ICRS 2019 - Conference Calendar
16.2.2 - A wood-derived biomimetic scaffold for segmental bone reconstruction: Pre-clinical safety and performance assessment in a sheep model
- E. Kon (Milano, IT)
- G. Filardo (Bologna, IT)
- F. Perdisa (Ozzano Dell'Emilia (BO), IT)
- J. Shani (Beit Berl, IL)
- N. Shabshin (Ra'anana, IL)
- F. Veronesi (Bologna, IT)
- F. Salamanna (bologna, IT)
- A. Parrilli (Bologna, IT)
- B. Di Matteo (Rozzano Milano, IT)
- M. Marcacci (Rozzano Milano, IT)
- S. Sprio (Faenza, IT)
- A. Ruffini (Faenza, IT)
- A. Tampieri (Faenza, IT)
Abstract
Purpose
To assess safety and performance of a bone substitute (GreenboneTM) obtained by biomorphic transformation of natural wood into 3D biomimetic substituted calcium phosphate (hydroxyapatite and b-tricalciumphosphate) scaffold for segmental bone reconstruction in sheep model
Methods and Materials
The study evaluated bone implant in 24 sheep randomized to three groups, Greenbone scaffold I, Greenbone scaffold II, and allograft, followed up to 6 months. Bony defects were created in the metatarsus and the scaffold was inserted. Safety assessment considered any AE, macroscopic presentation and treatment-related abnormalities (popliteal lymph nodes histopathology). Performance (cumulative score for callus formation, new bone and implant resorption) was assessed by X-ray or CT and by ex vivo analyses after implant retrieval including microCT, macroscopic, and histological and histomorphometric assessments. Further bone biopsies were conducted for assessing mechanical, osteogenic, osteoclastic and angiogenic characteristics of newly formed bone
Results
Observations confirmed osteoinductive properties for Greenbone scaffolds. Medullary bone formation was observed for the Greenbone groups only, already at month 3. Both interfaces of the scaffolds were covered by newly formed bone, without interposition of fibrous connective tissue in the most internal part of the scaffolds, with the new osteonic systems and newly formed blood vessels; many osteocytes and osteoblasts lining the edges of the calcified structures and outbreaks of osteoclastic resorption on the scaffolds. Presence of bone callous containing micro vessels and osteoblasts was seen in most of the samples. Cortical volume and thickness were similar among groups. Both GB I (p=0.0092) and GBII (p=0.014) scaffolds presented higher OS/BS values vs AG-allograft, with highest OSTh and Ob/BS for GBI-doped with ions compared to GBII HA only non doped and AG. Implant resorption started after the first month.
Conclusion
The present study has confirmed Greenbone's characteristics in terms of biocompatibility, mechanical properties and bioresorbability to provide a safe and highly performing bone substitute for segmental bone reconstruction
16.2.3 - Volume-by-volume bioprinting of chondrocytes-alginate bioinks in high temperature thermoplastic scaffolds for cartilage regeneration
Abstract
Purpose
The objective of this study was to develop a novel volume-by-volume 3D-biofabrication process that divides the printed part into different volumes and injects the cells after each volume has been printed, once the temperature of the printed thermoplastic fibers has decreased. This novel 3D-biofabrication procedure prints a mesh structure layer-by-layer with a high adhesion surface/volume ratio, driving a rapid decrease in the temperature, avoiding contact with cells in high temperature zones. In our study, chondrocytes survived the manufacturing process and after seven days in culture, chondrocytes proliferated and totally colonized the scaffold.
Methods and Materials
A bioprinter with three syringes and one FDM extruder consisting of hardware, designer software with the algorithms that allow the configuration of VbV, and an electronic control unit (ECU) that connects the software to the hardware, were used for the experiments
Results
Results showed an incremental growth in the number of cells, with a stabilization of growth between day 5 and day 7. After 24 h in culture, individual cells with rounded shape appeared; however, seven days later, chondrocytes were able to migrate and proliferate throughout the scaffolds, completely colonizing the PLA fibers, and forming a homogeneous surface. Cell density, was calculated over volume of the scaffold. At seven days, positive cells were found per 1 mm3 of scaffold, being a total of 1,423,000 of cells per 1000 mm3 (total volume of the scaffold).
