact, miRNA modulation has been shown to be involved in several musculoskeletal diseases¹. In those pathologies, they modulate the transcriptional activity of mRNAs important for differentiation, tissue-specific activity, extracellular matrix production, etc. Because of their function in inhibiting translation, miRNAs are being researched in many diseases and are already being used for interventional treatment². Bone tissue and its related conditions have been widely investigated up to this day^1,3. This talk will focus on the relevancy of miRNAs to bone tissue, its homeostasis, and disease. After, examples will be given of how miRNAs can be used in bone regeneration and diseases such as osteoporosis and osteosarcoma. The use of miRNAs in both, detection and therapy will be discussed.

The aim of the ongoing projects was to demonstrate the efficacy of autologous bone marrow derived stem cells (MSC) combined with biomaterial to induced new bone formation in a randomized multicenter controlled clinical trial.

Patients with a need for bone reconstruction of residual edentulous ridges in both the mandible and maxilla due to bone defects with a vertical loss of alveolar bone volume and/or knife edge ridges (≤ than 4,5 mm) unable to provide adequate primary stabilization for dental implants were included in the clinical study. Autologous bone marrow MSC were expanded, loaded on BCP and used to augment the alveolar ridges. After five months bone biopsies were harvested at the implant position site and implants were installed in the regenerated bone. The implants were loaded after 8–12 weeks. Safety, efficacy, quality of life and success/survival were assessed. Five clinical centers, 4 different countries participated. Bone grafts harvested from the ramus of the mandibles were used as control in the projects.

The use of mesenchymal stem cell (MSCs) for intervertebral disc (IVD) regeneration has been extensively explored in the last two decades. MSCs are potent cell types that can be easily and safely harvested due to their abundancy and availability. Moreover, they are characterized by the capacity to differentiate towards IVD cells as well as release growth factors to support resident cell metabolism and recruit local progenitor cells to induce endogenous repair of degenerated IVDs. This talk will outline the characteristics of the main MSC sources and their effect towards IVD regeneration based on available preclinical and clinical evidence. In addition, innovative aspects of MSC-derived cell-free therapies will also be discussed.

Developments in the field of additive manufacturing have allowed significant improvements in the design and production of scaffolds with biologically relevant features to treat bone defects. Unfortunately, the workflow to generate personalized scaffolds is source of inaccuracies leading to a poor fit between the implant and patients' bone defects. In addition, scaffolds are often brittle and fragile, uneasing their handling by surgeons, with significant risks of fracture during their insertion in the defect. Consequently, we developed organo-mineral cementitious scaffolds displaying evolutive mechanical properties which are currently being evaluated to treat maxillofacial bone deformities in veterinary clinics. Treatment of dog patients was approved by ethic and welfare committees (CERVO-2022-14-V). To date, 8 puppies with cleft palate/lip deformities received the following treatment. Two weeks prior surgery, CT-scan of patient's skull was performed to allow for surgical planning and scaffold designing. Organo-mineral printable pastes were formulated by mixing an inorganic cement precursor (α-Ca3(PO4)2) to a self-reticulating hydrogel (silanized hyaluronic acid) supplemented with a viscosifier (hydroxymethylpropylcellulose). Scaffolds were produced by robocasting of these pastes. Surgical interventions included the reconstruction of soft tissues, and the insertion of the scaffold soaked with autologous bone marrow. Bone formation was monitored 3 and 6 months after reconstruction, and a biopsy at 6 months was performed for more detailed analyses. Scaffolds displayed great handling properties and were inserted within bone defects without significant issue with a relevant bone edges/scaffold contact. Osteointegration of the scaffolds was observed after 3 months, and regeneration of the defect at 6 months seemed quite promising. Preliminary results have demonstrated a potential of the set-up strategy to treat cleft lip/palate deformities in real, spontaneous clinical setting. Translation of these innovative scaffolds to orthopedics is planned for a near future.

Our musculoskeletal system has a limited capacity for repair. This has led to increased interest in the development of tissue engineering and biofabrication strategies for the regeneration of musculoskeletal tissues such as bone, ligament, tendon, meniscus and articular cartilage. This talk will demonstrate how different musculoskeletal tissues, specifically cartilage, bone and osteochondral defects, can be repaired using emerging biofabrication and 3D bioprinting strategies. This will include examples from our lab where cells and/or growth factors are bioprinted into constructs that can be implanted directly into the body, to approaches where biomimetic tissues are first engineered in vitro before in vivo implantation. The efficacy of these different biofabrication strategies in different preclinical studies will be reviewed, and lessons from the relative successes and failures of these approaches to tissue regeneration will be discussed.

