Biomimetic antibacterial pro-osteogenic cu-sericin MOFs for osteomyelitis treatment

Osteomyelitis is an inflammation of the bone caused by bacterial infection. It usually develops from broken bones, decayed teeth, or heavily punctured wounds. Multi-drug-resistant bacteria are the major hurdle in the treatment of osteomyelitis. The ever-rising antibiotic resistance even leads to amputations or fatalities as a consequence of chronic osteomyelitis. Hence, a single agent with antibacterial activity as well as bone regenerative properties can serve as a potential off-the-shelf product in the treatment of osteomyelitis. Herein, the antibacterial and pro-osteogenic characteristics of copper sericin (Cu-SER) metal–organic frameworks (MOFs) are reported. Sericin, a silk protein with antibacterial activity and an osteoinduction property, acts as an organic template for the deposition of Cu-SER MOFs, similar to collagen during biomineralization in bone. The MOFs exhibit cytocompatibility and osteogenic activity in a dose-dependent manner, as revealed by cell proliferation (alamarBlue) and mineralization (Alizarin Red S and Energy Dispersive X-ray analysis). The bactericidal activity of Cu-SER MOFs was investigated by scanning electron microscopy and a growth kinetic analysis. Together, the report illuminates the unique phenomenon of Cu-SER MOFs that kill bacteria upon contact while being well-tolerated by primary human cells. Hence, Cu-SER MOFs hold the potential to minimize antibiotic dependence.This work is supported by the European Union Framework Programme for Research and Innovation Horizon 2020 (No. 668983—FoReCaST) and the BREAST-IT project (PTDC/BTM-ORG/28168/2017) to S.C.K., funded by the Programa Operacional Regional do Norte supported by European Regional Development Funds (ERDF), Portugal

In vitro cancer models: a closer look at limitations on translation

In vitro cancer models are envisioned as high-throughput screening platforms for potential new therapeutic discovery and/or validation. They also serve as tools to achieve personalized treatment strategies or real-time monitoring of disease propagation, providing effective treatments to patients. To battle the fatality of metastatic cancers, the development and commercialization of predictive and robust preclinical in vitro cancer models are of urgent need. In the past decades, the translation of cancer research from 2D to 3D platforms and the development of diverse in vitro cancer models have been well elaborated in an enormous number of reviews. However, the meagre clinical success rate of cancer therapeutics urges the critical introspection of currently available preclinical platforms, including patents, to hasten the development of precision medicine and commercialization of in vitro cancer models. Hence, the present article critically reflects the difficulty of translating cancer therapeutics from discovery to adoption and commercialization in the light of in vitro cancer models as predictive tools. The state of the art of in vitro cancer models is discussed first, followed by identifying the limitations of bench-to-bedside transition. This review tries to establish compatibility between the current findings and obstacles and indicates future directions to accelerate the market penetration, considering the niche market.This work is supported by FROnTHERA (NORTE-01-0145-FEDER-000023) and the European Union Framework Programme for Research and Innovation Horizon 2020 under grant agreement No. 668983 —FoReCaST. N. Antunes thanks the funds provided by FCT under the doctoral program in Tissue Engineering, Regenerative Medicine and Stem Cells (PD/BD/143050/2018). SCK also records the support of FCT through the BREAST-IT project (PTDC/BTM-ORG/28168/2017)

