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Glioblastoma multiforme (GBM) is the most prevalent malignant brain tumor, with an average survival time of 14 to 20 months. Its capacity to invade brain parenchyma leads to the failure of conventional treatments and subsequent tumor recurrence. Recent studies have explored new therapeutic strategies using a chemoattracting gradient to attract GBM cells into a soft hydrogel trap where they can be exposed to higher doses of radiation or chemotherapy. It has been demonstrated in vitro under static conditions, that nanoparticles (NPs) encapsulating the chemoattractant CXCL12 can create a gradient to attract GBM cell. However, GBM cell invasion is also largely dependent on interstitial fluid flow (IFF). In the present study, a custom-made in vitro 3D model with indirect perfusion to mimic IFF at flow rates of 0.5 μL/min and 3 μL/min was used to examine the invasive behavior of F98-rodent-derived and U87-human-derived GBM cells. This model simulated IFF and CXCL12 gradient within an alginate:matrigel-based hydrogel mimicking brain parenchyma. Findings revealed that CXCL12 (1600 ng/mL) released from NPs significantly increased the migration of F98 GBM cells after 72 hours under IFF conditions at both 0.5 and 3 μL/min. In contrast, U87 GBM cells required a higher CXCL12 concentration (2400 ng/mL) and longer incubation time for migration (120 hours). Unlike the F98 cells, U87 GBM cells showed a CXCL12 dose-dependent proliferation. Semi-quantitative qPCR showed higher CXCR4 mRNA levels in F98 cells than in U87 cells. CXCL12 significantly increased intracellular calcium levels via CXCR4 activation, with a 2.3-fold rise in F98 cells compared to U87, consistent with observed cell behavior during perfusion. This highlights the combined influence of IFF and CXCL12 on cell migration, dependent on cell line. This 3D dynamic model is a valuable tool to analyze parameters like interstitial fluid flow (IFF) and chemokine gradients, influenced by GBM tumor diversity., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 El Kheir et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Grade IV multiforme glioblastoma (GBM) is an aggressive cancer that remains incurable due to the GBM cells invading and proliferating in the surrounding healthy tissues, even after tumor resection. A new therapeutic paradigm to treat GBM is to attract and accumulate GBM cells in a macroporous hydrogel inserted in the surgical cavity after tumor resection, followed by a targeted high dose of radiotherapy. This work presents a molding-based method to prepare macroporous hydrogels composed of sodium alginate and chitosan, homogeneously mixed in solution using sodium bicarbonate, and subsequently crosslinked with genipin and calcium chloride. The gels display a blue color, the result of chitosan crosslinking with genipin, fully interconnected pores with an average diameter of 180 μm (and tunable over a wide range), with a compression modulus of 10 kPa, close to the value of brain tissues. The gels are stable in cell culture media and keep their integrity after radiation doses comparable to current GBM treatment levels. Finally, F98 GBM cells accumulate relatively homogeneously and are retained within the gels., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

The development of biomaterials such as synthetic scaffolds for peripheral nerve regeneration requires a precise knowledge of the mechanical properties of the nerve in physiological-like conditions. Mechanical properties (Young's modulus, maximum stress and strain at break) for peripheral nerves are scarce and large discrepancies are observed in between reports. This is due in part to the absence of a robust testing device for nerves. To overcome this limitation, a custom-made tensile device (CMTD) has been built. To evaluate its reproducibility and accuracy, the imposed speed and distance over measured speed and distance was performed, followed by a validation using poly(dimethylsiloxane) (PDMS), a commercial polymer with established mechanical properties. Finally, the mechanical characterization of rodents (mice and rats) sciatic nerves using the CMTD was performed. Mouse and rat sciatic nerves Young's modulus were 4.57 ± 2.04 and 19.2 ± 0.86 MPa respectively. Maximum stress was 1.26 ± 0.56 MPa for mice and 3.81 ± 1.84 MPa for rats. Strain at break was 53 ± 17% for mice and 32 ± 12% for rats. The number of axons per sciatic nerve was found to be twice higher for rats. Statistical analysis of the measured mechanical properties revealed no sex-related trends, for both mice and rats (except for mouse maximum stress with p=0.03). Histological evaluation of rat sciatic nerve corroborated these findings. By developing a robust CMTD to establish the key mechanical properties (Young's modulus, maximum stress and strain at break) values for rodents sciatic nerves, our work represent an essential step toward the development of better synthetic scaffolds for peripheral nerve regeneration., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this article., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Traumatic injuries to the peripheral nervous system (PNI) can lead to severe consequences such as paralysis. Unfortunately, current treatments rarely allow for satisfactory functional recovery. The high healthcare costs associated with PNS injuries, worker disability, and low patient satisfaction press for alternative solutions that surpass current standards. For the treatment of injuries with a deficit of less than 30 mm to bridge, the use of synthetic nerve conduits (NGC) is favored. However, to develop such promising therapeutic strategies, in vitro models that more faithfully mimic nerve physiology are needed. The absence of a clinically scaled model with essential elements such as a three-dimension environment and dynamic coculture has hindered progress in this field. The presented research focuses on the development of an in vitro coculture model of the peripheral nervous system (PNS) involving the use of functional biomaterial which microstructure replicates nerve topography. Initially, the behavior of neuron-derived cell lines (N) and Schwann cells (SC) in contact with a short section of biomaterial (5 mm) was studied. Subsequent investigations, using fluorescent markers and survival assays, demonstrated the synergistic effects of coculture. These optimized parameters were then applied to longer biomaterials (30 mm), equivalent to clinically used NGC. The results obtained demonstrated the possibility of maintaining an extended coculture of SC and N over a 7-day period on a clinically scaled biomaterial, observing some functionality. In the long term, the knowledge gained from this work will contribute to a better understanding of the PNS regeneration process and promote the development of future therapeutic approaches while reducing reliance on animal experimentation. This model can be used for drug screening and adapted for personalized medicine trials. Ultimately, this work fills a critical gap in current research, providing a transformative approach to study and advance treatments for PNS injuries., Competing Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s) 2024.)

