Periodic Report Summary 2 - FIBROGELNET (Network for Development of Soft Nanofibrous Construct for Cellular Therapy of Degenerative Skeletal Disorders)
In compliance with the major objective of FIBROGELNET project: “To bring together the expertise of academia and industry Partners for the development of an innovative strategy for targeting bone and cartilage regeneration”, the main activities during the reporting period (2016-2017) were as follows:
1. Efforts for the current and sufficient production of FIBROGELNET construct building blocks (gel-nanofibers discs) and their distribution to the Partners
2. Assuring the planned exchange of researchers between Academia (IBEC, UPV) and Industry (BioElpida and BulGen)
3. To concentrate their efforts on the updated research plan (established during discussions in the Midterm Consortium meeting (Lyon 2015), i.e. to switch the strategy of the project to the development of an osteochondral construct
4. Analysis and arrangement of the obtained results in a publishable quality
5. Publishing of the project results
The work on (Activity 1) was performed mainly at IBEC (Coordinator) and UPV, yet dedicated on the “material’s part” of the project (WP 1 and WP 2). UPV assured production of gel-slices (WP1), while IBEC was involved in the preparation of nanofibers and their deposition on these slides representing the building blocks of the construct (according WP2). In addition, following the decisions of the Midterm consortium meeting in Lyon, UPV organized production of polylactic aid (PLA) sponge-like inserts considered for inclusion in a putative osteochondral construct (Activity 3). It comprises the main experimental effort for the reported period dedicated on bringing the FIBROGELNET construct closer to the practical application. The idea was to introduce a sponge-like material within the environmental hole in the middle of construct that will contains stem cells (in 3D environment) previously committed to osteogenic lineage. Conversely, the covering nanofibers will contain cells (in 2D environment) that are further committed to a chondrogenic direction – therefore the construct is termed osteochondral. The rationale comes from the preceding results showing the best chondrogenesis on 2D random nanofibers, while the 3D environment is required for osteogenesis. An additional effort was dedicated on the direct inclusion of BMP-2 in the PLA sponge (according WP3). The main experiments and scientific results were obtained during the visits of IBEC and UPV researchers in BulGen (in compliance with Activity 2) - exchange of researchers. In this respect BulGen was the most visited laboratory in the reported period, while IBEC and UPV were involved in fulfilling the secondments program. The coordination was assured by IBEC through two separate visits of the Coordinator (Prof Altankov) in BulGen. Altogether in BulGen were performed 7 research visits (15 Mo), dedicated on the development of the osteochondral construct (Activity 3). BioElpida hosted 3 more researchers (7 Mo) to receive theoretical training in manufacturing, production, quality control (QC) and quality assurance (QA). During her visit Rosa Morales developed a GMP adapted protocol for the production of PLA sponge.
From scientific point of view, we will note on the most important conclusions resulting from the comparing of chondrogenic response of human adipose derived stem cells (ADMSCs) and umbilical vein derived ones (UDMSCs) in the presence and absence of PLA sponge in the construct. The stem cells in the sponges were previously committed to osteogenesis (21 days), while whole construct was subsequently committed to chondrogenesis (up to Day 51 in Chondrogenic medium). The efficiency of chondrogenic and osteogenic differentiation of whole construct was assayed through qPCR. The effect of preloading of sponge with BMP-2, performed by Partner MLU (Germany) according WP3, was also assayed in separate experiment. The main conclusions from these studies were: (i) inclusion of BMP-2 in the sponge significantly diminish the chondrogenic response of ADMSCs judged from the activity of COL 10, RUNX 2 and SOX 9 genes; (ii) the sponge itself increase the chondrogenic activity of ADSCs viewed by the activity of same genes; (iii) the chondrogenic environment strongly inhibit the osteogenic activity of ADMSCs in whole construct (coming from the sponge insert) judged by the ALP and BSP genes expression (iv) the chondrogenesis-supporting effect of the sponge insert on the chondrogenic activity was more prominent on random nanofibers; (v) for the first time was observed that gel-nanofibers environment itself provoke spontaneous chondrogenic differentiation of ADMSCs independently on the nanofibers orientation, while the osteo-committed sponge inhibit it; (vi) Umbilical stem cells UDMSCs show a bit different behaviour, increasing both spontaneous and induced chondrogenic activity with sponges and no effect of chondrogenic environment on the osteogenic response. These data support the view that loading the putative implants with growth factors or pre-committed stem cells may have competitive (cross-inhibitory) effect on their proper differentiation in situ. To relay on the spontaneous differentiation of stem cells under physiological conditions appears to be a very promising alternative.