AdipoSIGHT in therapeutic response: consequences in osteosarcoma treatment

Chemotherapeutic resistance is a major problem in effective cancer treatment. Cancer cells engage various cells or mechanisms to resist anti-cancer therapeutics, which results in metastasis and the recurrence of disease. Considering the cellular heterogeneity of cancer stroma, the involvement of stem cells is reported to affect the proliferation and metastasis of osteosarcoma. Hence, the duo (osteosarcoma: Saos 2 and human adipose-derived stem cells: ASCs) is co-cultured in present study to investigate the therapeutic response using a nonadherent, concave surface. Staining with a cell tracker allows real-time microscopic monitoring of the cell arrangement within the sphere. Cell–cell interaction is investigated by means of E-cadherin expression. Comparatively high expression of E-cadherin and compact organization is observed in heterotypic tumorspheres (Saos 2–ASCs) compared to homotypic ones (ASCs), limiting the infiltration of chemotherapeutic compound doxorubicin into the heterotypic tumorsphere, which in turn protects cells from the toxic effect of the chemotherapeutic. In addition, genes known to be associated with drug resistance, such as SOX2, OCT4, and CD44 are overexpressed in heterotypic tumorspheres post-chemotherapy, indicating that the duo collectively repels the effect of doxorubicin. The interaction between ASCs and Saos 2 in the present study points toward the growing oncological risk of using ASC-based regenerative therapy in cancer patients and warrants further investigation.This work is supported by the European Union Framework Programme for Research and Innovation Horizon 2020 (nº 668983 — FoReCaST; FROnTHERA—NORTE-01-0145-FEDER-000023), Investigator FCT program (IF/01214/2014—V.M.), FCT2015 (IF/01285/2015—J.M.O.) and PTDC/BTMORG/28168/2017 (V.B. and S.C.K.)

Tumor - stroma interactions alter the sensitivity of drug in breast cancer

Flat cell cultures or xenografts are inadequate tools to unravel cancer complex biology. 3D in vitro tumor models garnered interest since they recapitulate better dynamic mechanisms of cancer, but a gold standardmodel that faithfullymimics solid cancer is not available yet. 3D breast cancermodel is fabricated using freeze-dried silk fibroin scaffolds. Breast cancer cell lines (MCF-7 and MDA-MB231) are seeded with normal mammary fibroblasts onto silk fibroin scaffold (1 and 2mm thick). Cells proliferation is monitored by means of Alamar blue assay. 3D breast cancer models morphology is observed by confocal microscopy. Gene expression modulation concerning extracellular matrix markers is evaluated. Further, 3D bioengineered breast cancer models are treated with doxorubicin. Silk fibroin scaffolds allow the proliferation of cancer cells and fibroblasts. Cells growth is enhanced when cancer cells and fibroblasts are seeded together. Histological staining shows 3D cell organization. MMP-1, MMP-2, MMP-3, Col-1, and Fibronectin expression is upregulated in co-culture. After doxorubicin treatment, stronger reduction in cell activity is observed in 2mm SF scaffold in comparison to 1mm. The 3D in vitro breast cancer model obtained can easily be scaled-up and translated to the preclinical testing of novel chemotherapeutics.This work was supported by EU-Horizon 2020 grant FoReCaST—Forefront Research in 3D Disease Cancer Modelsasin vitroScreening Technologies (H2020-WIDESPREAD-2014-668983). The authors also acknowledge the FRONTHERA project (Frontiers of technology for theranostics of cancer,metabolic and neurodegenerative diseases) (NORTE-01-0145-FEDER-0000232) and Fundação Ciência e tecnologia (FCT grantagreement: PTDC/BTM-ORG/28168/2017 to VB and SK)

Silk fibroin scaffolds with muscle-like elasticity support in vitro differentiation of human skeletal muscle cells

Human adult skeletal muscle has a limited ability to regenerate after injury and therapeutic options for volumetric muscle loss are few. Technologies to enhance regeneration of tissues generally rely upon bioscaffolds to mimic aspects of the tissue extracellular matrix (ECM). In the present study, silk fibroins from four Lepidoptera (silkworm) species engineered into three-dimensional scaffolds were examined for their ability to support the differentiation of primary human skeletal muscle myoblasts. Human skeletal muscle myoblasts (HSMMs) adhered, spread and deposited extensive ECM on all the scaffolds, but immunofluorescence and quantitative polymerase chain reaction analysis of gene expression revealed that myotube formation occurred differently on the various scaffolds. Bombyx mori fibroin scaffolds supported formation of long, well-aligned myotubes, whereas on Antheraea mylitta fibroin scaffolds the myotubes were thicker and shorter. Myotubes were oriented in two perpendicular layers on Antheraea assamensis scaffolds, and scaffolds of Philosamia/Samia ricini (S. ricini) fibroin poorly supported myotube formation. These differences were not caused by fibroin composition per se, as HSMMs adhered to, proliferated on and formed striated myotubes on all four fibroins presented as two-dimensional fibroin films. The Young's modulus of A. mylitta and B. mori scaffolds mimicked that of normal skeletal muscle, but A. assamensis and S. ricini scaffolds were more flexible. The present study demonstrates that although myoblasts deposit matrix onto fibroin scaffolds and create a permissive environment for cell proliferation, a scaffold elasticity resembling that of normal muscle is required for optimal myotube length, alignment, and maturation