Glioblastoma (GBM) accounts for half of all central nervous system tumors. Once the tumor is removed, many GBM cells remain present near the surgical cavity and infiltrate the brain up to a distance of 20-30 mm, resulting in recurrence a few months later. GBM remains incurable due to the limited efficiency of current treatments, a result of the blood-brain barrier and sensitivity of healthy brain tissues to chemotherapy and radiation. A new therapeutic paradigm under development to treat GBM is to attract and accumulate GBM cells in a cancer cell trap inserted in the surgical cavity after tumor resection. In this work, porous gels were prepared using porous polylactide molds obtained from melt-processed co-continuous polymer blends of polystyrene and polylactide, with an average pore size ranging from 5 μm to over 500 μm. In order to efficiently accumulate and retain GBM brain cancer cells within a macroporous sodium alginate-based hydrogel trap, the pores must have an average diameter superior to 100 μm, with the best results obtained at 225 μm. In that case, the accumulation and retention of F98 GBM cells were more homogeneous, especially when functionalized with RGD adhesion peptides. At an alginate concentration of 1% w/v, the compression modulus reaches 15 kPa, close to the average value of 1-2 kPa reported for brain tissues, while adhesion and retention were also superior compared to 2% w/v gels. Overall, 1% w/v gels with 225 μm pores functionalized with the RGD peptide display the best performances., (Creative Commons Attribution license.)

Peripheral nerve injuries (PNIs) are detrimental to the quality of life of affected individuals. Patients are often left with life-long ailments that affect them physically and psychologically. Autologous nerve transplant is still the gold standard treatment for PNIs despite limited donor site and partial recovery of nerve functions. Nerve guidance conduits are used as a nerve graft substitute and are efficient for the repair of small nerve gaps but require further improvement for repairs exceeding 30 mm. Freeze-casting is an interesting fabrication method for the conception of scaffolds meant for nerve tissue engineering since the microstructure obtained comprises highly aligned micro-channels. The present work focuses on the fabrication and characterization of large scaffolds (35 mm length, 5 mm diameter) made of collagen/chitosan blends by freeze-casting via thermoelectric effect instead of traditional freezing solvents. As a freeze-casting microstructure reference, scaffolds made from pure collagen were used for comparison. Scaffolds were covalently crosslinked for better performance under load and laminins were further added to enhance cell interactions. Microstructural features of lamellar pores display an average aspect ratio of 0.67 ± 0.2 for all compositions. Longitudinally aligned micro-channels are reported as well as enhanced mechanical properties in traction under physiological-like conditions (37 °C, pH = 7.4) resulting from crosslinking treatment. Cell viability assays using a rat Schwann cell line derived from sciatic nerve (S16) indicate that scaffold cytocompatibility is similar between scaffolds made from collagen only and scaffolds made from collagen/chitosan blend with high collagen content. These results confirm that freeze-casting via thermoelectric effect is a reliable manufacturing strategy for the fabrication of biopolymer scaffolds for future peripheral nerve repair applications.

Recent studies have identified FoxL1 + -telocytes (TC FoxL1+ ) as key players in gut epithelial-mesenchymal interactions which can determine the colonic microenvironment. Bone morphogenetic protein signaling disruption in TC FoxL1+ alters the physical and cellular microenvironment and leads to colon pathophysiology. This suggests a role for TC FoxL1+ in stromagenesis, but it is hard to identify the specific contribution of TC FoxL1+ when analyzing whole tissue profiling studies. We performed ex vivo deconstruction of control and BmpR1a △FoxL1+ colon samples, isolated the mesenchyme-enriched fractions, and determined the protein composition of the in vivo extracellular matrix (ECM) to analyze microenvironment variation. Matrisomic analysis of mesenchyme fractions revealed modulations in ECM proteins with functions associated with innate immunity, epithelial wound healing, and the collagen network. These results show that TC FoxL1+ is critical in orchestrating the biodynamics of the colon ECM. TC FoxL1+ disfunction reprograms the gut's microenvironment and drives the intestinal epithelium toward colonic pathologies. SIGNIFICANCE: In this study, the method that was elected to isolate ECM proteins might not encompass the full extent of ECM proteins in a tissue, due to the protocol chosen, as this protocol by Naba et al., targets more the insoluble part of the matrisome and eliminates the more soluble components in the first steps. However, this ECM-enrichment strategy represents an improvement and interesting avenue to study ECM proteins in the colon compared to total tissue analysis with a background of abundant cellular protein. Thus, the matrisomic approach presented in this study, and its target validation delivered a broader evaluation of the matrix remodeling occurring in the colonic sub-epithelial mesenchyme of the BmpR1a △FoxL1+ mouse model., Competing Interests: Declaration of Competing Interest The authors make the statement that there is no conflict of interest to disclose., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

