Coumarin-Based Photodegradable Hydrogels Enable Two-Photon Subtractive Biofabrication at 300†mm s-1.

Spatiotemporally controlled two-photon photodegradation of hydrogels has gained increasing attention for high-precision subtractive tissue engineering. However, conventional photolabile hydrogels often have poor efficiency upon two-photon excitation in the near-infrared (NIR) region and thus require high laser dosage that may compromise cell activity. As a result, high-speed two-photon hydrogel erosion in the presence of cells remains challenging. Here we introduce the design and synthesis of efficient coumarin-based photodegradable hydrogels to overcome these limitations. A set of photolabile coumarin-functionalized polyethylene glycol linkers are synthesized through a Passerini multicomponent reaction. After mixing these linkers with thiolated hyaluronic acid, semi-synthetic photodegradable hydrogels are formed in situ via Michael addition crosslinking. The efficiency of photodegradation in these hydrogels is significantly higher than that in nitrobenzyl counterparts upon two-photon irradiation at 780 nm. A complex microfluidic network mimicking the bone microarchitecture is successfully fabricated in preformed coumarin hydrogels at high speeds of up to 300 mm s-1 and low laser dosage down to 10 mW. Further, we demonstrate fast two-photon printing of hollow microchannels inside a hydrogel to spatiotemporally direct cell migration in 3D. Collectively, these hydrogels may open new avenues for fast laser-guided tissue fabrication at high spatial resolution.

Enhanced bone regeneration in rat calvarial defects through BMP2 release from engineered poly(ethylene glycol) hydrogels.

The clinical standard therapy for large bone defects, typically addressed through autograft or allograft donor tissue, faces significant limitations. Tissue engineering offers a promising alternative strategy for the regeneration of substantial bone lesions. In this study, we harnessed poly(ethylene glycol) (PEG)-based hydrogels, optimizing critical parameters including stiffness, incorporation of arginine-glycine-aspartic acid (RGD) cell adhesion motifs, degradability, and the release of BMP2 to promote bone formation. In vitro we demonstrated that human bone marrow derived stromal cell (hBMSC) proliferation and spreading strongly correlates with hydrogel stiffness and adhesion to RGD peptide motifs. Moreover, the incorporation of the osteogenic growth factor BMP2 into the hydrogels enabled sustained release, effectively inducing bone regeneration in encapsulated progenitor cells. When used in vivo to treat calvarial defects in rats, we showed that hydrogels of low and intermediate stiffness optimally facilitated cell migration, proliferation, and differentiation promoting the efficient repair of bone defects. Our comprehensive in vitro and in vivo findings collectively suggest that the developed hydrogels hold significant promise for clinical translation for bone repair and regeneration by delivering sustained and controlled stimuli from active signaling molecules.

Synthetic biodegradable microporous hydrogels for in vitro 3D culture of functional human bone cell networks.

Generating 3D bone cell networks in vitro that mimic the dynamic process during early bone formation remains challenging. Here, we report a synthetic biodegradable microporous hydrogel for efficient formation of 3D networks from human primary cells, analysis of cell-secreted extracellular matrix (ECM) and microfluidic integration. Using polymerization-induced phase separation, we demonstrate dynamic in situ formation of microporosity (5-20 µm) within matrix metalloproteinase-degradable polyethylene glycol hydrogels in the presence of living cells. Pore formation is triggered by thiol-Michael-addition crosslinking of a viscous precursor solution supplemented with hyaluronic acid and dextran. The resulting microporous architecture can be fine-tuned by adjusting the concentration and molecular weight of dextran. After encapsulation in microporous hydrogels, human mesenchymal stromal cells and osteoblasts spread rapidly and form 3D networks within 24 hours. We demonstrate that matrix degradability controls cell-matrix remodeling, osteogenic differentiation, and deposition of ECM proteins such as collagen. Finally, we report microfluidic integration and proof-of-concept osteogenic differentiation of 3D cell networks under perfusion on chip. Altogether, this work introduces a synthetic microporous hydrogel to efficiently differentiate 3D human bone cell networks, facilitating future in vitro studies on early bone development.

