Periodic Reporting for period 1 - 3NTHESES (3D-printed boNes and Tendon-inspired Hierarchical Electrospun Scaffolds strategies to enhance the Enthesis regeneration via Stem cells)
Periodo di rendicontazione: 2022-10-01 al 2024-09-30
Tendon lesions are one of the most relevant musculoskeletal sources of injuries worldwide. Among them, the most frequent site of rupture is located at the enthesis (i.e. tendon-to-bone junction). This is caused by complex collagen fibrils’ structure and mechanics of this interfacial tissue region. In fact, the enthesis, designed by nature to connect soft (tendon) and hard (bone) tissues, is mainly composed of four distinct but continuous extracellular matrix (ECM) regions populated by different cells: tendon (aligned collagen fibrils; tenocytes), non-mineralized fibrocartilage (progressive loss of fibrils alignment and conical shape; fibrochondrocytes), mineralized fibrocartilage (increment of random orientation of fibrils; hypertrophic fibrochondrocytes) and bone (fibrils organized in trabecular bone tissue; osteocytes). Moreover, starting from the mineralized fibrocartilage, ECM assumes a progressive gradient of mineralization/stiffness approaching the bone. To avoid abrupt ruptures at the enthesis, the non-mineralized fibrocartilage assumes a typical conical structure, able to self-expand in response to a load (i.e. auxetic behavior), serving as a stress concentration reducer.
Due to the morphological/mechanical similarities with the human tissue, large animal models, especially the sheep ones, are often used to validate innovative implantable regenerative or prosthetic devices in vivo.
From a regenerative perspective, dedicated biofabrication strategies are often applied to regenerate the different regions of the enthesis. In particular, electrospinning (ES), and its polymeric nanofibers, is the most suitable technique to mimic the collagen fibril structure of tendons and fibrocartilage, while additive manufacturing (AM) is the perfect strategy to reproduce the bone tissue. These scaffolds have widely demonstrated to enhance stem cell proliferation and differentiation when these cells are cultured on them.
However, despite the preliminarily results shown in literature, a method to faithfully mimic the target tendon-enthesis-bone chain of interest, driving the stem cells fate, is unexplored so far.
The aim of 3NTHESES was to fill these scientific gaps by developing the first multi-layered hierarchical multi-material scaffold (MLHMMS) able to biomimetically replicate the multiscale structure/mechanics of a target sheep tendon-enthesis-bone complex of interest. This ambitious goal was achieved by combining the skills in the unique hierarchical ES technique developed by the applicant (Dr. Alberto Sensini) to mimic the tendon tissue and his skills in morphological/mechanical characterizations of biological/biofabricated tissues, with the top-notch AM, stem cells and enthesis knowledge of the host institution (Maastricht University, Profs. Lorenzo Moroni and Martijn van Griensven).
Specifically, the objectives of 3NTHESES were:
1. To study the morphology and mechanics of a relevant target animal model of interest (i.e. sheep calcaneal tendon) by using a mix of imaging techniques, biological evaluations and imaging-based mechanical characterizations. This to parallelly acquire data to replicate the tissue and increase the limited biomechanical knowledge concerning this relevant animal model.
2. To develop and characterize a panel of innovative ES scaffolds to mimic, with a bottom-up hierarchical approach, the whole target sheep tendon and enthesis tissues.
3. To develop procedures to match image-based biomimetic AM bone scaffolds and ES tendon-enthesis biomimetic scaffolds to produce innovative MLHMMS able to drive the stem cells fate.
3NTHESES reached all the objectives opening the way for new research to maximize its scientific impact.
Due to the morphological/mechanical similarities with the human tissue, large animal models, especially the sheep ones, are often used to validate innovative implantable regenerative or prosthetic devices in vivo.
From a regenerative perspective, dedicated biofabrication strategies are often applied to regenerate the different regions of the enthesis. In particular, electrospinning (ES), and its polymeric nanofibers, is the most suitable technique to mimic the collagen fibril structure of tendons and fibrocartilage, while additive manufacturing (AM) is the perfect strategy to reproduce the bone tissue. These scaffolds have widely demonstrated to enhance stem cell proliferation and differentiation when these cells are cultured on them.
However, despite the preliminarily results shown in literature, a method to faithfully mimic the target tendon-enthesis-bone chain of interest, driving the stem cells fate, is unexplored so far.
The aim of 3NTHESES was to fill these scientific gaps by developing the first multi-layered hierarchical multi-material scaffold (MLHMMS) able to biomimetically replicate the multiscale structure/mechanics of a target sheep tendon-enthesis-bone complex of interest. This ambitious goal was achieved by combining the skills in the unique hierarchical ES technique developed by the applicant (Dr. Alberto Sensini) to mimic the tendon tissue and his skills in morphological/mechanical characterizations of biological/biofabricated tissues, with the top-notch AM, stem cells and enthesis knowledge of the host institution (Maastricht University, Profs. Lorenzo Moroni and Martijn van Griensven).
