The sustained PGE2 release matrix improves neovascularization and skeletal muscle regeneration in a hindlimb ischemia model.

BACKGROUND: The promising therapeutic strategy for the treatment of peripheral artery disease (PAD) is to restore blood supply and promote regeneration of skeletal muscle regeneration. Increasing evidence revealed that prostaglandin E2 (PGE2), a lipid signaling molecule, has significant therapeutic potential for tissue repair and regeneration. Though PGE2 has been well reported in tissue regeneration, the application of PGE2 is hampered by its short half-life in vivo and the lack of a viable system for sustained release of PGE2. RESULTS: In this study, we designed and synthesized a new PGE2 release matrix by chemically bonding PGE2 to collagen. Our results revealed that the PGE2 matrix effectively extends the half-life of PGE2 in vitro and in vivo. Moreover, the PGE2 matrix markedly improved neovascularization by increasing angiogenesis, as confirmed by bioluminescence imaging (BLI). Furthermore, the PGE2 matrix exhibits superior therapeutic efficacy in the hindlimb ischemia model through the activation of MyoD1-mediated muscle stem cells, which is consistent with accelerated structural recovery of skeletal muscle, as evidenced by histological analysis. CONCLUSIONS: Our findings highlight the chemical bonding strategy of chemical bonding PGE2 to collagen for sustained release and may facilitate the development of PGE2-based therapies to significantly improve tissue regeneration.

Nano-Structured Ridged Micro-Filaments (≥100 µm Diameter) Produced Using a Single Step Strategy for Improved Bone Cell Adhesion and Proliferation in Textile Scaffolds.

Textile scaffolds that are either 2D or 3D with tunable shapes and pore sizes can be made through textile processing (weaving, knitting, braiding, nonwovens) using microfilaments. However, these filaments lack nano-topographical features to improve bone cell adhesion and proliferation. Moreover, the diameter of such filaments should be higher than that used for classical textiles (10−30 µm) to enable adhesion and the efficient spreading of the osteoblast cell (>30 µm diameter). We report, for the first time, the fabrication of biodegradable nanostructured cylindrical PLLA (poly-L-Lactic acid) microfilaments of diameters 100 µm and 230 µm, using a single step melt-spinning process for straightforward integration of nano-scale ridge-like structures oriented in the fiber length direction. Appropriate drawing speed and temperature used during the filament spinning allowed for the creation of instabilities giving rise to nanofibrillar ridges, as observed by AFM (Atomic Force Microscopy). These micro-filaments were hydrophobic, and had reduced crystallinity and mechanical strength, but could still be processed into 2D/3D textile scaffolds of various shapes. Biological tests carried out on the woven scaffolds made from these nano-structured micro filaments showed excellent human bone cell MG 63 adhesion and proliferation, better than on smooth 30 µm- diameter fibers. Elongated filopodia of the osteoblast, intimately anchored to the nano-structured filaments, was observed. The filaments also induced in vitro osteogenic expression, as shown by the expression of osteocalcin and bone sialoprotein after 21 days of culture. This work deals with the fabrication of a new generation of nano-structured micro-filament for use as scaffolds of different shapes suited for bone cell engineering.

Interests of Exosomes in Bone and Periodontal Regeneration: A Systematic Review.

Periodontitis is an infectious inflammatory disease characterized by clinical attachment loss and tooth supporting tissue destruction. As exosomes demonstrated pro-regenerative ability, their use in periodontal treatment has been suggested. The aim of this systematic review is to gather and summarize the most recent data regarding exosomes to determine their potential impact in bone and periodontal regeneration. Electronic databases (Pubmed, Web of Science) were searched up to February 2020. Studies assessing the impact of exosomes administration in experimental bone and periodontal defects have been identified according to PRISMA guidelines. Among the 183 identified articles, 16 met the inclusion criteria and were included in this systematic review. Experimental bone defects were mainly surgically induced with a dental bur or distraction tools. All studies considered bone healing after exosomes administration as the primary outcome. Results showed that mesenchymal stem cells derived exosomes administration promoted bone healing and neovascularization. Nevertheless, a dose-effect relationship was observed. Exosomes administration appears to promote significantly the bone healing and periodontal regeneration. However, only a limited number of studies have been carried out so far and the optimized protocols in this context need to be evaluated.