Multi-doped apatite: Strontium, magnesium, gallium and zinc ions synergistically affect osteogenic stimulation in human mesenchymal cells important for bone tissue engineering.

Functional calcium phosphate biomaterials can be designed as carriers of a balanced mixture of biologically relevant ions able to target critical processes in bone regeneration. They hold the potential to use mechanisms very similar to growth factors naturally produced during fracture healing, while circumventing some of their drawbacks. Here we present a novel phase of carbonated-apatite containing Mg2+, Sr2+, Zn2+ and Ga3+ ions (HApMgSrZnGa). While all dopants decrease the crystallinity, Ga3+ limits crystal growth and enables the formation of a nanosized apatite phase with enhanced specific surface area. Coexistence of the ions enhances degradability and controls solubility of low crystalline, distorted, multi-doped apatite structure, controlled by Ga3+ ions accumulated at the surface. Consequently, HApMgSrZnGa supports the viability of human mesenchymal stromal cells (MSCs) and induces their stimulation along the osteogenic lineage. In addition, the co-released ions has a synergistic antimicrobial effect, particularly within the HApMgSrZnGa-Au(arg) composite with Au(arg) as contact-based antimicrobial. The activity is stable up to two months in vitro. Osteogenic nature and antimicrobial activity, combined in a single biomaterial, are suggesting a well-balanced, multi-doped apatite design applicable as future option in bone regeneration and tissue engineering.

In situ laboratory for plastic degradation in the Red Sea.

Degradation and fragmentation of plastics in the environment are still poorly understood. This is partly caused by the lack of long-term studies and methods that determine weathering duration. We here present a novel study object that preserves information on plastic age: microplastic (MP) resin pellets from the wreck of the SS Hamada, a ship that foundered twenty-nine years ago at the coast of Wadi el Gemal national park, Egypt. Its sinking date enabled us to precisely determine how long MP rested in the wreck and a nearby beach, on which part of the load was washed off. Pellets from both sampling sites were analyzed by microscopy, X-ray tomography, spectroscopy, calorimetry, gel permeation chromatography, and rheology. Most pellets were made of low-density polyethylene, but a minor proportion also consisted of high-density polyethylene. MP from inside the wreck showed no signs of degradation compared to pristine reference samples. Contrary, beached plastics exhibited changes on all structural levels, which sometimes caused fragmentation. These findings provide further evidence that plastic degradation under saltwater conditions is comparatively slow, whereas UV radiation and high temperatures on beaches are major drivers of that process. Future long-term studies should focus on underlying mechanisms and timescales of plastic degradation.

The local and global geometry of trabecular bone.

The organization and shape of the microstructural elements of trabecular bone govern its physical properties, are implicated in bone disease, and serve as blueprints for biomaterial design. To devise fundamental structure-property relationships and design truly bone-mimicking biomaterials, it is essential to characterize trabecular bone structure from the perspective of geometry, the mathematical study of shape. Using micro-CT images from 70 donors at five different sites, we analyze the local and global geometry of human trabecular bone in detail, respectively by quantifying surface curvatures and Minkowski functionals. We find that curvature density maps provide distinct and sensitive shape fingerprints for bone from different sites. Contrary to a common assumption, these curvature maps also show that bone morphology does not approximate a minimal surface but exhibits a much more intricate curvature landscape. At the global (or integral) perspective, our Minkowski analysis illustrates that trabecular bone exhibits other types of anisotropy/ellipticity beyond interfacial orientation, and that anisotropy varies substantially within the trabecular structure. Moreover, we show that the Minkowski functionals unify several traditional morphometric indices. Our geometric approach to trabecular morphometry provides a fundamental language of shape that could be useful for bone failure prediction, understanding geometry-driven tissue growth, and the design of bone-mimicking tissue scaffolds. STATEMENT OF SIGNIFICANCE: The architecture of trabecular bone is key in determining bone properties, and is often a starting point for the design of bone-substitutes. Despite the substantial history of bone morphometry, a fundamental characterization of trabecular bone geometry is still lacking. Therefore, we introduce a robust framework to quantify local and global trabecular bone geometry, which we apply to hundreds of micro-CT scans. Our approach relies on quantifying surface curvatures and Minkowski functionals, which are the most fundamental local and global shape quantifiers. Our results show that these shape metrics are sensitive to differences between bone types and unify traditional metrics within a single mathematical framework. This geometrical framework could also be useful to design bone-mimicking scaffolds and understand geometry-driven tissue growth.