Growth factors produced by inflammatory cells and mesenchymal progenitors are required for proper bone regeneration. Signaling pathways activated downstream of these proteins work in concert and synergistically to drive osteoblast and/or chondrocyte differentiation. While dysregulation can result in abnormal healing, activating these pathways in the correct spatiotemporal context can enhance healing. Bone morphogenetic protein (BMP) signaling is well-recognized as being required for bone regeneration, and BMP is used clinically to enhance bone healing. However, it is imperative to develop new therapeutics that can be used alone or in conjunction with BMP to drive even more robust healing. Notch signaling is another highly conserved signaling pathway involved in tissue development and regeneration. Our work has explored Notch signaling during osteoblastogenesis and bone healing using both in vitro studies with human primary mesenchymal progenitor cells and in vivo studies with genetically modified mouse models. Notch signaling is required and sufficient for osteoblast differentiation, and is required for proper bone regeneration. Indeed, intact Notch signaling through the Jagged-1 ligand is required for BMP induced bone formation. On-going work continues to explore the intersection between BMP and Notch signaling, and determining cell types that express Notch receptors and Notch ligands during bone healing. Our long-term objective is to develop Notch signaling as a clinical therapy to repair bone.

Electrospinning is an advantageous technique for cartilage tissue engineering (CTE) applications due to its ability to produce nanofibers recapitulating the size and alignment of the collagen fibers present within the articular cartilage superficial zone. Moreover, coaxial electrospinning allows the fabrication of core-shell fibers able to encapsulate and release bioactive molecules in a sustained manner. Kartogenin (KTG) is a small heterocyclic molecule, which was demonstrated to promote the chondrogenic differentiation of human bone marrow-derived mesenchymal stem/stromal cells(hBMSCs)[1].

In this work, we developed and evaluated the biological performance of core-shell poly(glycerol sebacate)(PGS)/poly(caprolactone)(PCL) aligned nanofibers (core:PGS/shell:PCL) mimicking the native articular cartilage extracellular matrix(ECM) and able to promote the sustained release of the chondroinductive drug KTG[2].

The produced coaxial aligned PGS/PCL scaffolds were characterized in terms of their structure and fiber diameter, chemical composition, thermal properties, mechanical performance under tensile testing and in vitro degradation kinetics, in comparison to monoaxial PCL aligned fibers and respective non-aligned controls. KTG was incorporated into the core PGS solution to generate core-shell PGS-KTG/PCL nanofibers and its release kinetics was studied by HPLC analysis. KTG-loaded electrospun aligned scaffolds capacity to promote hBMSCs chondrogenic differentiation was evaluated by assessing cell proliferation, typical cartilage-ECM production (sulfated glycosaminiglycans(sGAG)) and chondrogenic marker genes expression in comparison to non-loaded controls. All the scaffolds fabricated showed average fiber diameters within the nanometer-scale and the core-shell structure of the fibers was clearly confirmed by TEM. The coaxial PGS-KTG/PCL nanofibers evidenced a more sustained drug release over 21 days. Remarkably, in the absence of the chondrogenic cytokine TGF-β3, KTG-loaded nanofibers promoted significantly the proliferation and chondrogenic differentiation of hBMSCs, as suggested by the increased cell numbers, higher sGAG amounts and up-regulation of the chondrogenic genes COL2A1, Sox9, ACAN and PRG4 expression. Overall, our results highlight the potential of core-shell PGS-KTG/PCL aligned nanofibers for the development of novel MSC-based CTE strategies.

Acknowledgements: The authors thank FCT for funding through the project InSilico4OCReg (PTDC/EME-SIS/0838/2021) and to institutions iBB (UID/BIO/04565/2020) and Associate Laboratory I4HB (LA/P/0140/2020).

Introduction

Tendon ruptures represent one of the most common acute tendon injuries in adults worldwide, affecting millions of people anually and becoming more prevalent due to longer life expectancies and sports activities. Current clinical treatments for full tears are unable to completely restore the torn tendons to their native composition, structure and mechanical properties.

To address this clinical challenge, tissue-engineered substitutes will be developed to serve as functional replacements for total tendon ruptures that closely resemble the original tissue, restoring functionality.

Low back pain resulting from Interertebral disc (IVD) degeneration is a serious worldwide problem, with poor treatment options available. Notochordal (NC) cells, are a promising therapeutic cell source with anti-catabolic and regenerative effect. However, their behaviour in the harsh degenerate environment is unknown.

Porcine NC cells (pNCs), and Human NP cells from degenerate IVDs were cultured in alginate beads to maintain phenotype. Cells were cultured alone or in combination, or co-stimulated with notochordal cell condition media (NCCM), in media to mimic the healthy and degenerate disc environment, together with controls for up to 1 week. Following culture viability, qPCR and proteomic analysis using Digiwest was performed.