According (Activity 4) connected with improving the publishing activity during the reporting period in IBEC were recruited two postdocs, the senior postdoc Firas Awaja (ER>10 years) from Italy and a young postdoc Dr. Salima Nedjari (ER<10 years) from France, both for 1 year. Dr Nedjary was working on the implication of specific honeycomb arranged nanofibers to support the osteogenic response of stem cells. This sub-project was successfully accomplished with submitting a research paper in Scientific reports (Nature group) entitled “Three Dimensional Honeycomb Patterned Fibrinogen Based Nanofibers Induce Substantial Osteogenic Response of Mesenchymal Stem Cells, authored by Salima Nedjari, Firas Awaja and George Altankov. This paper is under review.
Together with the Coordinator, Dr Awaja was mostly involved in the analysis of the project results and their publication (Activity 5). For the reporting period were published 4 papers, some of which contain results from previously reported period, namely:
(1) Gugutkov D, Gustavsson J, Cantini M, Salmeron-Sánchez M and Altankov G ( 2016) Electrospun Fibrinogen/PLA Nanofibers for vascular tissue engineering, Tissue Engineering and Regenerative Medicine (in press) published on line 30 May 2016:
Here we reported our new type of hybrid FBG–PLA nanofibres showing that randomly organized NFs could support the endothelization of implants while aligned NFs would rather direct cell locomotion for guided neovascularization.
(2) Forget J, Awaja F, Gugutkov D, Gustavsson J, Gallego Ferrer G, Coelho-Sampaio T, Hochman-Mendez C, Salmeron-Sánchez M, and Altankov G (2016) Differentiation of Human Mesenchymal Stem Cells Toward Quality Cartilage Using Fibrinogen-Based Nanofibers Macromol. Biosci. 2016 vol 16(9) 1348-1359
In this paper we show that human adipose derived mesenchymal stem cells (hADMSCs)
establishes significantly better chondrogenic differentiation and may generate quality cartilage when cultured on 2D environment of randomly oriented nanofibers.
(3) Gugutkov D, Awaja F, Belemezova K, Keremidarska M, Krasteva N, Kuyrkchiev S, Gallego Ferrer G, Seker S, Elcin AE, Elcin YM and Altankov G 2017 "Osteogenic Differentiation of Mesenchymal Stem Cells using Hybrid Nanofibers with Different Configurations and Dimensionality" Journal of Biomedical Materials Research Part A (in press) DOI: 10.1002/jbm.a.36065
Here we aim at producing bone-like tissue in vitro based on an engineered nanofibrous scaffold able to support the osteogenic differentiation of adult human MSCs. The role of nanofibers orientation (random vs. aligned) and dimensionality (2D vs. 3D environment) are clearly addressed.
(4) Hristova-Panusheva K, Keremidarska-Markova M, Altankov G, Krasteva N. Age-related Changes in Adhesive Phenotype of Bone Marrow-derived Mesenchymal Stem Cells on Extracellular Matrix Proteins Age-related Changes in Adhesive Phenotype of Bone Marrow-derived Mesenchimal stem cells Journal of New Results in Science (JNRS) 2017, 6(1) 11-19
This is a cooperative work showing the effect of aging on the adhesion behaviour of MSC and MG-63 osteoblasts within 21 days of culture suggesting that age-related changes have to be considered when expanding adult stem cells for clinical applications.