Spatio-temporal characterization of fracture healing patterns and assessment of biomaterials by time-lapsed in vivo micro-computed tomography.

Thorough preclinical evaluation of functionalized biomaterials for treatment of large bone defects is essential prior to clinical application. Using in vivo micro-computed tomography (micro-CT) and mouse femoral defect models with different defect sizes, we were able to detect spatio-temporal healing patterns indicative of physiological and impaired healing in three defect sub-volumes and the adjacent cortex. The time-lapsed in vivo micro-CT-based approach was then applied to evaluate the bone regeneration potential of functionalized biomaterials using collagen and bone morphogenetic protein (BMP-2). Both collagen and BMP-2 treatment led to distinct changes in bone turnover in the different healing phases. Despite increased periosteal bone formation, 87.5% of the defects treated with collagen scaffolds resulted in non-unions. Additional BMP-2 application significantly accelerated the healing process and increased the union rate to 100%. This study further shows potential of time-lapsed in vivo micro-CT for capturing spatio-temporal deviations preceding non-union formation and how this can be prevented by application of functionalized biomaterials. This study therefore supports the application of longitudinal in vivo micro-CT for discrimination of normal and disturbed healing patterns and for the spatio-temporal characterization of the bone regeneration capacity of functionalized biomaterials.

Bone substitute: transforming beta-tricalcium phosphate porous scaffolds into monetite.

The goal of the present study was to assess the possibility to change the composition of a calcium phosphate scaffold from a high-temperature phase to a phase only stable at or close to room temperature without macrostructural changes. For that purpose, macroporous beta-TCP scaffolds were converted into alpha-TCP by high-temperature thermal treatment and then dipped into a phosphoric acid solution to obtain a more acidic calcium phosphate phase called monetite or dicalcium phosphate (DCP; CaHPO4). Two different solid-to-liquid ratios (SLR: 0.067 and 0.200g/mL) and three different temperatures (T: 37, 60 and 80 degrees C) were used. The reaction was followed by measuring the change of sample size and weight, by determining the compositional changes by X-ray diffraction (Rietveld analysis), and by looking at the micro- and macrostructural changes by scanning electron microscopy and micro-computed tomography. The results revealed that the transformation proceeded faster at a higher temperature and a higher SLR value but was achieved within a few days in all cases. Morphologically, the porosity decreased by 10%, the pore size distribution became wider and the mean macro pore size was reduced from 0.28 to 0.19mm. The fastest conversion and the highest compressive strength (9MPa) were measured using an incubation temperature of 80 degrees C and an SLR value of 0.2g/mL.

Micro-computed tomography: a method for the non-destructive evaluation of the three-dimensional structure of biological specimens.

The large increase in interest in micro-computed tomography (micro-CT) over the last decade reflects the+ need for a method able to non-destructively visualize the internal three-dimensional structure of an object. Thereby, the real beauty of computed tomography lies in the fact that it is available for a large range of nominal resolutions, which allows hierarchical imaging from whole bodies down to the tissue level. Although micro-CT is currently mainly used for imaging of hard tissue (i.e., bone and tooth), future developments might also allow high soft tissue contrast either using appropriate contrast agents or x-ray contrast mechanisms. This chapter aims to review the steps necessary for a successful micro-CT measurement. Although the actual measurement is often machine dependent, the chapter does not describe a specific system but rather lists all steps that eventually have to be considered to set up a measurement, run the measurement, process the image data, and get morphometric indices as a result. The chapter provides an easy understandable manual that should allow newcomers to perform successful measurements and hence to best profit from this powerful technique.

