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Adequate sterilization procedures for soft biomaterials such as hydrogels are known to be challenging. These materials are delicate in structure, making them sensitive to harsh conditions and prone to damage. In this study, a suitable sterilization method for hydrogels composed of tri(ethylene glycol)-functionalized polyisocyanopeptides (PIC) was explored. These high biomimetic hydrogels are temperature and strain sensitive and have been presented as novel cell culturing matrices, wound dressings, and drug carriers. The methods that were investigated include autoclaving, γ-irradiation, ultraviolet (UV) light irradiation, and supercritical CO 2 (scCO 2 ) treatment. The results show that autoclaving and γ-irradiation have deleterious effects on the gelation behavior and mechanical characteristics of PIC. For γ-irradiation, cooling the gels on dry ice alleviated this negative impact, but not sufficiently enough to make the method viable. In contrast, UV light and scCO 2 treatment do not affect the mechanical properties of the PIC gels. Studies with gels inoculated with 10 7 CFU/mL Gram-positive bacteria Staphylococcus aureus show that only scCO 2 is capable of successfully sterilizing PIC hydrogels by achieving a 6-log reduction in bacterial load. It was concluded that, within the range of tested techniques, the sterilization of PIC is limited to scCO 2 .

The ultimate challenge of tissue engineering research is the translation of experimental knowledge into clinical application. In the preclinical testing phase of any new therapy, animal models remain the gold standard. Therefore, the methodological choice of a suitable model is critical to meet the requirements for a safe clinical application of the developed treatment. For instance, we have shown in rats that the application of calcium phosphate cement (CPC)/propylene glycol alginate (PGA) with bone morphogenetic protein (BMP)-2 or fibroblast growth factor (FGF)-2 resulted in the regeneration of periodontal defects. However, it is debated whether using small models form a predictive method for translation to larger species. At the same time, the 3R framework is encouraged as guiding principles of the ethical use of animal testing. Therefore, based on the successful rat study, the objective of this study was to further investigate the periodontal regenerative efficacy of the CPC/BMP and PGA/FGF system in a periodontal defect model with a low number of nonhuman primates (NHPs). Three Macaca fascicularis -overstocked from breeding for other purposes-were used (reuse of animals and appropriateness of the experimental animal species according to 3R framework). Three-wall periodontal defects were surgically created in the mandible. In total, 10 defects were created and distributed over two groups: (1) control group: PGA+CPC ( n  = 5) and (2) experimental group: PGA/FGF+CPC/BMP ( n  = 5). After 3 months, tissue regeneration was evaluated by histomorphometry and radiographic measurements. Data showed that epithelial downgrowth, cementum, and ligament regeneration were significantly enhanced in the experimental group compared with the control group ( n  = 5; p  = 0.013, p  = 0.028, and p  = 0.018, respectively). However, the amount of newly formed bone did not differ ( p  = 0.146). Overall, as a translational proof-of-principle study, the hybrid periodontal regenerative method of CPC/BMP+PGA/FGF promoted periodontal regeneration in NHPs. This study warrants the application of CPC/BMP/PGA/FGF in clinical trials. Impact Statement This study validated an earlier successful periodontal regeneration strategy from a rat model into a few spare nonhuman primates (NHPs). The hybrid periodontal regenerative method of calcium phosphate cement (CPC)/bone morphogenetic protein (BMP)-2/propylene glycol alginate (PGA)/fibroblast growth factor (FGF)-2 promoted periodontal regeneration in NHPs, which corroborated the previous rat results. This translational approach was a very practical option and thus reduced the number and species of experimental animals in translational research. These results found in NHPs indicate a consistent conclusion with the earlier findings in the rat model. It further warrants the application of CPC/BMP-2+PGA/FGF-2 in human clinical trials.

Pneumatic extrusion-based three-dimensional (3D) printing can be used to fabricate custom-made scaffolds to restore irregular bone defects. During the 3D printing process, therapeutic agents can be added to the scaffolds. This study aimed to develop a polycaprolactone (PCL) scaffold loaded with Ag 3 PO 4 to prevent infections and lidocaine for pain relief by one-step 3D printing. We hypothesized that the drug release could be controlled by varying the filament diameter of the 3D printed scaffolds. To this end, PCL slurry mixed with different amounts of silver phosphate and lidocaine was printed via differently sized nozzles. The obtained cylindric scaffolds displayed a porous interconnected microstructure with high fidelity. The Ag 3 PO 4 and lidocaine were distributed homogeneously. The lidocaine release could be controlled by adjusting the filament diameter while the silver release is correlated with the Ag 3 PO 4 loading amount. The released medium from silver-loaded scaffolds exhibited an obvious inhibition zone against Staphylococcus aureus and Escherichia coli upon loading with 1% Ag 3 PO 4 for up to 6 days and with 3% Ag 3 PO 4 for at least 7 days. Cytotoxicity of all scaffolds was screened by cell assay. In conclusion, the pneumatic extrusion-based 3D printing provides a practical technique to fabricate drug-loaded scaffolds. The Ag 3 PO 4 and lidocaine loaded PCL scaffolds showed the potential for infection prevention and pain relief.