Conclusion
In conclusion, we have shown that a novel VbV-based bioprinting method enhances chondrocyte survival and distribution within the bioprinted scaffolds, using high temperature thermoplastic without scaffold mesh geometry limitations. VbV solves the two main complications of common bioprinting techniques: 1. it can be used with already clinically approved biomaterials and, 2. this process does not have restrictions in geometries that could limit the clinical application of 3D bioprinting in cartilage TE.
16.2.4 - Structurally and Functionally Optimized Silk Fibroin-Gelatin Scaffold using 3DP to Repair Cartilage Injury
Abstract
Purpose
We want to design a structurally and functionally optimized scaffold for recruiting more autologous BMSCs and providing suitable microenvironment for cartilage regeneration in the knee joint.
Methods and Materials
We designed a structurally and functionally optimized scaffold by integrating silk fibroin with gelatin(SFG) in combination of BMSC-specific affinity peptide(SFG-E7) using three-dimensional printing (3DP) technology. In vitro experiments, we tested the viability and morphology of the BMSCs seeded on the SFG or SFG-E7 scaffolds, measured the recruitment capacity and chondrogenic differentiation of different scaffolds. In animal experiments, there were three groups : MF, MF+SFG and MF+SFG-E7. Then we evaluated the chondrogenic capacity of different groups by staining with hematoxylin and eosin (H&E), toluidine blue, immunostaining of collagen type II antibody and biomechanical analysis of repair cartilage after the rabbits were sacrificed at 6,12,24 weeks.
Results
The scaffolds with mass ratio of 1:2 (6.9 w/v %) were selected which might match the previously reported requirements of tissue engineered cartilage scaffold. In vitro, BMSCs grew well on the fashioned scaffolds and the typical fusiform morphology of BMSCs was observed the scaffolds. Also SFG-E7 scaffolds had higher chondrogenic capacity than SFG scaffolds, as evidenced by more HYP and GAG production and collagen type II expression in vitro. In vivo,SFG group had a better repair effect than MF group and neo-cartilage in SFG-E7 group was more similar to normal cartilage than SFG group through a series of assessment including gross observation, MRI, histology, SEM and biomechanics evaluation.
Conclusion
This dually optimized scaffold has shown superior performance for cartilage repair in knee joint. It appears to be a promising biomaterial for knee cartilage repair, and is worthy of further investigation in large animal studies and preclinical applications. Beyond knee cartilage, this dually optimized scaffold may also serve as an ideal biomaterial for the regeneration of other joint cartilages.
16.2.5 - Magneto-responsive hydrogel recapitulates cartilage-like cell distribution in engineered tissue
Abstract
Purpose
Instructive biomaterials may guide cell position and phenotype to engineer cartilage tissue with native-like depth dependent properties. In this study, an FDA-approved paramagnetic MRI contrast agent, Gadodiamide (Gd), was combined with a hyaluronic acid (HA) solution to create a cytocompatible magneto-responsive hydrogel. We investigated the movement of mesenchymal stem cells (MSCs) within this material under brief exposure to a static magnetic field and the biologic activity of these cells after positioning, to generate tissue constructs with a cell template similar to that of native cartilage.
Methods and Materials
Construct fabrication: A 1% w/v methacrylated HA solution (20 million bovine MSCs/mL) with 0.05% Lithium phenyl-2,4,6-trimethylbenzoylphosphinate, and 200mM Gd, was cast inside a polydimethylsiloxane ring (4mm inner diameter, 1.3mm thickness) on a glass slide (Fig.1A). After 0-minutes, 2-minutes, or 5-minutes of exposure to a static magnetic field (0.28-0.34T), the solution was UV crosslinked. Constructs (n = 8) were cultured for 15 days in chemically defined media with 10ng/mL TGFβ3. Cell distribution/matrix staining: After 9 days of culture, samples were processed, paraffin embedded, and sectioned for labeling of cell nuclei (DAPI) and matrix. Cell viability: Cell viability and metabolism was assessed throughout culture using the live/dead kit and the Alamar blue assay.