Glioblastoma multiforme is a type of brain cancer associated with a very low survival rate since a large number of cancer cells remain infiltrated in the brain despite the treatments currently available. This work presents a macroporous hydrogel trap, destined to be implanted in the surgical cavity following tumor resection and designed to attract and retain cancer cells, in order to eliminate them afterward with a lethal dose of stereotactic radiotherapy. The biocompatible hydrogel formulation comprises sodium alginate (SA) and chitosan (CHI) bearing complementary electrostatic charges and stabilizing the gels in saline and cell culture media, as compared to pristine SA gels. The highly controlled and interconnected porosity, characterized by X-ray microCT, yields mechanical properties comparable to those of brain tissues and allows F98 glioblastoma cells to penetrate the gels within the entire volume, as confirmed by fluorescence microscopy. The addition of a grafted -RGD peptide on SA, combined with CHI, significantly enhances the adhesion and retention of F98 cells within the gels. Overall, the best compromise between low proliferation and a high level of accumulation and retention of F98 cells was obtained with the hydrogel formulated with 1% SA and 0.2% CHI, without the -RGD adhesion peptide.

Docosahexaenoic acid (DHA) consumption reduces spatial memory impairment in mice carrying the human apolipoprotein E ε4 ( APOE4 ) allele. The current study evaluated whether astrocyte and microglia morphology contribute to the mechanism of this result. APOE3 and APOE4 mice were fed either a DHA-enriched diet or a control diet from 4 to 12 months of age. Coronal brain sections were immunostained for GFAP, Iba1, and NeuN. Astrocytes from APOE4 mice exhibited signs of reactive astrogliosis compared to APOE3 mice. Consumption of DHA exacerbated reactive astrocyte morphology in APOE4 carriers. Microglia from APOE4 -control mice exhibited characteristics of amoeboid morphology and other characteristics of ramified morphology (more processes, greater process complexity, and greater distance between neighboring microglia). DHA enhanced ramified microglia morphology in APOE4 mice. In addition, APOE4 mice fed the DHA diet had lower hippocampal concentrations of interleukin-7, lipopolysaccharide-induced CXC chemokine and monocyte chemoattractant protein 1, and higher concentration of interferon-gamma compared to APOE4 -control mice. Our results indicate that a diet rich in DHA enhances reactive astrogliosis and ramified microglia morphology in APOE4 mice., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Author(s).)

Glioblastoma multiforme (GBM) is a grade IV glioma considered the most fatal cancer of the central nervous system (CNS), with less than a 5% survival rate after five years. The tumor heterogeneity, the high infiltrative behavior of its cells, and the blood-brain barrier (BBB) that limits the access of therapeutic drugs to the brain are the main reasons hampering the current standard treatment efficiency. Following the tumor resection, the infiltrative remaining GBM cells, which are resistant to chemotherapy and radiotherapy, can further invade the surrounding brain parenchyma. Consequently, the development of new strategies to treat parenchyma-infiltrating GBM cells, such as vaccines, nanotherapies, and tumor cells traps including drug delivery systems, is required. For example, the chemoattractant CXCL12, by binding to its CXCR4 receptor, activates signaling pathways that play a critical role in tumor progression and invasion, making it an interesting therapeutic target to properly control the direction of GBM cell migration for treatment proposes. Moreover, the interstitial fluid flow (IFF) is also implicated in increasing the GBM cell migration through the activation of the CXCL12-CXCR4 signaling pathway. However, due to its complex and variable nature, the influence of the IFF on the efficiency of drug delivery systems is not well understood yet. Therefore, this review discusses novel drug delivery strategies to overcome the GBM treatment limitations, focusing on chemokines such as CXCL12 as an innovative approach to reverse the migration of infiltrated GBM. Furthermore, recent developments regarding in vitro 3D culture systems aiming to mimic the dynamic peritumoral environment for the optimization of new drug delivery technologies are highlighted.