Micro- and nanotextured biomaterial surfaces have been widely studied for their capacity to drive the regeneration of organized tissues. Nanotopographical features in the shape of groove-ridge patterns aim at mimicking the extracellular matrix organization. However, to date, a wide array of groove and ridge sizes has been described. In this work, we therefore tested a device composed of a multipatterned array consisting of six patterns of radially arranged parallel nanogrooves, with a pitch ranging from 0 to 1000 nm and a depth ranging from 0 to 170 nm, to be used as a tool for the expeditious and simultaneous screening of surface topographies aiming the regeneration of anisotropically organized tissues such as ligament. The topographies were reproduced in (1) epoxy resin or (2) membranes produced by the crosslinking of platelet lysate (PL) with genipin (gPL). Both materials were seeded with periodontal ligament cells (PDLCs) and the proliferation, migration, as well as cell alignment were assessed. The effect of topography in PDLCs was only evident in terms of cell organization, resulting in a highly anisotropic organization of the cells for the 1000 and 600 nm patterns, and in an increased isotropic organization for shallower topographies. Overall, our results suggest that this multipatterned system can be a valuable diagnostic tool for biomaterials aiming at the regeneration of anisotropically organized tissues, such as periodontal ligament.

Calcium phosphate cements (CPCs) are commonly used bone substitute materials, which closely resemble the composition of the mineral phase of bone. However, this high similarity to natural bone also results in difficult discrimination from the bone tissue by common imaging modalities, that is, plain X-ray radiography and three-dimensional computed tomography (CT). In addition, new imaging techniques introduced for bone tissue visualization, like magnetic resonance imaging (MRI), face a similar problem. Even at high MRI resolution, the lack of contrast between CPCs and surrounding bone is evident. Therefore, this study aimed to evaluate the feasibility of a dual contrast agent, traceable with both CT and MRI as enhancers of CPC/bone tissue contrast. Our formulation is based on the use of silica beads as vectors, which encapsulate and carry contrast-enhancing nanoparticles, in our case, colloidal Gold and Superparamagnetic Iron oxide particles (SPIO). The bead suspension was incorporated within a calcium phosphate powder. The resultant cements were then tested both in vitro and in vivo in a femoral condyle defect model in rats. Results showed that the mechanical properties of the cement were not significantly affected by the inclusion of the beads. Both in vitro and in vivo data proved the homogeneous incorporation of the contrast within the cement and its visual localization, characterized by a short-term CT contrast enhancement and a long-term MR effect recognizable by the characteristic blooming shape. Finally, no signs of adverse tissue reactions were noticed in vivo. In conclusion, this study proved the feasibility of a multimodal contrast agent as an inert and biocompatible enhancer of CaP cement versus bone tissue contrast.

Calcium phosphate cements (CPCs) are widely used bone substitutes. However, CPCs have similar radiopacity as natural bone, rendering them difficult to be differentiated in classical X-ray and computed tomography imaging. As conventional magnetic resonance imaging (MRI) of bone is cumbersome, due to low water content and very short T(2) relaxation time, ultra-short echo time (UTE) and zero echo time (ZTE) MRI have been explored for bone visualization. This study examined the possibility to differentiate bone and CPC by MRI. T(1) and T(2)* values determined with UTE MRI showed little difference between bone and CPC; hence, these materials were difficult to separate based on T(1) or T(2) alone. Incorporation of ultra-small particles of iron oxide and gadopentetatedimeglumine (Gd-DTPA; 1 weight percentage [wt%] and 5 wt% respectively) into CPC resulted in visualization of CPC with decreased intensity on ZTE images in in vitro and ex vivo experiments. However, these additions had unfavorable effects on the solidification time and/or mechanical properties of the CPC, with the exception of 1% Gd-DTPA alone. Therefore, we tested this material in an in vivo experiment. The contrast of CPC was enhanced at an early stage postimplantation, and was significantly reduced in the 8 weeks thereafter. This indicates that ZTE imaging with Gd-DTPA as a contrast agent could be a valid radiation-free method to visualize CPC degradation and bone regeneration in preclinical experiments.