Results
With increased exposure to the magnetic field, cells moved away from the surface of the construct closest to the magnet, creating a depth-dependent cartilage-like cell distribution (Fig.1B). Transient exposure to Gd had no deleterious effects on cell viability or metabolism, as assessed by live/dead staining, and Alamar blue assays (Fig.1C,D). By 15 days of culture, all constructs showed similar healthy metabolic activity and matrix staining that reflected the original positioning of cells (Fig. 1D,1E).
Conclusion
The developed magneto-responsive hydrogel eliminates the need for potentially harmful intracellular magnetic tags currently used for magnetic field-based cell positioning, and produces a cartilage-like cell template within a single material.
16.2.6 - Suspended manufacture as a method to produce complex tissue interfaces
Abstract
Purpose
Hydrogels are in many ways the perfect materials to support tissue growth. They typically consist of a high volume of water (approximately 99%) and can form through relatively mild physical changes or chemical reactions. One of the major issues with hydrogels, however, is that they are typically relatively weak and are liquid up to the point of gelation. As a consequence, they are very challenging to fabricate in elaborate morphologies. We have addressed this problem by suspending hydrogel structures within a self-healing structured gel material that provides support to the hydrogel during the gelation process (see figure). In this abstract we report on how we have used this suspended manufacture process to produce 3D structures that consist of multiple cells types.
Methods and Materials
We created an osteochondral defect in a harvested tibial plateau. The defect was subsequently CT scanned and a CAD model of the defect was created. Cells were isolated from the cartilage and bone within the core and expanded before being deposited with a gellan reproduction of the defect (boney section was augmented with nanocrystalline HA) using the suspended manufacuring process with the CAD model. The plug was then reinserted into the defect and cultured for 28 days before characterisation with respect to physicochemical properties and the phenotype of the encapsulated cells.
Results
The plug that was manufactured using the suspended manfacturing process was robust and could be handled and reinserted into the defect without fragmentation. On retrieval from the gellan, the cells deposited within the cartilaginous region of the construct retained their chondrogenic phenotype (ACAN and Coll II expression) and those in the boney region maintained an osteoblastic phenotype (see figure).
Conclusion
We succsesfully demonstrated that suspended manufacture can create complex multicellular structures and that these structures could support maintenance of both osteoblastic and chondrogenic phenotypes in spatially defined regions.
16.2.7 - Mechanisms of cell-free cartilage repair using a modified collagen type I matrix and additive fibrin glue
Abstract
Purpose
Cartilage repair using cell-free matrices has become popular as an alternative to matrix-associated autologous chondrocyte transplantation (MACT). Aim of the present investigation was to better understand the mechanisms behind cell-free cartilage regeneration and the impact of various matrix modifications (e.g. perforation, additive fibrin glue) on cell migration and colonization.
Methods and Materials
A cell-free cartilage matrix composed out of rat-tail-derived type I collagen (CaReS-1S, ArthroKinetics GmbH, Krems a.d. Donau, Austria) was used. The effect of additive fibrin glue on the colonization of CaReS-1S was investigated in a special cell culture insert model. Parameters of cell migration of bone marrow-derived mesenchymal stromal cells (BM-MSC) as well as cell counts and proliferation rates were recorded. Secondly, the colonization potential of various periarticular cell niches was tested to identify a preferable cell source. BM-MSC, cartilage MSC (C-MSC) and synovial MSC (S-MSC) were used. Proliferation (MTT) and migration assays were performed, as well as a matrix migration/penetration depth assessment using color coded confocal microscopy. Modification of the matrix with a self-developed micro-needle device was investigated under chondroinductive and standard conditions.
Results
Fibrin glue showed a good attraction potential to BM-MSC, but cell counts over time, as well as subsequent proliferation rates were significantly higher in CaReS-1S. Regarding colonization potential, CaReS-1S did not show a preference for either periarticular cell niche. Micro-perforation of the matrix led to a deeper cell penetration and increased migration of cells under standard culture conditions, but not under chondrogenically induced conditions. Non-induced MSC, regardless of cell niche, showed a deeper migration as compared to induced MSC without significant differences among groups.
Conclusion
Bone marrow, adjacent cartilage and synovium are all suitable sources for colonization of cell-free matrices. Fibrin glue may hinder trans-migration into matrices in vivo due to a high retention of cells. Micro-perforation of the matrix boosted its colonization, especially of deeper layers.