ERBB2b mRNA isoform encodes a nuclear variant of the ERBB2 oncogene in breast cancer.

The presence of nuclear ERBB2 receptor-type tyrosine kinase is one of the causes of the resistance to membrane ERBB2-targeted therapy in breast cancers. It has been previously reported that this nuclear location arises through at least two different mechanisms: proteolytic shedding of the extracellular domain of the full-length receptor and translation of the messenger RNA (mRNA)-encoding ERBB2 from internal initiation codons. Here, we report a new mechanism and function where a significant portion of nuclear ERBB2 results from the translation of the variant ERBB2 mRNA under the transcriptional control of a distal promoter that is actively used in breast cancer cells. We show that both membrane ERBB2a and nuclear ERBB2b isoforms are prevalently expressed in breast cancer cell lines and carcinoma samples. The ERBB2b isoform, which is translated from mRNA variant 2, can directly translocate into the nucleus due to the lack of the signal peptide which is required for an intermediate membrane location. Small interfering RNA-mediated gene silencing showed that ERBB2b can repress ERBB2a expression, encoded by variant 1, whereas ERBB2a activates ERBB2b. Nuclear ERBB2 binding to its own promoter was revealed by chromatin immunoprecipitation assay. Altogether, our results provide new insights into the origin and function of nuclear ERBB2 where it can participate at the same time in a positive or a negative feedback autoregulatory loop, dependent on which of its promoters this bona fide transcription factor is acting. They also provide a new understanding for the resistance to therapies targeting the membrane-anchored ERBB2 in breast cancer.

Assessment of plasma microvesicles to monitor pancreatic islet graft dysfunction: Beta cell- and leukocyte-derived microvesicles as specific features in a pilot longitudinal study.

Markers of early pancreatic islet graft dysfunction and its causes are lacking. We monitored 19 type 1 diabetes islet-transplanted patients for up to 36 months following last islet injection. Patients were categorized as Partial (PS) or complete (S) Success, or Graft Failure (F), using the ß-score as an indicator of graft function. F was the subset reference of maximum worsened graft outcome. To identify the immune, pancreatic, and liver contribution to the graft dysfunction, the cell origin and concentration of circulating microvesicles (MVs) were assessed, including MVs from insulin-secreting ß-cells typified by polysialic acid of neural cell adhesion molecule (PSA-NCAM), and data were compared with values of the ß-score. Similar ranges of PSA-NCAM+ -MVs were found in healthy volunteers and S patients, indicating minimal cell damage. In PS, a 2-fold elevation in PSA-NCAM+ -MVs preceded each ß-score drop along with a concomitant rise in insulin needs, suggesting ß-cell damage or altered function. Significant elevation of liver asialoglycoprotein receptor (ASGPR)+ -MVs, endothelial CD105+ -MVs, neutrophil CD66b+ -MVs, monocyte CD 14+ -MVs, and T4 lymphocyte CD4+ -MVs occurred before each ß-score drop, CD8+ -MVs increased only in F, and B lymphocyte CD19+ -MVs remained undetectable. In conclusion, PSA-NCAM+ -MVs are noninvasive early markers of transplant dysfunction, while ASGPR+ -MVs signal host tissue remodeling. Leukocyte MVs could identify the cause of graft dysfunction.

Osteochondral repair combining therapeutics implant with mesenchymal stem cells spheroids.

Functional articular cartilage regeneration remains challenging, and it is essential to restore focal osteochondral defects and prevent secondary osteoarthritis. Combining autologous stem cells with therapeutic medical device, we developed a bi-compartmented implant that could promote both articular cartilage and subchondral bone regeneration. The first compartment based on therapeutic collagen associated with bone morphogenetic protein 2, provides structural support and promotes subchondral bone regeneration. The second compartment contains bone marrow-derived mesenchymal stem cell spheroids to support the regeneration of the articular cartilage. Six-month post-implantation, the regenerated articular cartilage surface was 3 times larger than that of untreated animals, and the regeneration of the osteochondral tissue occurred during the formation of hyaline-like cartilage. Our results demonstrate the positive impact of this combined advanced therapy medicinal product, meeting the needs of promising osteochondral regeneration in critical size articular defects in a large animal model combining not only therapeutic implant but also stem cells.