Periodontitis is a disease affecting the supporting structures of the teeth, which can eventually result in tooth loss. A three-dimensional (3D) tissue culture model was developed that may serve to grow a 3D construct that not only transplants into defective periodontal sites, but also allows to examine the effect of mechanical load in vitro. In the current in vitro study, green fluorescent protein labeled periodontal ligament (PDL) cells form rat incisors were embedded in a 3D matrix and exposed to mechanical loading alone, to a chemical stimulus (Emdogain; enamel matrix derivative [EMD]) alone, or a combination of both. Loading consisted of unilateral stretching (8%, 1 Hz) and was applied for 1, 3, or 5 days. Results showed that PDL cells were distributed and randomly oriented within the artificial PDL space in static culture. On mechanical loading, the cells showed higher cell numbers. Moreover, cells realigned perpendicular to the stretching force depending on time and position, with great analogy to natural PDL tissue. EMD application gave a significant effect on growth and upregulated bone sialoprotein (BSP) and collagen type-I (Col-I), whereas Runx-2 was downregulated. This implies that PDL cells under loading might tend to act similar to bone-like cells (BSP and Col-I) but at the same time, react tendon like (Runx-2). The combination of chemical and mechanical stimulation seems possible, but does not show synergistic effects. In this study, a new model was successfully introduced in the field of PDL-related regenerative research. Besides validating the 3D model to mimic an authentic PDL space, it also provided a useful and well-controlled approach to study cell response to mechanical loading and other stimuli.

In tissue engineering research, various three-dimensional (3D) techniques are available to study cell morphology, biomaterials, and their relations. To overcome disadvantages of frequently used imaging techniques, in the current study stereoimaging scanning electron microscopy (SEM) is proposed. First, the 3D SEM application was validated using a series of standardized microspheres. Thereafter, MC-3T3 cell morphology was visualized and cell parameters as cell height were quantified on titanium and calcium-phosphate materials using 3D reconstruction software. Besides 3D visualization of the cells, quantitative assessment showed significant substrate dependency of cell spreading in time. Such quantification of cell spreading kinetics can be used for optimization of tissue engineering scaffold surface properties. However, further standardization of SEM image acquisition and 3D SEM software settings are still essential for 3D cell analysis.

With the advance of nanotechnology in biomaterials science and tissue engineering, it is essential that new techniques become available to observe processes that take place at the direct interface between tissue and scaffold materials. Here, Cryo DualBeam focused ion beam-scanning electron microscopy (FIB-SEM) was used as a novel approach to observe the interactions between frozen hydrated cells and nanometric structures in high detail. Through a comparison of images acquired with transmission electron microscopy (TEM), conventional FIB-SEM operated at ambient temperature, and Cryo DualBeam FIB-SEM, the advantages and disadvantages of each technique were evaluated. Ultrastructural details of both (extra)cellular components and cell organelles were best observe with TEM. However, processing artifacts such as shrinkage of cells at the substrate interface were introduced in both TEM and conventional FIB-SEM. In addition, the cellular contrast in conventional FIB-SEM was low; consequently, cells were difficult to distinguish from the adjoining substrate. Cryo DualBeam FIB-SEM did preserve (extra)cellular details like the contour, cell membrane, and mineralized matrix. The three described techniques have proven to be complementary for the evaluation of processes that take place at the interface between tissue and substrate.

To improve the mechanical properties of polymers used in bone repair, it has been suggested to incorporate single-walled carbon nanotubes (CNTs). However, concern exists about the biosafety of the CNTs in vivo. Therefore, the aim of this study was to develop a magnetic resonance imaging technique to examine the distribution pattern of CNTs after release from a degrading poly(lactic-co-glycolic acid) (PLGA) scaffold in vivo. Five rats received a PLGA scaffold with incorporated gadolinium-labeled single-walled CNTs ("gadonanotubes") subcutaneously. The rats were analyzed up to 5 weeks, subsequently euthanized, followed by histological evaluation of the explanted scaffolds with their surrounding tissue. A significant increase in intensity of the scaffold surrounding tissue was shown in the time period around 3 weeks, as compared to internal control areas. The intensity declined soon thereafter. This is suggested to be caused by the release of gadonanotubes from the degrading scaffold into the surrounding tissue. Histological imaging showed encapsulation by connective fibrous tissue and some mild inflammation around the scaffolds. In conclusion, magnetic resonance imaging is an excellent technique to study the biological fate of gadonanotubes. However, to formulate solid conclusions on the distribution pattern of gadonanotubes in vivo the experimental setup requires further optimization.