To overcome the radioresistance of glioblastoma (GBM) cells infiltrated in the brain, we propose to attract these cancer cells into a trap to which a lethal radiation dose can be delivered safely. Herein, we have prepared and characterized a sodium alginate-based macroporous hydrogel as a potential cancer cell trap. Microcomputed X-ray tomography shows that the hydrogel matrices comprise interconnected pores with an average diameter of 300 μm. The F98 GBM cells migrated in the pores and mainly accumulated in the center of the matrix. Depending on the number of cancer cells added, the grafting of RGD cell-adhesion peptides to the alginate resulted in a 4 to 10 times increase in the number of F98 cells (which overexpress the associated αvβ3 and αvβ5 binding integrins) retained in the matrix. Finally, a radiation dose of 25 Gy eliminated all F98 cells trapped in the matrix, without significantly altering the matrix mechanical properties., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Biomaterials that can control the behaviour of stem cells play a major role in regenerative medicine and tissue engineering. We previously showed that poly(epsilon)caprolactone (PCL) films functionalized with adhesive peptides containing sequences of both cell binding domain (RGD) and synergistic site (PHSRN) of the fibronectin (pFibro) enhanced the osteoblastic commitment of C3H10T1/2 mesenchymal progenitor cells (C3H10T1/2 cells) induced by soluble BMP-9 or its derived peptide SpBMP-9. Here, the effect of PCL films functionalized with pFibro and/or SpBMP-9 or its negative peptide NSpBMP-9 on adhesion and intracellular signalling of C3H10T1/2 cells was determined. The differentiation of adherent C3H10T1/2 cells and MC3T3-E1 preosteoblasts into osteoblasts was also analysed with or without IGF-2, since this growth factor can favour the osteoblastic differentiation induced by BMP-9. We found that pFibro and SpBMP-9 co-functionalization on PCL films promoted the adhesion of C3H10T1/2 cells with well-organized focal adhesion points and FAK activation. In these mesenchymal progenitor cells, PCL-SpBMP-9 and PCL-pFibro/SpBMP-9 induced the activation and nuclear translocation of Smad 1/5 after 4 h, and enhanced the protein expression of RUNX2 (3 d) and alkaline phosphatase (ALP) activity (7 d), two markers of osteoblast differentiation. No PPARγ, a marker of adipogenic differentiation, was detected. C3H10T1/2 cells attached to PCL-SpBMP-9 also contained more SOX9, a marker of chondroblastic lineage, compared with other experimental conditions. The use of inactive peptides NSpBMP-9 confirmed the specificity and effectiveness of SpBMP-9 on cell adhesion, intracellular signalling and osteoblastic differentiation. Adding IGF-2 only significantly improved the differentiation of MC3T3-E1 preosteoblasts into osteoblasts as shown by the increase in gene expression encoding Osterix (mRNA Sp7) and ALP (mRNA Alpl), probably because of the lack of serum in the medium. Therefore, material surface co-functionalized with pFibro and SpBMP-9 could be most useful for developing scaffolds with both osseointegrative and osteoinductive properties for bone application and tissue engineering strategy when combined with IGF-2 in serum free medium., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Hematoma and skeletal muscles play a crucial role in bone fracture healing. The muscle resident mesenchymal stromal cells (mrSCs) can promote bone formation by differentiating into osteoblasts upon treatment by bone morphogenetic proteins (BMP), such as BMP9. However, the influence of hematoma fracture extracts (Hema) on human mrSC (hmrSC) response to BMP9 is still unknown. We therefore determined the influence of Hema, human healthy serum (HH), and fetal bovine serum (FBS, control) on BMP9-induced osteoblast commitment of hmrSC by measuring alkaline phosphatase activity. Multiplex assays of 90 cytokines were performed to characterize HH and Hema composition and allow their classification by a multivariate statistical approach depending on their expression levels. We confirmed that BMP9 had a greater effect on osteoblastic differentiation of hmrSCs than BMP2 in presence of FBS. The hmrSCs response to BMP9 was enhanced by both Hema and HH, even though several cytokines were upregulated (IL-6, IL-8, MCP-1, VEGF-A and osteopontin), downregulated (BMP9, PDGF) or similar (TNF-alpha) in Hema compared with HH. Thus, hematoma may potentiate BMP9-induced osteogenic differentiation of hmrSCs during bone fracture healing. The multivariate statistical analyses will help to identify the cytokines involved in such phenomenon leading to normal or pathological bone healing.

Chitosan (Chit) currently used to prepare nanoparticles (NPs) for brain application can be complexed with negatively charged polymers such as alginate (Alg) to better entrap positively charged molecules such as CXCL12. A sustained CXCL12 gradient created by a delivery system can be used, as a therapeutic approach, to control the migration of cancerous cells infiltrated in peri-tumoral tissues similar to those of glioblastoma multiforme (GBM). For this purpose, we prepared Alg/Chit NPs entrapping CXCL12 and characterized them. We demonstrated that Alg/Chit NPs, with an average size of ~250 nm, entrapped CXCL12 with ~98% efficiency for initial mass loadings varying from 0.372 to 1.490 µg/mg NPs. The release kinetic profiles of CXCL12 were dependent on the initial mass loading, and the released chemokine from NPs after seven days reached 12.6%, 32.3%, and 59.9% of cumulative release for initial contents of 0.372, 0.744, and 1.490 µg CXCL12/mg NPs, respectively. Mathematical modeling of released kinetics showed a predominant diffusive process with strong interactions between Alg and CXCL12. The CXCL12-NPs were not toxic and did not promote F98 GBM cell proliferation, while the released CXCL12 kept its chemotaxis effect. Thus, we developed an efficient and tunable CXCL12 delivery system as a promising therapeutic strategy that aims to be injected into a hydrogel used to fill the cavity after surgical tumor resection. This system will be used to attract infiltrated GBM cells prior to their elimination by conventional treatment without affecting a large zone of healthy brain tissue.