Thus, all planned scientific activities of FIBROGELNET project were successfully accomplished.
http://fibrogelnet.org/
1. Efforts for the current and sufficient production of FIBROGELNET construct building blocks (gel-nanofibers discs) and their distribution to the Partners
2. Assuring the planned exchange of researchers between Academia (IBEC, UPV) and Industry (BioElpida and BulGen)
3. To concentrate their efforts on the updated research plan (established during discussions in the Midterm Consortium meeting (Lyon 2015), i.e. to switch the strategy of the project to the development of an osteochondral construct
4. Analysis and arrangement of the obtained results in a publishable quality
5. Publishing of the project results
The work on (Activity 1) was performed mainly at IBEC (Coordinator) and UPV, yet dedicated on the “material’s part” of the project (WP 1 and WP 2). UPV assured production of gel-slices (WP1), while IBEC was involved in the preparation of nanofibers and their deposition on these slides representing the building blocks of the construct (according WP2). In addition, following the decisions of the Midterm consortium meeting in Lyon, UPV organized production of polylactic aid (PLA) sponge-like inserts considered for inclusion in a putative osteochondral construct (Activity 3). It comprises the main experimental effort for the reported period dedicated on bringing the FIBROGELNET construct closer to the practical application. The idea was to introduce a sponge-like material within the environmental hole in the middle of construct that will contains stem cells (in 3D environment) previously committed to osteogenic lineage. Conversely, the covering nanofibers will contain cells (in 2D environment) that are further committed to a chondrogenic direction – therefore the construct is termed osteochondral. The rationale comes from the preceding results showing the best chondrogenesis on 2D random nanofibers, while the 3D environment is required for osteogenesis. An additional effort was dedicated on the direct inclusion of BMP-2 in the PLA sponge (according WP3). The main experiments and scientific results were obtained during the visits of IBEC and UPV researchers in BulGen (in compliance with Activity 2) - exchange of researchers. In this respect BulGen was the most visited laboratory in the reported period, while IBEC and UPV were involved in fulfilling the secondments program. The coordination was assured by IBEC through two separate visits of the Coordinator (Prof Altankov) in BulGen. Altogether in BulGen were performed 7 research visits (15 Mo), dedicated on the development of the osteochondral construct (Activity 3). BioElpida hosted 3 more researchers (7 Mo) to receive theoretical training in manufacturing, production, quality control (QC) and quality assurance (QA). During her visit Rosa Morales developed a GMP adapted protocol for the production of PLA sponge.
From scientific point of view, we will note on the most important conclusions resulting from the comparing of chondrogenic response of human adipose derived stem cells (ADMSCs) and umbilical vein derived ones (UDMSCs) in the presence and absence of PLA sponge in the construct. The stem cells in the sponges were previously committed to osteogenesis (21 days), while whole construct was subsequently committed to chondrogenesis (up to Day 51 in Chondrogenic medium). The efficiency of chondrogenic and osteogenic differentiation of whole construct was assayed through qPCR. The effect of preloading of sponge with BMP-2, performed by Partner MLU (Germany) according WP3, was also assayed in separate experiment. The main conclusions from these studies were: (i) inclusion of BMP-2 in the sponge significantly diminish the chondrogenic response of ADMSCs judged from the activity of COL 10, RUNX 2 and SOX 9 genes; (ii) the sponge itself increase the chondrogenic activity of ADSCs viewed by the activity of same genes; (iii) the chondrogenic environment strongly inhibit the osteogenic activity of ADMSCs in whole construct (coming from the sponge insert) judged by the ALP and BSP genes expression (iv) the chondrogenesis-supporting effect of the sponge insert on the chondrogenic activity was more prominent on random nanofibers; (v) for the first time was observed that gel-nanofibers environment itself provoke spontaneous chondrogenic differentiation of ADMSCs independently on the nanofibers orientation, while the osteo-committed sponge inhibit it; (vi) Umbilical stem cells UDMSCs show a bit different behaviour, increasing both spontaneous and induced chondrogenic activity with sponges and no effect of chondrogenic environment on the osteogenic response. These data support the view that loading the putative implants with growth factors or pre-committed stem cells may have competitive (cross-inhibitory) effect on their proper differentiation in situ. To relay on the spontaneous differentiation of stem cells under physiological conditions appears to be a very promising alternative.