Periodontitis is an inflammatory disease affecting the support of the teeth, eventually leading to loosening and subsequent loss of teeth. Effective procedures for periodontal tissue engineering or regeneration require preclinical models before market introduction. Research has been performed in either small or large animals. Unfortunately, there is no intermediate-sized in vivo model available for periodontal regeneration studies, such as, for instance, rabbits. The objective of this study was to evaluate the rabbit as a new experimental model to study periodontal regeneration. In 12 rabbits, periodontal defects were created in a 4 x 6 mm bone window. The animals were sacrificed after 2, 4, 6, 8, 10, and 12 weeks. Up to 6 weeks, the fenestration defects healed partly by repair and partly by regeneration. After 6 weeks the root had erupted to such an extent that the original root defect shifted into the oral cavity. This signifies that the periodontal ligament (PDL) bordering the original bone defect site is newly formed during the natural eruption process and not locally regenerated. Apparently, the new PDL originates from mesenchymal cells that arise from the apical part (sheath of Hertwig) and subsequently developed into PDL fibroblasts. At 12 weeks, no signs of surgery were present anymore. On the basis of our observation that the defect of the PDL was replaced rather than restored, we conclude that the rabbit model has disadvantages and is less suitable for studies of regeneration of PDL.

For adequate histological processing of implanted biomaterials or tissue-engineered constructs, it is sometimes essential to obtain insight into the localization of structures inside the tissue samples. Observation of three-dimensional (3D) surface reconstruction, including basic photorealistic texture characteristics as surface pattern and color combined with X-ray computed tomography 3D reconstruction at different levels, is a useful approach to localize anatomical or implanted structures within experimental tissue samples. Because of the possible observation of structures of interest in a 3D environment, fusion of these techniques can greatly facilitate histological processing.

In this study we present a new three-dimensional (3D) model to study effects of mechanical loading on tendon/ligament formation in vitro. The model mimics a functional periodontal ligament (PDL), which anchors dental roots to the jaw bone and transfers the axial load of mastication to the jaw bone. A collagen gel containing human PDL fibroblasts was seeded in a PDL space between an artificial root and bone surface. The effects of 3-day loading on the fibroblasts were studied in vitro by axial and intermittent displacement of the root to which the gel was attached. Cell responses were recorded by measuring expression of three sets of genes: (i) cyclooxygenase 1 and 2 (COX-1, COX-2) producing prostaglandins (signaling molecules); (ii) Runx2, a transcription factor for the osteogenic lineage; and (iii) the extracellular matrix proteins osteopontin, dentin matrix protein 1, and collagen type I (COL1). Loading for 3 days resulted in magnitude-dependent changes in the expression of COX-2 and COL1. A low loading magnitude significantly decreased COX-2 expression, an intermediate magnitude increased its expression, while a high magnitude increased COL1 expression. We concluded that the 3D loading model provides a useful, well-controlled method to examine ligament fibroblast responses to mechanical loading. The model may serve to explore the application of mechanical loading as an anabolic factor for ligament reconstruction.

The aim of this work was to introduce high-resolution computed tomography (micro-CT) for scaffolds made from soft natural biomaterials, and to compare these data with the conventional techniques scanning electron microscopy and light microscopy. Collagen-based scaffolds were used as examples. Unlike mineralized tissues, collagen scaffolds do not provide enough X-ray attenuation for micro-CT imaging. Therefore, various metal-based contrast agents were applied and evaluated using two structurally distinct scaffolds, one with round pores and one with unidirectional lamellae. The optimal contrast techniques for obtaining high-resolution three-dimensional images were either a combination of osmium tetroxide and uranyl acetate, or a combination of uranyl acetate and lead citrate. The data obtained by micro-CT analysis were in line with data obtained by light and electron microscopy. However, small structures (less than a few mum) could not be visualized due to limitation of the spot size of the micro-CT apparatus. In conclusion, reliable three-dimensional images of scaffolds prepared from soft natural biomaterials can be obtained using appropriate contrast protocols. This extends the use of micro-CT analysis to soft materials, such as protein-based biomaterials.