Background: Trauma-induced heterotopic ossification (HO) is a complication that develops under three conditions: the presence of an osteogenic progenitor cell, an inducing factor, and a permissive environment. We previously showed that a mouse multipotent Sca1 + CD31 - Lin - muscle resident stromal cell (mrSC) population is involved in the development of HO in the presence of inducing factors, members of the bone morphogenetic protein family. Interestingly, BMP9 unlike BMP2 causes HO only if the muscle is damaged by injection of cardiotoxin. Because acute trauma often results in blood vessel breakdown, we hypothesized that a hypoxic state in damaged muscles may foster mrSCs activation and proliferation and trigger differentiation toward an osteogenic lineage, thus promoting the development of HO., Methods: Three- to - six-month-old male C57Bl/6 mice were used to induce muscle damage by injection of cardiotoxin intramuscularly into the tibialis anterior and gastrocnemius muscles. mrSCs were isolated from damaged (hypoxic state) and contralateral healthy muscles and counted, and their osteoblastic differentiation with or without BMP2 and BMP9 was determined by alkaline phosphatase activity measurement. The proliferation and differentiation of mrSCs isolated from healthy muscles was also studied in normoxic incubator and hypoxic conditions. The effect of hypoxia on BMP synthesis and Smad pathway activation was determined by qPCR and/or Western blot analyses. Differences between normally distributed groups were compared using a Student's paired t test or an unpaired t test., Results: The hypoxic state of a severely damaged muscle increased the proliferation and osteogenic differentiation of mrSCs. mrSCs isolated from damaged muscles also displayed greater sensitivity to osteogenic signals, especially BMP9, than did mrSCs from a healthy muscle. In hypoxic conditions, mrSCs isolated from a control muscle were more proliferative and were more prone to osteogenic differentiation. Interestingly, Smad1/5/8 activation was detected in hypoxic conditions and was still present after 5 days, while Smad1/5/8 phosphorylation could not be detected after 3 h of normoxic incubator condition. BMP9 mRNA transcripts and protein levels were higher in mrSCs cultured in hypoxic conditions. Our results suggest that low-oxygen levels in damaged muscle influence mrSC behavior by facilitating their differentiation into osteoblasts. This effect may be mediated partly through the activation of the Smad pathway and the expression of osteoinductive growth factors such as BMP9 by mrSCs., Conclusion: Hypoxia should be considered a key factor in the microenvironment of damaged muscle that triggers HO.