A small increase in pNC cell death was observed in degenerated media compared to standard and healthy media, with a further decrease seen when cultured with IL-1β. Whilst no significant differences were seen in phenotypic marker expression in pNCs cultured in any media at gene level (ACAN, KRT8, KRT18, FOXA2, COL1A1 and Brachyury). Preliminary Digiwest analysis showed increased protein production for Cytokeratin 18, src and phosphorylated PKC but a decrease in fibronectin in degenerated media compared to standard media. Human NP cells cultured with NCCM, showed a decrease in IL-8 production compared to human NP cells alone when cultured in healthy media. However, gene expression analysis (ACAN, VEGF, MMP3 and IL-1β) demonstrated no significant difference between NP only and NP+NCCM groups.

Studying the behaviour of the NCs in in vitro conditions that mimic the in vivo healthy or degenerate niche will help us to better understand their potential for therapeutic approaches. The potential use of NC cell sources for regenerative therapies can then be translated to investigate the potential use of iPSCs differentiated into NC cells as a regenerative cell source.

Stem cells are known to have low levels of intracellular reactive oxygen species (ROS) and high levels of glutathione. ROS are thought to interact with several pathways that affect the transcription machinery required for stem cell differentiation, and are critical for maintaining stem cell function. In this study, we are developing a new fluorescent probe that rapidly and reversibly reacts with glutathione (GSH), the most abundant non-protein thiol in living cells that acts as an antioxidant and redox regulator.

Multipotent perivascular progenitor cells derived from human ESCs (hESC-PVPCs): Differentiated ESCs as embryoid bodies in the presence of BMP4 to induce mesoderm differentiation followed by a simple cell selection strategy using attachment of single cells onto collagen-coated dishes. Differential gene expression profiling was performed among H9 hESCs, EBs induced by BMP4 and naturally selected CD140B+CD44+ population at Day 7 (PVPCs). Colony-forming assay: GSHhigh and GSHlow PVPCs were plated on 10-cm tissue culture-treated polystyrene dishes in triplicate in growth medium and cultured for 14 days. Transwell migration assay: GSHhigh and GSHlow PVPCs at passage 4 were resuspended at 1 × 10⁶/mL in the migration medium and seeded in the upper chamber. The following human recombinant SDF-1 and PDGF-AA proteins were used as chemoattractants in the lower compartment.

Probe-GSH conjugate shows shifts in fluorescence excitation and emission spectra that enables ratiometric measurement of GSH levels. Using these properties, stem cells can be purified by FACS-based technology according to intracellular GSH level. We are developing a protocol both for comparing GSH level in stem cell from different culture conditions and for preparing stem cells with high-GSH level . Our results reveal that GSHhigh PVPC purified by FACS show increased colony forming ability compared with that GSHlow PVPC, indicating that intracellular GSH contributes to the maintenance of stemness. Moreover, transplantation of GSHlow PVPC is more effective than that of GSHlow PVPC for cartilage regeneration in osteochondral defect.

This technique enable FACS-based sorting of stem cells according to intracellular GSH levels and thus investigation of functional role of GSH (high antioxidant capacity) in the stem cell maintenance and chondrogenic differentiation.

Intervertebral disc (IVD) degeneration occurs with aging, leading to low back pain (LBP), which is one of the leading conditions of disability worldwide. With the lack of effective treatment, decellularized extracellular matrix (dECM) – based biomaterials have been proposed for IVD regeneration. However, the impact of donor ages on tissue repair had never been explored before in the disc field. Therefore, we aimed to address this question.

For that, a decellularization protocol for bovine nucleus pulposus (NP) of different aged donors (fetus, young and old) was optimized by testing several detergents (SDS and Triton). The process efficiency was evaluated in terms of DNA and cell removal, as well as ECM preservation. Afterwards, dECMs were repopulated with bovine NP cells and cultured ex vivo. At day 7, cell behavior, ECM de novo synthesis and remodeling were evaluated [1]. Moreover, dECMs’ inflammatory response was assessed after in vivo CAM assay. Finally, inflammatory and angiogenic cytokines were analyzed in the conditioned media-derived from dECMs by using a cytokine array.

As results, an optimal decellularization protocol (SDS 0.1%, 1h), efficient at removing cells and DNA from bovine NPs, while preserving ECM cues of native tissues, was developed. After repopulation, aggrecan increased in younger NPs, while collagen 2 decreased which may be indicative of matrix remodeling [1]. After in vivo CAM assay, fetal dECMs showed the highest inflammatory response. Finally, no statistically significant changes of cytokines were detected in the matrices, despite for a trend of higher IFN-α, IFN-γ and LIF in fetal dECMs, IL-1β in young dECMs and Decorin in old dECMs.

Overall, this work uncovered the importance of tissue donor ages for tissue regenerative purpose, opening new avenues for the development of appropriate therapeutic strategies for IVD degeneration.

Acknowledgments: FCT, EUROSPINE, ON Foundation.