Specifically, the objectives of 3NTHESES were:
1. To study the morphology and mechanics of a relevant target animal model of interest (i.e. sheep calcaneal tendon) by using a mix of imaging techniques, biological evaluations and imaging-based mechanical characterizations. This to parallelly acquire data to replicate the tissue and increase the limited biomechanical knowledge concerning this relevant animal model.
2. To develop and characterize a panel of innovative ES scaffolds to mimic, with a bottom-up hierarchical approach, the whole target sheep tendon and enthesis tissues.
3. To develop procedures to match image-based biomimetic AM bone scaffolds and ES tendon-enthesis biomimetic scaffolds to produce innovative MLHMMS able to drive the stem cells fate.
3NTHESES reached all the objectives opening the way for new research to maximize its scientific impact.
3NTHESES was organized in three interconnected work packages (WP). In WP1 we carried out a comprehensive overview of the morphology and mechanics of the sheep tendon of triceps surae muscle (TTSM). This tendon fascicle is one of the two principal fascicles which compose the calcaneal bone. The morphology was studied by using scanning electron microscopy (SEM) on different regions of the decellularized tissue. This procedure allowed to detect the specific dimensions/organization of the ECM collagen fibrils and trabecular bone of both the tendon, fibrocartilage and bone tissue. A synchrotron micro-computed x-ray tomography of the calcaneal bone enthesis was acquired to investigate the 3D-volume structure of the tissue and produce STL models to be subsequently 3D-printed obtaining the bone scaffolds. Histology was performed to study the composition and structure of the ECM of the different regions of the TTSM. Quantitative backscatter images (qBEI) were acquired to quantify the percentage of mineral content at the mineralized fibrocartilage and bone tissue. Then, qBEI images were used to investigate via nanoindentation the nano-mechanics of these different regions. Finally, mechanical tensile tests coupled with Digital Image Correlation (DIC) were performed to study the TTSM mechanics and the superficial strain distribution. Peaks of load were registered during cyclic tests, to study the evolution of the progressive loss of load of the tissue during the cycles.
The results of WP1 will be soon summarized in a dedicated preprint and in a scientific paper.
In WP2 at first, we designed innovative electrospun PLLA/collagen-based enthesis fascicle-inspired multimaterial conical bundles (mimicking the non-mineralized fibrocartilage) of nanofibers resembling the structure and mechanics of all the different regions of the enthesis tissue. We morphologically verified the biomimicry of bundles with the natural tissues with SEM and synchrotron micro-/nanoCT. The biomechanical mimicry was confirmed via an extensive mechanical characterization at physiological strain-rates. Human mesenchymal stromal cells (hMSCs) proliferation and differentiation was assessed via a parallel spheroid culture up to 28 days in basic media of both an as spun and a nano-mineralized version of bundles at the mineralized fibrocartilage region, showing a more balanced expression of all the tendon, enthesis and bone markers in the as spun ones. This data was obtained by using qPCR, SEM, confocal microscopy, immunostaining and ELISA immunolabeling assays. Finally, the auxetic volumetric expansion of the conical junction of bundles was confirmed by a cutting-edge and unpreceded synchrotron multiscale in situ tests coupled with Digital Volume Correlation (DVC), further verifying the biomimicry of the enthesis tissue. This complex experiment was possible thanks the previous optimization of a protocol to apply DVC on electrospun scaffolds described in Sensini et al., Heliyon, 2024 (https://doi.org/10.1016/j.heliyon.2024.e26796(si apre in una nuova finestra)). The results of the work are at the moment under revision in a scientific paper and summarized in the preprint Sensini et al., BioRxiv, 2024 (DOI: https://doi.org/10.1101/2024.08.12.607645(si apre in una nuova finestra)).
Subsequently bundles were used as building blocks to assembly biomimetic ES hierarchical scaffolds (EHS), resembling the dimensions of the tendon-enthesis side of TTSM, by using the hierarchical ES technique developed by Dr. Sensini. EHS reproduced the whole hierarchical structure of the tendon-enthesis side of sheep TTSM. EHS were matched with PLA-based 3D-printed calcaneal bone scaffolds, during the printing, obtaining the final TTSM-inspired MLHMMS. Scaffolds well resembled the structure and mechanics of TTSM (both in tensile and in cyclic tests).
In WP3 portions of the bone STL model were printed and matched with scaled versions of the whole MLHMMS previously obtained and will be cultured with hMSCs up to 28 days in static and dynamic conditions in a mechanical bioreactor. The biomechanical properties of the as spun MLHMMS will be compared with ones after 28 days of culture. Moreover, hMSCs differentiation and proliferation will be investigated by using qPCR, SEM and immunostaining. We are currently performing these last experiments due to some delays caused by some adjustments of the electrospinning machine at MERLN.