Endothelial cell (EC) dysfunction contributes to atherosclerosis, which is associated with arterial stiffening and fibronectin (FN) deposition, by ECs and smooth muscle cells (SMCs). The effect of stiffness on the EC/FN interaction and fibrillar adhesion formation has been poorly studied. An in vitro model was prepared that included FN-coated polydimethylsiloxane (PDMS) films with similar hydrophobicity and roughness but distinct Young's modulus values, mimicking healthy (1.0 MPa) and atherosclerotic (2.8 MPa) arteries. Human aortic abdominal endothelial cells (HAAECs) seeded on 1.0 MPa PDMS films spread over time and reached their maximum surface area faster than on 2.8 MPa PDMS films. In addition, HAAECs appeared to organize focal adhesion more rapidly on 1.0 MPa PDMS films, despite the similar cell binding domain accessibility to adsorbed FN. Interestingly, we also observed up to a ~5-fold increase in the percentage of HAAECs that had a well-developed fibrillar adhesion on 1.0 MPa compared to 2.8 MPa PDMS films as verified by integrin α 5 subunits, tensin, and FN staining. This variation did not affect EC migration. These results suggest that there are favourable conditions for FN matrix assembly by ECs in early atherosclerosis rather than at advanced stages. Our in vitro model will therefore be helpful to understand the influence of bulk stiffness on cells involved in atherosclerosis., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The current aging of the world population will increase the number of people suffering from brain degenerative diseases such as Alzheimer's disease (AD). There are evidence showing that the use of growth factors such as BMP-9 could restored cognitive function as it acts on many AD hallmarks at the same time. However, BMP-9 is a big protein expensive to produce that can hardly access the central nervous system. We have therefore developed a small peptide, SpBMP-9, derived from the knuckle epitope of BMP-9 and showed its therapeutic potential in a previous study. Since it is known that the native protein, BMP-9, can act in synergy with other growth factors in the context of AD, here we study the potential synergistic effect of various combinations of SpBMP-9 with bFGF, EGF, IGF-2 or NGF on the cholinergic differentiation of human neuroblastoma cells SH-SY5Y. We found that, in opposition to IGF-2 or EGF, the combination of SpBMP-9 with bFGF or NGF can stimulate to a greater extent the neurite outgrowth and neuronal differentiation toward the cholinergic phenotype as shown by expression and localization of the neuronal markers NSE and VAchT and the staining of intracellular calcium. Those results strongly suggest that SpBMP-9 plus NGF or bFGF are promising therapeutic combinations against AD that required further attention., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The incidence of brain degenerative disease such as Alzheimer's disease (AD) will increase as the world population is ageing. While current AD treatments have only a transient effect, there are many evidences indicating that some growth factors, such as BMP-9, may be used to treat AD. However, growth factors cannot readily access the brain because of their size and the presence of the blood brain barrier. We have therefore developed a small peptide derived from BMP-9, SpBMP-9, which can promote the differentiation of cholinergic neurons and inactivate GSK3beta, a Tau kinase. Here, we investigated the potential of a nanoparticle-based delivery system of SpBMP-9, made of alginate and chitosan (Alg/Chit NPs), as a new therapeutic strategy against AD. The Alg/Chit NPs size distribution revealed NPs with an average diameter of ∼240nm. The encapsulation efficiency of SpBMP-9 was ∼70% of the initial peptide mass loading. Release kinetics of SpBMP-9 were performed in physiological conditions and modelled with a mechanistic framework that took into account the size distribution of Alg/Chit NPs. The release of SpBMP-9 revealed to be mostly diffusive, but there were interactions between the peptide and the alginate chains. The Alg/Chit NPs could also increase the viability of SH-SY5Y cells in comparison to the control. Finally, the SpBMP-9 released from Alg/Chit NPs promoted the SH-SY5Y differentiation into mature neurons as demonstrated by a higher neurite outgrowth and an increased expression of the neuronal markers NSE and VAchT. In conclusion, the nano-scale SpBMP-9 delivery system made of Alg/Chit may be a promising therapeutic strategy against AD., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The incidence of brain degenerative disorders like Alzheimer's disease (AD) will increase as the world population ages. While there is presently no known cure for AD and current treatments having only a transient effect, an increasing number of publications indicate that growth factors (GF) may be used to treat AD. GFs like the bone morphogenetic proteins (BMPs), especially BMP-9, affect many aspects of AD. However, BMP-9 is a big protein that cannot readily cross the blood-brain barrier. We have therefore studied the effects of two small peptides derived from BMP-9 (pBMP-9 and SpBMP-9). We investigated their capacity to differentiate SH-SY5Y human neuroblastoma cells into neurons with or without retinoic acid (RA). Both peptides induced Smad 1/5 phosphorylation and their nuclear translocation. They increased the number and length of neurites and the expression of neuronal markers MAP-2, NeuN and NSE better than did BMP-9. They also promoted differentiation to the cholinergic phenotype more actively than BMP-9, SpBMP-9 being the most effective as shown by increases in intracellular acetylcholine, ChAT and VAchT. Finally, both peptides activated the PI3K/Akt pathway and inhibited GSK3beta, a current AD therapeutic target. BMP-9-derived peptides, especially SpBMP-9, with or without RA, are promising molecules that warrant further investigation.

The bone morphogenetic proteins (BMPs), which are involved in bone formation and repair, play an important role in tissue engineering. For example, BMP-9 and BMP-2, which are members of different BMP subfamilies, are osteoinductive factors. However, several studies have recently shown that BMP-9 is more osteogenic than BMP-2. We have previously shown that fetal bovine serum (FBS) strongly enhances the osteoblast differentiation of murine preosteoblasts (MC3T3-E1) to BMP-9 but not to BMP-2. This effect is mimicked by IGF-2, which primarily activates the PI3K/Akt pathway, but how Akt phosphorylation sites are implicated in such differentiation is unclear. The effects of BMP-9 and BMP-2 with or without FBS or IGF-2 on Akt phosphorylation sites and subsequent osteoblastic differentiation were determined, respectively, by western blot analysis and alkaline phosphatase activity measurements. The involvement of phosphorylated Akt at Thr308 and/or Ser473 on BMP-mediated osteoblast differentiation was further studied using specific inhibitors. In MC3T3-E1 incubated with or without FBS, BMP-9 and BMP-2 activate Akt on Ser473 and Thr308 very differently in a time and dose-dependent manner. Using inhibitors specific to each Akt phosphorylation site, we showed that both Ser473 and Thr308 must be phosphorylated for BMP-9 and/or IGF-2-induced osteoblast differentiation, whereas BMP-2 requires phosphorylation of only Ser473. Furthermore, cells stimulated with BMP-2 in the presence of FBS require the phosphorylation of Akt at Ser473 and the dephosphorylation of Akt at Thr308 to increase the osteoblast differentiation with alkaline phosphatase activity similar to that of BMP-9 plus FBS. These results provide a better understanding into how BMP-9 induces osteoblast differentiation and its synergy with IGF-2 at the signaling level. This knowledge is essential for preparing the serum-free osteogenic media required for bone tissue engineering or developing growth factor delivery systems to improve bone formation.

As the populations of the Western world become older, they will suffer more and more from bone defects related to osteoporosis (non-union fractures, vertebral damages), cancers (malignant osteolysis) and infections (osteomyelitis). Autografts are usually used to fill these defects, but they have several drawbacks such as morbidity at the donor site and the amount and quality of bone that can be harvested. Recent scientific milestones made in biomaterials development were shown to be promising to overcome these limitations. Cell interactions with biomaterials can be improved by adding at their surface functional groups such as adhesive peptides and/or growth factors. The development of such biomimetic materials able to control bone cell responses can only proceed if it is based on a sound understanding of bone cell behavior and regulation. This review focuses on bone physiology and the regulation of bone cell differentiation and function, and how the latest advances in biomimetic materials can be translated within promising clinical outcomes.