According (Activity 4) connected with improving the publishing activity during the reporting period in IBEC were recruited two postdocs, the senior postdoc Firas Awaja (ER>10 years) from Italy and a young postdoc Dr. Salima Nedjari (ER<10 years) from France, both for 1 year. Dr Nedjary was working on the implication of specific honeycomb arranged nanofibers to support the osteogenic response of stem cells. This sub-project was successfully accomplished with submitting a research paper in Scientific reports (Nature group) entitled “Three Dimensional Honeycomb Patterned Fibrinogen Based Nanofibers Induce Substantial Osteogenic Response of Mesenchymal Stem Cells, authored by Salima Nedjari, Firas Awaja and George Altankov. This paper is under review.
Together with the Coordinator, Dr Awaja was mostly involved in the analysis of the project results and their publication (Activity 5). For the reporting period were published 4 papers, some of which contain results from previously reported period, namely:
(1) Gugutkov D, Gustavsson J, Cantini M, Salmeron-Sánchez M and Altankov G ( 2016) Electrospun Fibrinogen/PLA Nanofibers for vascular tissue engineering, Tissue Engineering and Regenerative Medicine (in press) published on line 30 May 2016:
Here we reported our new type of hybrid FBG–PLA nanofibres showing that randomly organized NFs could support the endothelization of implants while aligned NFs would rather direct cell locomotion for guided neovascularization.
(2) Forget J, Awaja F, Gugutkov D, Gustavsson J, Gallego Ferrer G, Coelho-Sampaio T, Hochman-Mendez C, Salmeron-Sánchez M, and Altankov G (2016) Differentiation of Human Mesenchymal Stem Cells Toward Quality Cartilage Using Fibrinogen-Based Nanofibers Macromol. Biosci. 2016 vol 16(9) 1348-1359
In this paper we show that human adipose derived mesenchymal stem cells (hADMSCs)
establishes significantly better chondrogenic differentiation and may generate quality cartilage when cultured on 2D environment of randomly oriented nanofibers.
(3) Gugutkov D, Awaja F, Belemezova K, Keremidarska M, Krasteva N, Kuyrkchiev S, Gallego Ferrer G, Seker S, Elcin AE, Elcin YM and Altankov G 2017 "Osteogenic Differentiation of Mesenchymal Stem Cells using Hybrid Nanofibers with Different Configurations and Dimensionality" Journal of Biomedical Materials Research Part A (in press) DOI: 10.1002/jbm.a.36065
Here we aim at producing bone-like tissue in vitro based on an engineered nanofibrous scaffold able to support the osteogenic differentiation of adult human MSCs. The role of nanofibers orientation (random vs. aligned) and dimensionality (2D vs. 3D environment) are clearly addressed.
(4) Hristova-Panusheva K, Keremidarska-Markova M, Altankov G, Krasteva N. Age-related Changes in Adhesive Phenotype of Bone Marrow-derived Mesenchymal Stem Cells on Extracellular Matrix Proteins Age-related Changes in Adhesive Phenotype of Bone Marrow-derived Mesenchimal stem cells Journal of New Results in Science (JNRS) 2017, 6(1) 11-19
This is a cooperative work showing the effect of aging on the adhesion behaviour of MSC and MG-63 osteoblasts within 21 days of culture suggesting that age-related changes have to be considered when expanding adult stem cells for clinical applications.
Thus, all planned scientific activities of FIBROGELNET project were successfully accomplished.
http://fibrogelnet.org/