Emerging tumor spheroids technologies for 3D in vitro cancer modeling

"Article in Press, Available online 31 October 2017" ; "S0163-7258(17)30268-1"Cancer is a leading cause of mortality and morbidity worldwide. Around 90% of deaths are caused by metastasis and just 10% by primary tumor. The advancement of treatment approaches is not at the same rhythm of the disease; making cancer a focal target of biomedical research. To enhance the understanding and promts the therapeutic delivery; concepts of tissue engineering are applied in the development of in vitro models that can bridge between 2D cell culture and animal models, mimicking tissue microenvironment. Tumor spheroid represents highly suitable 3D organoid-like framework elucidiating the intra and inter cellular signaling of cancer, like that formed in physiological niche. However, spheroids are of limited value in studying critical biological phenomenon such as tumor-stroma interactons involving extra cellular matrix or immune system. Therefore, a compelling need of tailoring spheroid technologies with physiologically relevant biomaterials or in silico models, is ever emerging. The diagnostic and prognostic role of spheroids rearrangements within biomaterials or microfluidic channel is indicative of patient management; particularly for the decision of targated therapy. Fragmented information on available in vitro spheroid models and lack of critical analysis on transformation aspects of these strategies; pushes the urge to comprehensively overview the recent technological advancements (e.g. bioprinting, micro-fluidic technologies or use of biomaterials to attain the third dimension) in the shed of tranlationable cancer research. In present article, relationships between current models and their possible exploitation in clinical success is explored with the highlight of existing challenges in defining therapeutic targets and screening of drug efficacy.The authors are thankful to European Union (Horizon 2020) funded project FoReCaST (No. 668983), the FCT fellowship to J. Silva-Correia (Grant No. SFRH/BPD/100590/2014), distinctions to J.M.O. under the Investigator FCT program (IF/00423/2012) and V.M.C. under the Investigator FCT program (IF/01214/2014) for supporting this work financially.info:eu-repo/semantics/publishedVersio

Cytotoxicity and sustained release of modified divinylsulfone from silk based 3D construct

Monovinylsulfones have been extensively studied for its biological activities but modified divinylsulfones (mDVS2) were largely neglected due to the non-availability of appropriate synthetic routes. The present report describes the potential of a unique derivative of divinylsulfone as a remedial molecule. The mDVS2, available in reasonably large amount through an easy synthesis route, incites necrosis in invasive and non-invasive breast cancer cells in a time and concentration dependent manner. This molecule is further used to fabricate mDVS2 embedded silk based 3D scaffolds in order to achieve sustained release. The entrapped molecules retain their activity over time, as 100 % cell death is observed within 7 days. The findings demonstrate the cytotoxic property of mDVS and highlight the importance of under utilized mDVSs as potential therapeutic agents

The tumor microenvironment: an introduction to the development of microfluidic devices