Biomimetic materials were developed to regulate stem cell behaviour. We have analyzed the influence of polycaprolactone (PCL) films, functionalized with adhesive peptides derived from fibronectin (pFibro) or bone sialoprotein (pBSP), on the response of murine multipotent C3H10T1/2 cells to bone morphogenetic protein-9 (BMP-9) and its derived peptides (pBMP-9 and SpBMP-9). PCL-pFibro promoted better cell cytoskeleton organization and faster focal adhesion kinase activation than did PCL-pBSP. PCL-pFibro also promoted MAPK signalling to improve the cell response to BMP-9 by inactivating ERK1/2 and stimulating p38 and JNK. BMP-9, pBMP-9 and SpBMP-9 induced greater phosphorylation of Smad1/5/8 in cells attached to PCL-pFibro than in cells on PCL-pBSP. These phosphorylated Smad1/5/8 were translocated to the nucleus. BMP-9 and its derived peptides restored the phosphorylation of JNK in cells on PCL-pBSP, but it remained less phosphorylated than in cells on PCL-pFibro stimulated with pBMP-9 and SpBMP-9. Cells attached to PCL-pFibro contained more Runx2, essential for stem cell commitment to become osteoblasts, than did cells on PCL-pBSP when incubated with BMP-9 and its derived peptides. Runx2 was no longer detected when the cells were pre-treated with JNK inhibitor. Therefore pFibro plus BMP-9 and its derived peptides may be a promising strategy to develop biomimetic materials., Statement of Significance: Biomaterials functionalized with adhesive peptides to favour bone repair have generated a great interest over the past decade. However, the effect of these materials on the ability of cells to respond to growth factors remains poorly known. One major growth factor subfamily involved in bone formation is the bone morphogenetic protein (BMP). However, these BMPs are expensive. We therefore developed less costly derived molecules. We showed how adhesive peptides derived from bone matrix proteins grafted onto polymer films affect the intracellular signalling and thus the ability of stem cells to be activated by BMP and its derived molecules. We have therefore identified a combination of bioactive polymers and BMP molecules that direct the stem cells towards bone forming cells., (Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

The number of people suffering from Alzheimer's disease (AD) will increase as the world population ages, creating a huge socio-economic burden. The three pathophysiological hallmarks of AD are the cholinergic system dysfunction, the β-amyloid peptide deposition and the Tau protein hyperphosphorylation. Current treatments have only transient effects and each tends to concentrate on a single pathophysiological aspect of AD. This review first provides an overall view of AD in terms of its pathophysiological symptoms and signaling dysfunction. We then examine the therapeutic potential of growth factors (GFs) by showing how they can overcome the dysfunctional cell signaling that occurs in AD. Finally, we discuss new alternatives to GFs that help overcome the problem of brain uptake, such as small peptides, with evidence from some of our unpublished data on human neuronal cell line., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The number of people diagnosed with Alzheimer's disease (AD) is increasing steadily as the world population ages, thus creating a huge socio-economic burden. Current treatments have only transient effects and concentrate on a single aspect of AD. There is much evidence suggesting that growth factors (GFs) have a great therapeutic potential and can play on all AD hallmarks. Because GFs are prone to denaturation and clearance, a delivery system is required to ensure protection and a sustainable delivery. This review provides information about the latest advances in the development of GF delivery systems (GFDS) targeting the brain in terms of in vitro and in vivo effects in the context of AD and discusses new strategies designed to increase the availability and the specificity of GFs to the brain. This paper also discusses, on a mechanistic level, the different delivery hurdles encountered by the carrier or the GF itself from its injection site up to the brain tissue. The major mass transport phenomena influencing the delivery systems targeting the brain are addressed and insights are given about how mechanistic mathematical frameworks can be developed to use and optimize them., (Copyright © 2015. Published by Elsevier B.V.)

The bone morphogenetic proteins (BMPs) are potent osteogenic molecules that are used for bone repair in delivery systems and in regenerative medicine. We studied the responses of murine MC3T3-E1 preosteoblasts to doses of recombinant human (rh)BMP-9 with and without fetal bovine serum (FBS). rhBMP-2 was used as a control since it is currently approved by the Food and Drug Administration for bone application. We analyzed the major cell signaling pathways and the expression of osteogenic markers. Without FBS, BMP-9 had a similar effect on MC3T3-E1 preosteoblast differentiation in comparison to BMP-2. In contrast, FBS reduced the EC50 of BMP-9 fourfold to sixfold, as determined by osterix gene expression and alkaline phosphatase (ALP) activity, while it had no influence on EC50 of BMP-2. As suggested by MAPK inhibitor assays, FBS could induce an intracellular signaling environment that favors cell response to BMP-9 by inhibiting ERK1/2 activation and increasing p38 phosphorylation. Finally, IGF-2 (100 ng/mL) could mimic the effect of FBS on BMP-9 cell response in terms of MAPK signaling and ALP activity. Thus, the action of BMP-9 on preosteoblast differentiation can be greatly improved by IGF-2. This finding may well be critical for developing optimal growth factor delivery systems and bone tissue engineering strategies.