Porphyromonas gingivalis Differentially Modulates Apoptosome Apoptotic Peptidase Activating Factor 1 in Epithelial Cells and Fibroblasts.

Porphyromonas gingivalis is able to invade and modulate host-immune response to promote its survival. This bacterium modulates the cell cycle and programed cell death, contributing to periodontal lesion worsening. Several molecular pathways have been identified as key triggers of apoptosis, including apoptosome apoptotic peptidase activating factor 1 (APAF-1). Apaf-1 and X-linked inhibitor of apoptosis protein (Xiap) mRNA were differentially expressed between gingival samples harvested from human healthy and chronic periodontitis tissues (Apaf-1, 19.2-fold; caspase-9, 14.5-fold; caspase-3, 6.8-fold; Xiap: 2.5-fold in chronic periodontitis) (P < 0.05), highlighting their potential role in periodontitis. An increased proteic expression of APAF-1 was also observed in a murine experimental periodontitis model induced by P. gingivalis-soaked ligatures. In vitro, it was observed that P. gingivalis targets APAF-1, XIAP, caspase-3, and caspase-9, to inhibit epithelial cell death at both mRNA and protein levels. Opposite effect was observed in fibroblasts in which P. gingivalis increased cell death and apoptosis. To assess if the observed effects were associated to APAF-1, epithelial cells and fibroblasts were transfected with siRNA targeting Apaf-1. Herein, we confirmed that APAF-1 is targeted by P. gingivalis in both cell types. This study identified APAF-1 apoptosome and XIAP as intracellular targets of P. gingivalis, contributing to the deterioration of periodontal lesion through an increased persistence of the bacteria within tissues and the subversion of host-immune response.

Contribution of Statins towards Periodontal Treatment: A Review.

The pleiotropic effects of statins have been evaluated to assess their potential benefit in the treatment of various inflammatory and immune-mediated diseases including periodontitis. Herein, the adjunctive use of statins in periodontal therapy in vitro, in vivo, and in clinical trials was reviewed. Statins act through several pathways to modulate inflammation, immune response, bone metabolism, and bacterial clearance. They control periodontal inflammation through inhibition of proinflammatory cytokines and promotion of anti-inflammatory and/or proresolution molecule release, mainly, through the ERK, MAPK, PI3-Akt, and NF-κB pathways. Moreover, they are able to modulate the host response activated by bacterial challenge, to prevent inflammation-mediated bone resorption and to promote bone formation. Furthermore, they reduce bacterial growth, disrupt bacterial membrane stability, and increase bacterial clearance, thus averting the exacerbation of infection. Local statin delivery as adjunct to both nonsurgical and surgical periodontal therapies results in better periodontal treatment outcomes compared to systemic delivery. Moreover, combination of statin therapy with other regenerative agents improves periodontal healing response. Therefore, statins could be proposed as a potential adjuvant to periodontal therapy. However, optimization of the combination of their dose, type, and carrier could be instrumental in achieving the best treatment response.

Application of Chitosan in Bone and Dental Engineering.

Chitosan is a deacetylated polysaccharide from chitin, the natural biopolymer primarily found in shells of marine crustaceans and fungi cell walls. Upon deacetylation, the protonation of free amino groups of the d-glucosamine residues of chitosan turns it into a polycation, which can easily interact with DNA, proteins, lipids, or negatively charged synthetic polymers. This positive-charged characteristic of chitosan not only increases its solubility, biodegradability, and biocompatibility, but also directly contributes to the muco-adhesion, hemostasis, and antimicrobial properties of chitosan. Combined with its low-cost and economic nature, chitosan has been extensively studied and widely used in biopharmaceutical and biomedical applications for several decades. In this review, we summarize the current chitosan-based applications for bone and dental engineering. Combining chitosan-based scaffolds with other nature or synthetic polymers and biomaterials induces their mechanical properties and bioactivities, as well as promoting osteogenesis. Incorporating the bioactive molecules into these biocomposite scaffolds accelerates new bone regeneration and enhances neovascularization in vivo.