Nondestructive micro-computed tomography for biological imaging and quantification of scaffold-bone interaction in vivo.

Scaffolds, also called bioscaffolds, are needed in all tissue engineering applications as carriers for cells and biochemical factors, as constructs providing appropriate mechanical conditions, or as a combination of the two. The aim of this paper is to present recent developments in micro-computed tomography (microCT) analyses of scaffolds. The focus will be on imaging and quantification aspects in bone research, and will deal with the assessment of scaffold architecture and how it interacts with bone tissue. We show that micro-architectural imaging is a nondestructive and noninvasive procedure that allows a precise three-dimensional (3D) measurement of scaffold architecture. Direct microCT-based image analysis allows to accurately quantify scaffold porosity, surface area, and 3D measures such as pore size, pore distribution, and strut thickness; furthermore, it allows for a precise measurement of bone growth into the scaffold and onto its surface. This methodology is useful for quality control of scaffold fabrication processes, to assess scaffold degradation kinetics, and to assess bone tissue response. Even more so, in combination with bioreactors or in vivo animal models, microCT allows to qualitatively and quantitatively assess the spatial and temporal mineralization of bone tissue formation in scaffolds; such longitudinal studies improve the assessment of bone response due to scaffold architecture. Computational models will be helpful in further analyses of these data in order to improve our understanding of mechanical and biochemical stimuli on bone formation, and are likely to provide valuable knowledge to optimize scaffold design.

Control of in vitro tissue-engineered bone-like structures using human mesenchymal stem cells and porous silk scaffolds.

Natural bone consists of cortical and trabecular morphologies, the latter having variable pore sizes. This study aims at engineering different bone-like structures using scaffolds with small pores (112-224 microm) in diameter on one side and large pores (400-500 microm) on the other, while keeping scaffold porosities constant among groups. We hypothesized that tissue engineered bone-like structure resulting from silk fibroin (SF) implants is pre-determined by the scaffolds' geometry. To test this hypothesis, SF scaffolds with different pore diameters were prepared and seeded with human mesenchymal stem cells (hMSC). As compared to static seeding, dynamic cell seeding in spinner flasks resulted in equal cell viability and proliferation, and better cell distribution throughout the scaffold as visualized by histology and confocal microscopy, and was, therefore, selected for subsequent differentiation studies. Differentiation of hMSC in osteogenic cell culture medium in spinner flasks for 3 and 5 weeks resulted in increased alkaline phosphatase activity and calcium deposition when compared to control medium. Micro-computed tomography (microCT) detailed the pore structures of the newly formed tissue and suggested that the structure of tissue-engineered bone was controlled by the underlying scaffold geometry.

Repair of bone defects using synthetic mimetics of collagenous extracellular matrices.

We have engineered synthetic poly(ethylene glycol) (PEG)-based hydrogels as cell-ingrowth matrices for in situ bone regeneration. These networks contain a combination of pendant oligopeptide ligands for cell adhesion (RGDSP) and substrates for matrix metalloproteinase (MMP) as linkers between PEG chains. Primary human fibroblasts were shown to migrate within these matrices by integrin- and MMP-dependent mechanisms. Gels used to deliver recombinant human bone morphogenetic protein-2 (rhBMP-2) to the site of critical- sized defects in rat crania were completely infiltrated by cells and were remodeled into bony tissue within five weeks. Bone regeneration was dependent on the proteolytic sensitivity of the matrices and their architecture. The cell-mediated proteolytic invasiveness of the gels and entrapment of rhBMP-2 resulted in efficient and highly localized bone regeneration.

