affordable-oxygen-microscopy-assisted-biofabrication-multicellular

Affordable Oxygen Microscopy-Assisted Biofabrication of Multicellular Spheroids

Multicellular spheroids are important tools for studying tissue and cancer physiology in 3D and are frequently used in tissue engineering as tissue ...

Ghent University

The present study aimed to compare the curing depth of polyacid-modified composite resins (PAM-C) and some representative composite resins as a function of shade and post-cure using a scraping method and a penetrometer.

Curing depth of (polyacid-modified) composite resins determined by scraping and a penetrometer.

To determine whether injection of substance P into the paratendinous region of a ruptured and subsequently sutured rat Achilles' tendon alters the biomechanic properties of the tendon.

The effects of substance P on the biomechanic properties of ruptured rat Achilles' tendon.

To determine the hardness versus depth profile of several polyacid-modified composite resins (PAM-Cs) as a function of shade (A2, A4) and compare the depth of cure (DoC) based on these profiles with that previously obtained with the scraping and penetrometer methods.

Knoop hardness depth profile of polyacid-modified composite resins.

Optimal pit and fissure sealing is determined by surface preparation techniques and choice of materials.

Microleakage and penetration of a hydrophilic sealant and a conventional resin-based sealant as a function of preparation techniques: a laboratory study.

Tricalcium silicate cements (TSC) are used in dental traumatology and endodontics for their bioactivity which is mostly attributed to formation of calcium hydroxide during TSC hydration and its subsequent release of calcium and hydroxide ions. The aim of this study was to determine the effect of volume (Vol), exposed surface area (ESA) and pH of surrounding medium on calcium ion release. Three commercially available hydraulic alkaline dental cements were mixed and condensed into cylindrical tubes of varying length and diameter ( = 6/group). For the effect of ESA and Vol, tubes were immersed in 10 mL of deionized water. To analyze the effect of environmental pH, the tubes were randomly immersed in 10 mL of buffer solutions with varying pH (10.4, 7.4 or 4.4). The solutions were collected and renewed at various time intervals. pH and/or calcium ion release was measured using a pH glass electrode and atomic absorption spectrophotometer respectively. The change of pH, short-term calcium ion release and rate at which calcium ion release reaches maximum were dependent on ESA ( < 0.05) while maximum calcium ion release was dependent on Vol of TSC ( < 0.05). Maximum calcium ion release was significantly higher in acidic solution followed by neutral and alkaline solution ( < 0.05).

Effect of Exposed Surface Area, Volume and Environmental pH on the Calcium Ion Release of Three Commercially Available Tricalcium Silicate Based Dental Cements.

The aim of this in vitro study was to determine the effects of remineralization promoting agents containing casein phosphopeptide-stabilized amorphous calcium phosphate (CPP-ACP), or CPP-ACP in combination with fluoride (CPP-ACPF) on artificial white spot lesions (WSLs) after 6 and 12 weeks.

The in vitro remineralizing effect of CPP-ACP and CPP-ACPF after 6 and 12 weeks on initial caries lesion.

For patients with soft tissue defects, repair with autologous in vitro engineered adipose tissue could be a promising alternative to current surgical therapies. A volume-persistent engineered adipose tissue construct under in vivo conditions can only be achieved by early vascularization after transplantation. The combination of 3D bioprinting technology with self-assembling microvascularized units as building blocks can potentially answer the need for a microvascular network. In the present study, co-culture spheroids combining adipose-derived stem cells (ASC) and human umbilical vein endothelial cells (HUVEC) were created with an ideal geometry for bioprinting. When applying the favourable seeding technique and condition, compact viable spheroids were obtained, demonstrating high adipogenic differentiation and capillary-like network formation after 7 and 14 days of culture, as shown by live/dead analysis, immunohistochemistry and RT-qPCR. Moreover, we were able to successfully 3D bioprint the encapsulated spheroids, resulting in compact viable spheroids presenting capillary-like structures, lipid droplets and spheroid outgrowth after 14 days of culture. This is the first study that generates viable high-throughput (pre-)vascularized adipose microtissues as building blocks for bioprinting applications using a novel ASC/HUVEC co-culture spheroid model, which enables both adipogenic differentiation while simultaneously supporting the formation of prevascular-like structures within engineered tissues in vitro.

High-throughput fabrication of vascularized adipose microtissues for 3D bioprinting.