Combining 2D angiogenesis and 3D osteosarcoma microtissues to improve vascularization.

Angiogenesis is now well known for being involved in tumor progression, aggressiveness, emergence of metastases, and also resistance to cancer therapies. In this study, to better mimic tumor angiogenesis encountered in vivo, we used 3D culture of osteosarcoma cells (MG-63) that we deposited on 2D endothelial cells (HUVEC) grown in monolayer. We report that endothelial cells combined with tumor cells were able to form a well-organized network, and that tubule-like structures corresponding to new vessels infiltrate tumor spheroids. These vessels presented a lumen and expressed specific markers as CD31 and collagen IV. The combination of 2D endothelial cells and 3D microtissues of tumor cells also increased expression of angiogenic factors as VEGF, CXCR4 and ICAM1. The cell environment is the key point to develop tumor vascularization in vitro and to be closer to tumor encountered in vivo.

Temporomandibular Joint Regenerative Medicine.

The temporomandibular joint (TMJ) is an articulation formed between the temporal bone and the mandibular condyle which is commonly affected. These affections are often so painful during fundamental oral activities that patients have lower quality of life. Limitations of therapeutics for severe TMJ diseases have led to increased interest in regenerative strategies combining stem cells, implantable scaffolds and well-targeting bioactive molecules. To succeed in functional and structural regeneration of TMJ is very challenging. Innovative strategies and biomaterials are absolutely crucial because TMJ can be considered as one of the most difficult tissues to regenerate due to its limited healing capacity, its unique histological and structural properties and the necessity for long-term prevention of its ossified or fibrous adhesions. The ideal approach for TMJ regeneration is a unique scaffold functionalized with an osteochondral molecular gradient containing a single stem cell population able to undergo osteogenic and chondrogenic differentiation such as BMSCs, ADSCs or DPSCs. The key for this complex regeneration is the functionalization with active molecules such as IGF-1, TGF-ß1 or bFGF. This regeneration can be optimized by nano/micro-assisted functionalization and by spatiotemporal drug delivery systems orchestrating the 3D formation of TMJ tissues.

Well-organized spheroids as a new platform to examine cell interaction and behaviour during organ development.

We present an experimental method allowing the production of three-dimensional organ-like structures, namely microtissues (MTs), in vitro without the need for exogenous extracellular matrix (ECM) or growth factors. Submandibular salivary glands (embryonic day ED14), kidneys (ED13) and lungs (ED13) were harvested from mouse embryos and dissociated into single cells by enzyme treatment. Single cells were seeded into special hanging drop culture plates (InSphero) and cultured for up to 14 days to obtain MTs. This strategy permitted full control of the quantity of seeded cells. The development of the MTs into organs was followed histologically and immunohistochemically. Well-organized epithelial structures surrounded by a basal lamina were formed, as confirmed by transmission electron microscopy. Expression of E-cadherin, vimentin, fibronectin and α-SMA was compared in organs and corresponding MTs by real-time quantitative polymerase chain reaction. Branching morphogenesis was induced in MTs (as shown by histology and immunostaining for fibronectin and perlecan) and was conserved even after 14 days of culture. MTs continued their development and their epithelial structures were comparable with those of the physiological organ at postnatal day 2 (PN2). Expression of aquaporins was investigated to obtain better support for the functional differentiation of epithelial cells. Histogenesis proceeded and led to the start of organogenesis. This experimental model might improve our knowledge of epithelial-mesenchymal histogenesis and can be employed to study development or cellular organization during the embryonic formation of organs.

iPSCs chondrogenic differentiation for personalized regenerative medicine: a literature review.