Parathyroid hormone 1-34 enhances titanium implant anchorage in low-density trabecular bone: a correlative micro-computed tomographic and biomechanical analysis.

The use of endosseous titanium implants is the standard of care in dentistry and orthopaedic surgery. Nevertheless, implantation in low-density bone has a poor prognosis and experimental studies show delayed implant anchorage following gonadectomy-induced bone loss. Intermittently administered human parathyroid hormone 1-34 [iahPTH(1-34)] is the leading bone anabolic therapy. Hence, this study assessed whether iahPTH(1-34) enhances titanium implant integration in low-density bone. Threaded titanium implants, 0.9 mm in diameter, were inserted horizontally into the proximal tibial metaphysis of 5-month-old rats, 7 weeks postorchiectomy (ORX). Subcutaneous administration of iahPTH(1-34), at 5, 25 and 75 microg/kg/day commenced immediately thereafter and lasted for 8 weeks. Quantitative micro-computed tomography (muCT) at the implantation site was carried out at 15 microm resolution using high energy and long integration time to minimize artifacts resulting from the high implant radiopacity. Osseointegration (OI) was calculated as percent implant surface in contact with bone (%OI) quantified as the ratio of "bone"-to-total voxels in contact with the implant. Additionally, the trabecular bone volume density (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N) and connectivity density (Conn.D) were measured in the peri-implant bone. All microCT parameters were stimulated by iahPTH(1-34) dose-dependently; the percent maximal enhancement was %OI = 143, BV/TV = 257, Tb.Th = 150, Tb.N = 140 and Conn.D = 193. The maximal values of %OI, BV/TV and Tb.Th in iahPTH(1-34)-treated ORX rats exceeded significantly those measured in the implantation site of untreated sham-ORX controls. The same specimens were then subjected to pullout biomechanical testing. The biomechanical parameters were also enhanced by iahPTH(1-34) dose-dependently, exceeding the values recorded in the sham-ORX controls. The percent iahPTH(1-34)-induced maximal enhancement was: ultimate force = 315, stiffness = 270 and toughness = 395. Except for the BV/TV and Tb.Th, there was no significant difference between the effect of the 25 and 75 microg/kg/day doses. There was a highly significant correlation between the morphometric and biomechanical parameters suggesting the use of quantitative CT as predictive of the implant mechanical properties. These findings demonstrate that iahPTH(1-34) effectively stimulates implant anchorage in low-density trabecular bone and thus the feasibility of administering iahPTH(1-34) to improve the clinical prognosis in low-density trabecular bone sites.

The influence of surface coatings of dicalcium phosphate (DCPD) and growth and differentiation factor-5 (GDF-5) on the stability of titanium implants in vivo.

Mechanical stability of implants is usually tested by pull out or push out tests which destroy the interface between the implant and bone. Pull out tests do not ideally reflect the clinical situation. In contrast, applying submaximal load leads to more physiologic micro-displacement between implant and bone. The aim of this study was to evaluate a new non-destructive mechanical testing device on different modifications of titanium implants. In 18 rabbits we investigated the influence of a dicalcium phosphate (DCPD) coating, or of a growth and differentiation factor-5 (GDF-5) coating, or a combination of both on the stability of titanium implants. The stability of implant was assessed by a non-destructive micro-measurement. In the same specimens the interface was investigated by micro-CT and histological evaluation. Surface modifications had a positive effect on the implant stability regarding displacement (p=0.001). Mechanical stability correlated with the quality of peri-implant tissue. Micro-displacement correlated negatively with the bone formation around the implants in histomorphometric evaluation (p=0.02). Amount of peri-prosthetic soft tissue showed a positive correlation with micro-displacement (p=0.01). Our findings indicate the positive effect of DCPD and GDF-5 coatings on stability of titanium implants. Results demonstrate the non-destructive testing to be an effective method to evaluate mechanical stability of implants.

In vivo behavior of calcium phosphate scaffolds with four different pore sizes.