To date, the treatment of articular cartilage lesions remains challenging. A promising strategy for the development of new regenerative therapies is hybrid bioprinting, combining the principles of developmental biology, biomaterial science, and 3D bioprinting. In this approach, scaffold-free cartilage microtissues with small diameters are used as building blocks, combined with a photo-crosslinkable hydrogel and subsequently bioprinted. Spheroids of human bone marrow-derived mesenchymal stem cells (hBM-MSC) are created using a high-throughput microwell system and chondrogenic differentiation is induced during 42 days by applying chondrogenic culture medium and low oxygen tension (5%). Stable and homogeneous cartilage spheroids with a mean diameter of 116 ± 2.80 μm, which is compatible with bioprinting, were created after 14 days of culture and a glycosaminoglycans (GAG)- and collagen II-positive extracellular matrix (ECM) was observed. Spheroids were able to assemble at random into a macrotissue, driven by developmental biology tissue fusion processes, and after 72 h of culture, a compact macrotissue was formed. In a directed assembly approach, spheroids were assembled with high spatial control using the bio-ink based extrusion bioprinting approach. Therefore, 14-day spheroids were combined with a photo-crosslinkable methacrylamide-modified gelatin (gelMA) as viscous printing medium to ensure shape fidelity of the printed construct. The photo-initiators Irgacure 2959 and Li-TPO-L were evaluated by assessing their effect on bio-ink properties and the chondrogenic phenotype. The encapsulation in gelMA resulted in further chondrogenic maturation observed by an increased production of GAG and a reduction of collagen I. Moreover, the use of Li-TPO-L lead to constructs with lower stiffness which induced a decrease of collagen I and an increase in GAG and collagen II production. After 3D bioprinting, spheroids remained viable and the cartilage phenotype was maintained. Our findings demonstrate that hBM-MSC spheroids are able to differentiate into cartilage microtissues and display a geometry compatible with 3D bioprinting. Furthermore, for hybrid bioprinting of these spheroids, gelMA is a promising material as it exhibits favorable properties in terms of printability and it supports the viability and chondrogenic phenotype of hBM-MSC microtissues. Moreover, it was shown that a lower hydrogel stiffness enhances further chondrogenic maturation after bioprinting.

Hybrid Bioprinting of Chondrogenically Induced Human Mesenchymal Stem Cell Spheroids.

The aim was to investigate the effect of cryopreservation on the enamel bonding properties of orthodontic brackets. Sixty-six human premolars were randomly allocated to a control group or a cryopreserved group. Conventional stainless-steel orthodontic brackets were bonded with a light cure adhesive on the buccal side of the premolars. The shear bond strength (SBS) was determined at a crosshead speed of 1 mm/min. The SBS and adhesive remnant index (ARI) were evaluated respectively by an independent samples t test and Fisher's exact test (α≤0.05). The mean failure load was lower in the cryopreserved group. However, this difference in SBS was not significant (p=0.443). In both groups, the ARI mostly indicated a failure at the enamel-adhesive interface. The mean ARI scores for both groups were not significantly different (p=0.099). Within the limitations of this macro bond strength testing, it can be concluded that cryopreservation does not significantly affect the bonding properties of enamel.

The orthodontic bonding properties of human enamel after cryopreservation.

Given the complex calcified nature of the fibrous bone tissue, a combinatorial approach merging specific topographical/biochemical cues was adopted to design bone tissue-engineered scaffolds. Coral having a Ca-enriched structure was added to electrospun chitosan (CS)/polyethylene oxide (PEO) nanofibers that were subjected to plasma surface modifications using a medium pressure Ar, air or N dielectric barrier discharge. Plasma incorporated oxygen- and nitrogen-containing functionalities onto the nanofibers surface thus enhancing their wettability. Plasma treatment enhanced the performance of osteoblasts and the interplay between plasma treatment and coral was shown to boost initial cell adhesion. The fibers capacity to trigger calcium phosphate growth was predicted via immersion in simulated body fluid. Globular carbonate apatite nanocrystals were deposited on plasma-treated CS/PEO NFs while thicker layers of flake-like nanocrystals were covering plasma-treated Coral/CS/PEO fibers without blocking the interfibrous pores. Overall, the exclusive multifaceted plasma-treated Coral/CS/PEO nanofibers are believed to revolutionize the bone tissue engineering field.

Combinatorial effects of coral addition and plasma treatment on the properties of chitosan/polyethylene oxide nanofibers intended for bone tissue engineering.

The authors wish to make the following correction to the paper [...].

Correction: Rajasekharan, S., et al. Effect of Exposed Surface Area, Volume and Environmental pH on the Calcium Ion Release of Three Commercially Available Tricalcium Silicate Based Dental Cements.  2018, , 123.

In hybrid bioprinting of cartilage tissue constructs, spheroids are used as cellular building blocks and combined with biomaterials for dispensing. However, biomaterial intrinsic cues can deeply affect cell fate and to date, the influence of hydrogel encapsulation on spheroid viability and phenotype has received limited attention. This study assesses this need and unravels 1) how the phenotype of spheroid-laden constructs can be tuned through adjusting the hydrogel physico-chemical properties and 2) if the spheroid maturation stage prior to encapsulation is a determining factor for the construct phenotype. Articular chondrocyte spheroids with a cartilage specific extracellular matrix (ECM) are generated and different maturation stages, early-, mid-, and late-stage (3, 7, and 14 days, respectively), are harvested and encapsulated in 10, 15, or 20 w/v% methacrylamide-modified gelatin (gelMA) for 14 days. The encapsulation of immature spheroids do not lead to a cartilage-like ECM production but when more mature mid- or late-stage spheroids are combined with a certain concentration of gelMA, a fibrocartilage-like as well as a hyaline cartilage-like phenotype can be induced. As a proof of concept, late-stage spheroids are bioprinted using a 10 w/v% gelMA-Irgacure 2959 solution with the aim to test the processing potential of the spheroid-laden bioink.