Cartilage, an important connective tissue, provides structural support to other body tissues, and serves as a cushion against impacts throughout the body. Found at the end of the bones, cartilage decreases friction and averts bone-on-bone contact during joint movement. Therefore, defects of cartilage can result from natural wear and tear, or from traumatic events, such as injuries or sudden changes in direction during sports activities. Overtime, these cartilage defects which do not always produce immediate symptoms, could lead to severe clinical pathologies. The emergence of induced pluripotent stem cells (iPSCs) has revolutionized the field of regenerative medicine, providing a promising platform for generating various cell types for therapeutic applications. Thus, chondrocytes differentiated from iPSCs become a promising avenue for non-invasive clinical interventions for cartilage injuries and diseases. In this review, we aim to highlight the current strategies used for in vitro chondrogenic differentiation of iPSCs and to explore their multifaceted applications in disease modeling, drug screening, and personalized regenerative medicine. Achieving abundant functional iPSC-derived chondrocytes requires optimization of culture conditions, incorporating specific growth factors, and precise temporal control. Continual improvements in differentiation methods and integration of emerging genome editing, organoids, and 3D bioprinting technologies will enhance the translational applications of iPSC-derived chondrocytes. Finally, to unlock the benefits for patients suffering from cartilage diseases through iPSCs-derived technologies in chondrogenesis, automatic cell therapy manufacturing systems will not only reduce human intervention and ensure sterile processes within isolator-like platforms to minimize contamination risks, but also provide customized production processes with enhanced scalability and efficiency.

Patient-derived tumoroids and proteomic signatures: tools for early drug discovery.

Onco-virotherapy is an emergent treatment for cancer based on viral vectors. The therapeutic activity is based on two different mechanisms including tumor-specific oncolysis and immunostimulatory properties. In this study, we evaluated onco-virotherapy in vitro responses on immunocompetent non-small cell lung cancer (NSCLC) patient-derived tumoroids (PDTs) and healthy organoids. PDTs are accurate tools to predict patient's clinical responses at the in vitro stage. We showed that onco-virotherapy could exert specific antitumoral effects by producing a higher number of viral particles in PDTs than in healthy organoids. In the present work, we used multiplex protein screening, based on proximity extension assay to highlight different response profiles. Our results pointed to the increase of proteins implied in T cell activation, such as IFN-γ following onco-virotherapy treatment. Based on our observation, oncolytic viruses-based therapy responders are dependent on several factors: a high PD-L1 expression, which is a biomarker of greater immune response under immunotherapies, and the number of viral particles present in tumor tissue, which is dependent to the metabolic state of tumoral cells. Herein, we highlight the use of PDTs as an alternative in vitro model to assess patient-specific responses to onco-virotherapy at the early stage of the preclinical phases.

Custom-made meniscus biofabrication.

Reconstructing the meniscus is not currently possible due to its intricate and heterogeneous structure. In this forum, we first discuss the shortcomings of current clinical strategies in meniscus repair. Then, we describe a new promising cell-based, ink-free 3D biofabrication technology to produce tailor-made large-scale functional menisci.

In vitro vascularized immunocompetent patient-derived model to test cancer therapies.

This work describes a patient-derived tumoroid model (PDTs) to support precision medicine in lung oncology. The use of human adipose tissue-derived microvasculature and patient-derived peripheral blood mononuclear cells (PBMCs) permits to achieve a physiologically relevant tumor microenvironment. This study involved ten patients at various stages of tumor progression. The vascularized, immune-infiltrated PDT model could be obtained within two weeks, matching the requirements of the therapeutic decision. Histological and transcriptomic analyses confirmed that the main features from the original tumor were reproduced. The 3D tumor model could be used to determine the dynamics of response to antiangiogenic therapy and platinum-based chemotherapy. Antiangiogenic therapy showed a significant decrease in vascular endothelial growth factor (VEGF)-A expression, reflecting its therapeutic effect in the model. In an immune-infiltrated PDT model, chemotherapy showed the ability to decrease the levels of lymphocyte activation gene-3 protein (LAG-3), B and T lymphocyte attenuator (BTLA), and inhibitory receptors of T cells functions.

Patient-Derived Tumoroid for the Prediction of Radiotherapy and Chemotherapy Responses in Non-Small-Cell Lung Cancer.