The results of 3NTHESES were/will be presented at sevaral national/international conferences. The results will be published in three major preprints and subsequent publications. Moreover, additional papers will be produced to describe the results of several additional related scientific discoveries done during the project.
The results of WP1 will be soon summarized in a dedicated preprint and in a scientific paper.
In WP2 at first, we designed innovative electrospun PLLA/collagen-based enthesis fascicle-inspired multimaterial conical bundles (mimicking the non-mineralized fibrocartilage) of nanofibers resembling the structure and mechanics of all the different regions of the enthesis tissue. We morphologically verified the biomimicry of bundles with the natural tissues with SEM and synchrotron micro-/nanoCT. The biomechanical mimicry was confirmed via an extensive mechanical characterization at physiological strain-rates. Human mesenchymal stromal cells (hMSCs) proliferation and differentiation was assessed via a parallel spheroid culture up to 28 days in basic media of both an as spun and a nano-mineralized version of bundles at the mineralized fibrocartilage region, showing a more balanced expression of all the tendon, enthesis and bone markers in the as spun ones. This data was obtained by using qPCR, SEM, confocal microscopy, immunostaining and ELISA immunolabeling assays. Finally, the auxetic volumetric expansion of the conical junction of bundles was confirmed by a cutting-edge and unpreceded synchrotron multiscale in situ tests coupled with Digital Volume Correlation (DVC), further verifying the biomimicry of the enthesis tissue. This complex experiment was possible thanks the previous optimization of a protocol to apply DVC on electrospun scaffolds described in Sensini et al., Heliyon, 2024 (https://doi.org/10.1016/j.heliyon.2024.e26796(si apre in una nuova finestra)). The results of the work are at the moment under revision in a scientific paper and summarized in the preprint Sensini et al., BioRxiv, 2024 (DOI: https://doi.org/10.1101/2024.08.12.607645(si apre in una nuova finestra)).
Subsequently bundles were used as building blocks to assembly biomimetic ES hierarchical scaffolds (EHS), resembling the dimensions of the tendon-enthesis side of TTSM, by using the hierarchical ES technique developed by Dr. Sensini. EHS reproduced the whole hierarchical structure of the tendon-enthesis side of sheep TTSM. EHS were matched with PLA-based 3D-printed calcaneal bone scaffolds, during the printing, obtaining the final TTSM-inspired MLHMMS. Scaffolds well resembled the structure and mechanics of TTSM (both in tensile and in cyclic tests).
In WP3 portions of the bone STL model were printed and matched with scaled versions of the whole MLHMMS previously obtained and will be cultured with hMSCs up to 28 days in static and dynamic conditions in a mechanical bioreactor. The biomechanical properties of the as spun MLHMMS will be compared with ones after 28 days of culture. Moreover, hMSCs differentiation and proliferation will be investigated by using qPCR, SEM and immunostaining. We are currently performing these last experiments due to some delays caused by some adjustments of the electrospinning machine at MERLN.
The results of 3NTHESES were/will be presented at sevaral national/international conferences. The results will be published in three major preprints and subsequent publications. Moreover, additional papers will be produced to describe the results of several additional related scientific discoveries done during the project.
3NTHESES is producing a panel of substantial progress beyond the state of the art of the design and biofabrication of a new generation of multimaterial biomimetic scaffolds for the regeneration of the whole tendon-enthesis-bone chain. The innovative pipelines for the assembly of scaffolds and the developed biofabrication protocols will open the way for a new generation of biomimetic, mechanical competent hierarchical scaffolds pushing the boundaries of the biofabrication of soft-hard tissues towards a credible personalized medicine. This also supported by a consistent differentiation of hMSCs towards the tendon, fibrocartilage and bone markers.
Most of the developed protocols were based on open-source softwares and commercial 3D-printers making the production of these devices easily replicable in each laboratory/hospital. Moreover, the innovative auxetic enthesis fascicle-inspired bundles developed, constitute the first example in literature of a material able to replicate the auxetic strain pattern of enthesis from the nano- up to the macroscale in a structural biomimetic manner. Furthermore, the cutting-edge synchrotron multiscale DVC protocols developed allowed, for the first time to describe the reorganization of electrospun nanofibers inside a scaffold producing a remarkable step forward in several research fields.
Most of the developed protocols were based on open-source softwares and commercial 3D-printers making the production of these devices easily replicable in each laboratory/hospital. Moreover, the innovative auxetic enthesis fascicle-inspired bundles developed, constitute the first example in literature of a material able to replicate the auxetic strain pattern of enthesis from the nano- up to the macroscale in a structural biomimetic manner. Furthermore, the cutting-edge synchrotron multiscale DVC protocols developed allowed, for the first time to describe the reorganization of electrospun nanofibers inside a scaffold producing a remarkable step forward in several research fields.