16.2.8 - The Development of an Anisotropic Composite Silk Nanofibre Bioink for Cartilage Tissue Engineering
Abstract
Purpose
To date, the majority of bioinks comprise highly isotropic structures with poor mechanical properties, which in turn gives rise to engineered tissue with simple unnatural extracellular matrices. This is particularly pertinent to the fabrication of cartilage tissue, which has a highly organised ultrastructure, supporting its anisotropic response to mechanical loading. Accordingly, we present on the development of an anisotropic composite bioink, comprising silk nanofibers aligned in a microporous alginate hydrogel.
Methods and Materials
Silk fibroin was electrospun and processed to generate monodispersed nanofibres with high aspect ratios. The nanofibres were combined with a sodium alginate and Pluronic F127 bioink (Armstrong et al., Adv.Health.Mat, 2016) and the mechanical and rheological properties elucidated using uniaxial compression and rotational rheometry, respectively. The composite bioink was then used to bioprint human mesenchymal stem cells (hMSCs) and the impact of this mechanical reinforcement on the chondrogenic differentiation of the stem cells was investigated.
Results
Reinforcement of the bioink using silk nanofibres increased its compressive modulus (Figure 1), and rheological analysis confirmed its shear-thinning properties. Furthermore, the extrusion printing process drove shear alignment of the nanofibres and the resulting bioink supported the viability of hMSCs (Figure 2). Moreover, analysis of the extracellular matrix produced by the hMSCs in the reinforced bioink confirmed its chondroinductive potential.
Conclusion
Incorporating high aspect ratio silk nanofibres in to an alginate-based bioink improved its mechanical properties. Bioprinting with hMSCs resulted in shear-alignment of the nanofibres and improved the chondrogenic potential of the stem cells. Functionalising these nanofibres with inherent cues could serve as a mechanism to tailor the behaviour of stem cells and fabricate biomimetic zonal cartilage tissue.
16.2.9 - An injectable hydrogel scaffold with Kartogenin-encapsulated nanoparticles for porcine cartilage regeneration – a six months follow-up
Abstract
Purpose
Based on our previous study, the utilization of an ultraviolet (UV) light photo-cross-linkable Hyaluronic acid (HA) hydrogel integrated with Kartogenin-encapsulated nanoparticles obtained good reconstruction of osteochondral defects in rabbit model. To further evaluate the safety and efficacy of this technique, the focal cartilage defects with critical size of a porcine model was used. Moreover, the defects with different sizes were created to assess which defect size it is suitable for repairing in future clinical applications.
Methods and Materials
24 skeletally mature minipigs were randomly divided into three groups, including m-HA hydrogel with Kartogenin-encapsulated nanoparticles treated (m-HA + KGN group), m-HA hydrogel treated (m-HA group) and untreated (Blank group). Focal defects were created in medial femoral condyle in both knees. Full-thickness (FT) cartilage defects (6.5 mm in diameter, 8.5 mm in diameter) and osteochondral defects (6.5 mm and 8.5 mm in diameter, 5mm in depth) were included. At 6 months, all minipigs were sacrificed for assessment of macroscopic appearance, MRI, micro-computed tomography (µCT), histology staining and the evaluation of elastic modulus, hardness and COL2, glycosaminoglycan (GAG) contents of the regenerated tissue.
Results
m-HA + KGN group had improved gross healing and histological scores, compared to m-HA and Blank group. The improved-quality repaired cartilage demonstrated by MRI and better subchondral bone reconstruction assessed by µCT were also observed in m-HA + KGN group. The m-HA + KGN group showed hyaline-like cartilage exhibited by histological staining in terms of extracellular matrix, cartilage lacuna and COL2. The mechanical properties were also improved in m-HA + KGN group, but remained inferior to normal cartilage, while COL2 and GAG contents remained same condition.
Conclusion
The HA hydrogel integrated with Kartogenin-encapsulated nanoparticles appeared to improve cartilage healing in critical sized defects in the minipig model evaluated for 6 months, with better cartilage healing in Full-thickness cartilage defects compared to osteochondral defects.