Radiation therapy and platinum-based chemotherapy are common treatments for lung cancer patients. Several factors are considered for the low overall survival rate of lung cancer, such as the patient's physical state and the complex heterogeneity of the tumor, which leads to resistance to the treatment. Consequently, precision medicines are needed for the patients to improve their survival and their quality of life. Until now, no patient-derived tumoroid model has been reported to predict the efficiency of radiation therapy in non-small-cell lung cancer. Using our patient-derived tumoroid model, we report that this model could be used to evaluate the efficiency of radiation therapy and cisplatin-based chemotherapy in non-small-cell lung cancer. In addition, these results can be correlated to clinical outcomes of patients, indicating that this patient-derived tumoroid model can predict the response to radiotherapy and chemotherapy in non-small-cell lung cancer.

Patient-Derived Lung Tumoroids-An Emerging Technology in Drug Development and Precision Medicine.

Synthetic 3D multicellular systems derived from patient tumors, or tumoroids, have been developed to complete the cancer research arsenal and overcome the limits of current preclinical models. They aim to represent the molecular and structural heterogeneity of the tumor micro-environment, and its complex network of interactions, with greater accuracy. They are more predictive of clinical outcomes, of adverse events, and of resistance mechanisms. Thus, they increase the success rate of drug development, and help clinicians in their decision-making process. Lung cancer remains amongst the deadliest of diseases, and still requires intensive research. In this review, we analyze the merits and drawbacks of the current preclinical models used in lung cancer research, and the position of tumoroids. The introduction of immune cells and healthy regulatory cells in autologous tumoroid models has enabled their application to most recent therapeutic concepts. The possibility of deriving tumoroids from primary tumors within reasonable time has opened a direct approach to patient-specific features, supporting their future role in precision medicine.

Nanomedicine and Periodontal Regenerative Treatment.

Current periodontal treatments aim to control bacterial infection and decrease inflammation. To optimize contemporary conventional treatments that present limitations owing to an inability to reach the lesion site, new methods are based on nanomedicine. Nanomedecine allows delivery of host-modulatory drugs or antibacterial molecules at the lesion site in an optimal concentration with decreased toxicity and risk of systemic side effects. Chitosan and polylactic-co-glycolic acid-loaded nanoparticles, carbon quantum dots, and mesoporous silicates open new perspectives in periodontitis management. The potential therapeutic impact of the main nanocarriers is discussed.

M101, a therapeutic oxygen carrier derived from Arenicola marina, decreased Porphyromonas gingivalis-induced hypoxia and improved periodontal healing.

BACKGROUND: Porphyromonas gingivalis exacerbates tissue hypoxia and worsens periodontal inflammation. This study investigated the effect of a therapeutic oxygen carrier (M101), derived from Arenicola marina, on hypoxia and associated inflammation in the context of periodontitis. METHODS: The effect of M101 on GLUT-1, GLUT-3, HIF-1α, and MMP-9 expression, hypoxia, and antioxidant status in oral epithelial cells (EC) exposed to CoCl2 (1000 µM), P. gingivalis (MOI 100), and CoCl2 + P. gingivalis was evaluated through hypoxia detection fluorescence assay, antioxidant concentration colorimetric assay, and RTqPCR. Evaluation of M101 on EC proliferation was evaluated in an in vitro wound assay. In experimental periodontitis, periodontal wound healing and osteoclastic activity were compared among natural wound healing, placebo, and gels containing M101 (1  and 2 g/L) groups through histomorphometry and TRAP (tartrate-resistant acid phosphatase activity assay) assay respectively. The expression of HIF-1α, MMP-9, and NFκB in periodontal tissues was also evaluated through immunofluorescence studies. RESULTS: M101 downregulated GLUT-1, GLUT-3, HIF-1α, and MMP-9 levels in EC exposed to CoCl2 , P. gingivalis, and CoCl2 + P. gingivalis (p < 0.05). Fluorescence and colorimetric analyses confirmed hypoxia reduction and antioxidant capacity improvement in such EC upon M101 treatment. Moreover, M101 improved significantly the in vitro wound closure. In vivo, the attachment level was significantly improved, and osteoclastic activity was reduced in mice treated with M101 gels compared to placebo and natural wound healing groups (p < 0.05). HIF-1α, MMP-9, and NFκB expression in periodontal tissues was reduced in M101 gels treated mice compared to the controls. CONCLUSION: M101 showed promise in resolving hypoxia and associated inflammation-mediated tissue degradation. Its potential in the clinical management of periodontitis must be further investigated.