Induced pluripotent stem cell seeded active osteochondral nanofibrous scaffolds

Project Information

iP-OSTEO

Grant agreement ID: 824007

DOI

10.3030/824007

Project closed

EC signature date 14 November 2018

Start date 1 February 2019

End date 31 July 2024

Funded under

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Total cost

€ 1 099 400,00

EU contribution

€ 1 099 400,00

Coordinated by

USTAV EXPERIMENTALNI MEDICINY AKADEMIE VED CESKE REPUBLIKY VEREJNA VYZKUMNA INSTITUCE
Czechia

Periodic Reporting for period 2 - iP-OSTEO (Induced pluripotent stem cell seeded active osteochondral nanofibrous scaffolds)

Reporting period: 2021-02-01 to 2024-07-31

Summary of the context and overall objectives of the project

Scientific objective of the project is the development of Induced Pluripotent Stem cells (iPSC)-based biodegradable implants for old patients affected by osteochondral defects. iPSCs are derived from terminally differentiated patient cells and re-programmed to pluripotent cells, which could be differentiated to wide range range of cell types. Specific scaffolds are being developed for patient with osteoarthritis, both fractures/osteochondral defect and osteoporosis. For this purpose, we have to develop scaffolds for both bone and cartilage. The scaffolds should be biocompatible, degradable, with a porous structure, should allow cell ingrowth, mainly mesenchymal stem cells (MSCs) from bone marrow. The scaffolds should control MSCs or iPSC differentiation into osteoblasts, present in bone, and to chondrocytes which are present in cartilage. In order to control cell differentiation, the scaffolds contain bioactive compounds, such as phosphates, hormones and growth factors. and release them for several days or weeks.
The impact of the new composite scaffold will be on older population where the incidence of trauma, osteoarthritis and osteoporosis is increased. The disability of movement associated with joint impairment leads to decreased quality of life and socioeconomic loss. With ageing of western population the problem becomes more evident and is projected that 25% of adults will have problem with joint disease (Hootman JR, 2006). Similarly, the number of osteoporotic patients (22 mio. woman and 5.5 mio. men in the EU) is rising and results in increased numbers of serious fractures. The effective therapy will decrease the number of their reoperation, will decrease the costs for surgery as well as indirect costs and will reduce the morbidity of patient and will increase the life quality of patients.
OBJECTIVES:
- To develop an international, intersectorial and multidisciplinary network to boost progress in the regeneration of osteochondral defects
- Optimized isolation and differentiation protocols for iPSCs) for musculoskeletal use – protocols for iPSC source will be optimized to achieve reproducible reprogramming and regenerative potential with 90% reproducibility.
- Nanofibrous scaffolds with optimized biological and mechanical properties – iPSCs will show complete scaffold colonization, the scaffold will enable vascular ingrowth and mechanical properties respecting needs of particular tissues.
- Up-scaled and stabilized manufacturing of implants – technology will be up-scaled to form compliant with GMP manufacturing, the production rate will be at least 100 standard scaffold/day and standard-operational protocols (SOPs) will be delivered for commercialization.
- Decreased re-operation of complex bone fractures and osteoarticular injuries by 20% measured by regeneration outcomes of pre-clinical studies.
-Enhancement of healing of bone fractures and osteochondral defects in pre-clinical studies.
-Development of a Business Plan. The Consortium will define the best strategy for future commercialization and exploitation, with special focus on the translational process for introduction of the new therapy into the European and world markets.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

We have developed composite scaffold for bone regeneration based on polycaprolactone (PCL) nanofibres enriched with either hydroxyapatite (HA) or bioactive molecules. Scaffolds were optimized for the desired release of compounds and for osteogenic differentiation of mesenchymal stem cells (MSCs). Similarly, induced pluripotent stem cells (iPCs) were studied for their osteogenic and chondrogenic differentiation.
We have developed cell-free composite nanofibrous scaffolds stimulating osteochondral regeneration in rabbits. Different 3D scaffolds based on hydrogels were tested with bioactive supplements in order to control chondrogenic differentiation of MSCs. Moreover, SmartBonePep®, xeno-hybrid organic graft with bioactive peptides for bone regeneration stimulated osteogenic differentiation of mesenchymal stem cells. We prepared hybrid nanofibers functionalized with plasmonic TiN NPs. They open up new avenues for theranostic applications. Advances in hybrid nanofibers for biomedical applications were studied. A new 3D-printed chamber for a bioreactor for dynamic cell culture on scaffolds was developed.
Nano-and microfibres from a block copolymer of PCL and poly(lactic acid) and HA. Fibres exhibited pores with a diameter in nanoscale range and supported osteosarcoma cells´growth. We have developed microparticles containing kartogenin using electrospraying. The tuneable release was observed from PLGA particles or as a blend of PLGA with with polyethyleneglycol or polyvinylpyrrolidone.
Both blend and coaxial electrospinning equally preserved ALP bioactivity at ca.100%. ALP activity decreased by coaxial electrospraying, but not blend electrospraying. Electrospinning and electrospraying of scaffolds containing proteins were studied. Electrospun scaffolds from PCL containing alendronate (ALN), an anti-osteoporotic drug, and HA nanoparticles were developed. Scaffolds with ALN implanted into defects of osteoporotic rats and control animals supported formation of bone tissue in the vicinity of scaffold residues and in the tissue surrounding the former defect.
MSCs and fibroblasts were cultures in medium containing platelet proteins in plasma, pure platelet proteins in deionized water, and pure plasma. The highest proliferation and viability of cells were found in the group containing both plasma and plasma proteins.
Catalase was covalently bound to the surface of PCL nanofibres, and retained its enzymatic activity; loading remained attached after six days. Osteochondral defects treatment using nanofibres and 3D printed scaffolds and cells, and piezoelectric materials in bone regeneration were studied.

SmartBonePep® for bone regeneration represents a significant advancement resulting from the iP-OSTEO project, with potential applications in clinical settings pending further development and regulatory approvals. The design of 3D chamber for dynamic culture was protected and will be offered commercially. Alcian blue and PAS histological staining were successfully combined and applied on samples prepared by laser cutting. Laser cutting and the combined staining are available commercially. The other results, including 2D and 3D bioactive scaffolds, obtained during the project are present in protocols, including SOPs, demonstrators, reports or know-how and will be implemented in further research or business. The cooperation led to novel projects submission.
Results were presented at conferences, seminars, webinars, and during different events for the public e.g Night of Scientists, Week of Academy of Sciences, Science Fest, on website, and published in 13 articles with impact factor.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

We expect development od 3D composite scaffold delivering osteogenic and chondrogenic supplements or growth factors for osteochondral regeneration. We expect enhanced synthesis of tissue specific matrix. Moreover, iPSC can be differentiated into the desired cell type and then can stimulate the formation of either bone or cartilage. The scaffold will be tested in vitro in a bioreactor and in vivo. In the first two years of the project we have developed drug delivery systems based on nanofibres, microfibres or microparticles. Moreover 3D scaffolds enhancing formation of cartilage have been prepared.

Nanofibres from polycaprolactone

Spray dried microparticles for the delivery of proteins

Collagen II (chondrogenic marker - green) produced by mesenchymal stem cells (red nuclei) in gel

Design of perfusion bioreactor for 3D cell culture developed in OSPIN

Osteogenic protei osteocalcin (red) produced by mesenchymal stem cells (blue nuclei) in a scaffold

Chamber for dynamic cell culture on scaffolds designed by Biofabics