Type I collagen is one of the most widely used materials for drug delivery in tissue repair. It is the reference carrier for delivering growth factors like bone morphogenetic proteins (BMPs such as BMP-2 and BMP-7) for bone repair. Since BMPs are expensive to produce, we have developed a peptide derived from BMP-9 (pBMP-9) that is 300 times less expensive than the entire protein while still promoting osteogenic differentiation. We have now evaluated the effects of the collagen concentration and the initial pBMP-9 load on peptide release. We then developed a model of pBMP-9 release kinetics by finite differences using a system based on Fick's second law in which the interactions between the peptide and collagen fibers are assumed to follow Langmuir adsorption kinetics. The Langmuir isotherms suggest that the structure of the collagen gel influences the strength of its electrostatic interaction with the peptide, since increasing the collagen concentration decreased the affinity of pBMP-9 for the collagen. The resulting model of the mechanism accurately reflects the experimental data and the parameters estimated indicate that the diffusivities with the different collagen concentrations are similar, whereas the mass transfer coefficient increases with the collagen concentration. The results also indicate that perfect sink conditions cannot be assumed and suggest the presence of an optimal collagen concentration. Finally, we have correlated our conclusions with the differences in collagen fiber organization observed by transmission electron microscopy., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Biomaterials functionalized by adhesive peptides improve the cell-substratum interaction. However, their influence on the response of cells to growth factors is still poorly understood. We have shown that bone morphogenetic protein (BMP) 2 activates the Smad pathway only in murine MC3T3-E1 preosteoblasts attached to polycaprolactone (PCL) film functionalized by RGD peptides derived from bone sialoprotein (pRGD). We have now analysed the way recombinant human BMP-2 and/or BMP-9 (0.38 nM) influence the signal transduction and differentiation of MC3T3-E1 preosteoblasts attached to PCL-pRGD. While kinetics of MAPK activation were similar in cells treated by BMP-2 and BMP-9, different kinetics of Smad activation and β-catenin stabilization were observed. BMP-2 induced Smad1/5/8 phosphorylation within 0.5 and BMP-9 within 4 h, while the β-catenin was lower at 2 h only in cells treated with BMP-9. However, both BMPs induced the translocation of phosphorylated Smad1/5/8 to the nucleus at 4 h and increased Dlx5, osterix and osteocalcin transcripts as well as alkaline phosphatase activity at 72 h. A BMP-2/BMP-9 combination that maintained the β-catenin amount constant but reduced that of phosphorylated Smad within 4 h had quite similar effect than BMP-2 alone. It is therefore important to determine how biomimetic materials influence the response of cells to BMPs., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

More and more of our aging populations will suffer from large bone defects in the next few years. But the growth factor (GF) delivery systems (DSs) currently under investigation will help overcome the limitations of the bone grafts presently used. Some GFDSs accredited by the Food and Drug Administration (FDA) are commercially available, but they have mechanical, structural and GF retention weaknesses. New studies focus on polymers and the composition of GFs in order to mimic as closely as possible the physiological environment of healing bone. This review first summarizes the process of endochondral bone healing and the major cytokines involved. We then review the latest GFDSs, with their combinations of organic, inorganic, natural and synthetic biomaterials, the kinetics of GF release and their biological effects. We will explore new research avenues such as the use of peptides derived from bone morphogenetic proteins, including our own results, and the sequential release of bone-inducing GFs. We then review the latest mathematical models of drug delivery systems (DDSs) for several transport phenomena that may be encountered when using GFDS. The final section discusses new improvements for GFDS modeling., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The efficiency of biomaterials used in bone repair depends greatly on their ability to interact with bone cells. Hence, we have functionalized polycaprolactone (PCL) films by peptides derived from the bone sialoprotein containing RGD sequence (pRGD), to increase their ability to interact with murine MC3T3-E1 preosteoblasts, and favour cell response to recombinant human bone morphogenetic protein-2 (rhBMP-2). RGE peptides (pRGE) were used as negative controls. The PCL films were hydrolyzed with NaOH and then carboxylic acid groups were activated to allow chemisorption of the peptides. Alkaline treatment increased the hydrophilicity of PCL films without significantly change their roughness. Peptide immobilization on PCL was checked by X-ray photoelectron spectroscopy. Hydrolyzed PCL films (Hydro PCL), which adsorbed fibronectin and vitronectin from serum after 1 h incubation, prevented the spreading of MC3T3-E1 preosteoblasts, while films bearing pRGD or pRGE did not. In contrast, MC3T3-E1 preosteoblasts attached to pRGD and incubated for 1 h in serum-free medium spread better than cells on Hydro PCL or pRGE. Only cells on pRGD had organized cytoskeleton, phosphorylated focal adhesion kinase on Y(397) and responded to rhBMP-2 by activating Smad pathway. Thus, pRGD PCL may be used to favour bone cell cytoskeletal organization and response to rhBMP-2., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)