The tumor microenvironment (TME) is like the Referee of a soccer match who has constant eyes on the activity of all players, such as cells, acellular stroma components, and signaling molecules for the successful completion of the game, that is, tumorigenesis. The cooperation among all the "team members" determines the characteristics of tumor, such as the hypoxic and acidic niche, stiffer mechanical properties, or dilated vasculature. Like in soccer, each TME is different. This heterogeneity makes it challenging to fully understand the intratumor dynamics, particularly among different tumor subpopulations and their role in therapeutic response or resistance. Further, during metastasis, tumor cells can disseminate to a secondary organ, a critical event responsible for approximately 90% of the deaths in cancer patients. The recapitulation of the rapidly changing TME in the laboratory is crucial to improve patients' prognosis for unraveling key mechanisms of tumorigenesis and developing better drugs. Hence, in this chapter, we provide an overview of the characteristic features of the TME and how to model them, followed by a brief description of the limitations of existing in vitro platforms. Finally, various attempts at simulating the TME using microfluidic platforms are highlighted. The chapter ends with the concerns that need to be addressed for designing more realistic and predictive tumor-on-a-chip platforms.The work was supported by the European Union Framework Programme for Research and Innovation Horizon 2020 under grant agreement nº 668983 — FoReCaST and the Portuguese Foundation for Science and Technology (FCT) under the BREAST-IT project (PTDC/BTMORG/28168/2017) to SCK, 2MATCH project (PTDC/BTM-ORG/28070/2017) to D.C., C.M.A., and S.C.K,, and OncoNeoTreat (PTDC/CTM-REF/0022/2020) to C.M.A. D.C. also thanks the financial support from FCT for the CEEC Individual 2017 (CEECIND/00352/2017)

The diagnostic value of magnetic resonance imaging measurements for assessing cervical spinal canal stenosis in relationship with the measurement of normal reference values of spinal canal diameter and space available for cord (SAC) at mid sagittal level

Background: Assessment of cervical spinal stenosis, which is not very uncommon presentation among adult age group, is necessary for planning of the management protocol, especially regarding surgical intervention, if necessary. Specific measurements used for assessing spinal canal stenosis, the spinal canal diameter and space available for cord (SAC) at mid sagittal level are considered  to be very important ones. To determine the normal range of the absolute values of these two parameters in the local population and their importance in predicting cervical canal stenosis, we selected 100 asymptomatic adult subjects of each of both sexes and 50 symptomatic subjects of each of both sexes. Materials & Methods: In the present study, for asymptomatic subjects selected from the patients referred to MRI Center, IPGME&R for MRI of brain due to some unrelated ailment or from the patients of adult age group needing spinal MRI screening without any manifestation related to diseases involving the cervical part of spine & cord. For symptomatic subjects selected from the patients referred to the MRI center of IPGME&R for cervical spinal MRI study to evaluate for cervical spinal canal stenosis with various symptoms such as neck or shoulder pain and stiffness, paresthesia of hands & feet, slowly progressive spastic paraparesis, other upper motor neuron signs of lower limb, dermatomal sensory loss, weakness of small muscles of hands etc. Results: Sensitivity of cervical canal diameter as a marker of canal stenosis in predicting symptoms was 62% (95%CI 47.17-75.35) with specificity 91%, positive predictive value (PPV) 77.5% and negative predictive value (NPV) 82.73%. Sensitivity of SAC as a maker of cervical canal stenosis in predicting symptoms was 66 % (51.23-78.79) with specificity 93%, PPV 82.5 %, and NPV 84.55%. Finally at C7 level, r is>0.8 so strong +ve correlation between canal diameter (CAD) & space available for cord (SAC) at C7 cervical level. Sensitivity of cervical canal diameter & SAC both as the marker of canal stenosis in predicting symptoms was 82% (68.56- 91.42) with specificity 87%, PPV 75.93 % and NPV 90.60 %. Conclusion: It is well recognized that mid sagittal spinal canal diameter and space available for the cord (SAC) in cervical vertebrae (C3 to C7) varies considerably in normal adult population of both the sexes and decrease in them will result in cervical stenosis symptoms. We know that when sensitivity more than 70%, it indicates predictability. In present study we find that sensitivity is 82%. So we may conclude that in case of patient having symptoms of cervical stenosis, canal diameter and space available for cord both measurements are important