Tuning the Phenotype of Cartilage Tissue Mimics by Varying Spheroid Maturation and Methacrylamide-Modified Gelatin Hydrogel Characteristics.

To engineer tissues with clinically relevant dimensions by three-dimensional bioprinting, an extended vascular network with diameters ranging from the macro- to micro-scale needs to be integrated. Extrusion-based bioprinting is the most commonly applied bioprinting technique but due to the limited resolution of conventional bioprinters, the establishment of a microvascular network for the transfer of oxygen, nutrients and metabolic waste products remains challenging. To answer this need, this study assessed the potential and processability of spheroids, containing a capillary-like network, to be used as micron-sized prevascularized units for incorporation throughout the bioprinted construct. Prevascularized spheroids were generated by combining endothelial cells with fibroblasts and adipose tissue-derived mesenchymal stem cells as supporting cells. To serve as a viscous medium for the bioink-based deposition by extrusion printing, spheroids were combined with a photo-crosslinkable methacrylamide-modified gelatin (gelMA) and Irgacure 2959. The influence of gelMA encapsulation, the printing process and photo-crosslinking conditions on spheroid viability, proliferation and vascularization were analyzed by live/dead staining, immunohistochemistry, gene expression analysis and sprouting analysis. Stable spheroid-laden constructs, allowing spheroid outgrowth, were achieved by applying 10 min UV-A photo-curing (365 nm, 4 mW cm), while the construct was incubated in an additional Irgacure 2959 immersion solution. Following implantationonto a chick chorioallantoic membrane, the prevascular engineered constructs showed anastomosis with the host vasculature. This study demonstrated (a) the potential of triculture prevascularized spheroids for application as multicellular building blocks, (b) the processability of the spheroid-laden gelMA bioink by extrusion bioprinting and (c) the importance of photo-crosslinking parameters post printing, as prolonged photo-curing intervals showed to be detrimental for the angiogenic potential and complete vascularization of the construct post printing.

Article	Year
Curing depth of (polyacid-modified) composite resins determined by scraping and a penetrometer. Dental materials : official publication of the Academy of Dental Materials\| PubMed ID: 15501318	2004
The effects of substance P on the biomechanic properties of ruptured rat Achilles' tendon. Archives of physical medicine and rehabilitation\| PubMed ID: 16442981	2006
Knoop hardness depth profile of polyacid-modified composite resins. Quintessence international (Berlin, Germany : 1985)\| PubMed ID: 19093045	2008
Microleakage and penetration of a hydrophilic sealant and a conventional resin-based sealant as a function of preparation techniques: a laboratory study. International journal of paediatric dentistry\| PubMed ID: 22276649	2013
Effect of Exposed Surface Area, Volume and Environmental pH on the Calcium Ion Release of Three Commercially Available Tricalcium Silicate Based Dental Cements. Materials (Basel, Switzerland)\| PubMed ID: 29342837	2018
The in vitro remineralizing effect of CPP-ACP and CPP-ACPF after 6 and 12 weeks on initial caries lesion. Journal of applied oral science : revista FOB\| PubMed ID: 31116280	2019
High-throughput fabrication of vascularized adipose microtissues for 3D bioprinting. Journal of tissue engineering and regenerative medicine\| PubMed ID: 32336037	2020
Hybrid Bioprinting of Chondrogenically Induced Human Mesenchymal Stem Cell Spheroids. Frontiers in bioengineering and biotechnology\| PubMed ID: 32523941	2020
The orthodontic bonding properties of human enamel after cryopreservation. Dental materials journal\| PubMed ID: 32595189	2020
Combinatorial effects of coral addition and plasma treatment on the properties of chitosan/polyethylene oxide nanofibers intended for bone tissue engineering. Carbohydrate polymers\| PubMed ID: 33278977	2021
Correction: Rajasekharan, S., et al. Effect of Exposed Surface Area, Volume and Environmental pH on the Calcium Ion Release of Three Commercially Available Tricalcium Silicate Based Dental C... Materials (Basel, Switzerland)\| PubMed ID: 33445818	2021
Tuning the Phenotype of Cartilage Tissue Mimics by Varying Spheroid Maturation and Methacrylamide-Modified Gelatin Hydrogel Characteristics. Macromolecular bioscience\| PubMed ID: 33729714	2021
Engineering microvasculature by 3D bioprinting of prevascularized spheroids in photo-crosslinkable gelatin. Biofabrication\| PubMed ID: 34496350	2021

Chris Vercruysse

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