The goal of the present study was to assess the effect of macropore size on the in vivo behavior of ceramic scaffolds. For that purpose, beta-tricalcium phosphate (beta-TCP) cylinders with four different macropore sizes (150, 260, 510, and 1220 microm) were implanted into drill hole defects in cancellous bone of sheep and their resorption behavior was followed for 6, 12 and 24 weeks. The scaffolds were evaluated for biocompatibility, and new bone formation was observed macroscopically, histologically and histomorphometrically. Histomorphometrical measurements were performed for the whole defect area and for the area subdivided into three concentric rings (outer, medial, and inner ring). All implants were tolerated very well as evidenced by the low amount of inflammatory cells and the absence of macroscopic signs of inflammation. Resorption proceeded fast since less than 5% ceramic remained at 24-week implantation. Hardly any effect of macropore size was observed on the in vivo response. Samples with an intermediate macropore size (510 microm) were resorbed significantly faster than samples with smaller macropore sizes (150 and 260 microm). However, this fast resorption was associated with a lower bone content and a higher soft tissue content. At 12 and 24 weeks, the latter differences had disappeared. Bone was more abundant in the outer ring than in the rest of the blocks at 6 weeks, and in the outer and medial ring compared to the inner ring at 12 weeks.

Architecture and properties of anisotropic polymer composite scaffolds for bone tissue engineering.

Bone is a complex porous composite structure with specific characteristics such as viscoelasticity and anisotropy, both in morphology and mechanical properties. Bone defects are regularly filled with artificial tissue grafts, which should ideally have properties similar to those of natural bone. Open cell composite foams made of bioresorbable poly(L-lactic acid) (PLA) and ceramic fillers, hydroxyapatite (HA) or beta-tricalcium phosphate (beta-TCP), were processed by supercritical CO2 foaming. Their internal 3D-structure was then analysed by micro-computed tomography (microCT), which evidenced anisotropy in morphology with pores oriented in the foaming direction. Furthermore compressive tests demonstrated anisotropy in mechanical behaviour, with an axial modulus up to 1.5 times greater than the transverse modulus. Composite scaffolds also showed viscoelastic behaviour with increased modulus for higher strain rates. Such scaffolds prepared by gas foaming of polymer composite materials therefore possess suitable architecture and properties for bone tissue engineering applications.

Effect of scaffold design on bone morphology in vitro.

Silk fibroin is an important polymer for scaffold designs, forming biocompatible and mechanically robust biomaterials for bone, cartilage, and ligament tissue engineering. In the present work, 3D biomaterial matrices were fabricated from silk fibroin with controlled pore diameter and pore interconnectivity, and utilized to engineer bone starting from human mesenchymal stem cells (hMSC). Osteogenic differentiation of hMSC seeded on these scaffolds resulted in extensive mineralization, alkaline phosphatase activity, and the formation of interconnected trabecular- or cortical-like mineralized networks as a function of the scaffold design utilized; allowing mineralized features of the tissue engineered bone to be dictated by the scaffold features used initially in the cell culture process. This approach to scaffold predictors of tissue structure expands the window of applications for silk fibroin-based biomaterials into the realm of directing the formation of complex tissue architecture. As a result of slow degradation inherent to silk fibroin, scaffolds preserved their initial morphology and provided a stable template during the mineralization phase of stem cells progressing through osteogenic differentiation and new extracellular matrix formation. The slow degradation feature also facilitated transport throughout the 3D scaffolds to foster improved homogeneity of new tissue, avoiding regions with decreased cellular density. The ability to direct bone morphology via scaffold design suggests new options in the use of biodegradable scaffolds to control in vitro engineered bone tissue outcomes.

Local delivery of bisphosphonate from coated orthopedic implants increases implants mechanical stability in osteoporotic rats.

Patients with osteoporosis and joint disabilities represent a constant growing and challenging population to be treated in the musculoskeletal clinical field. Especially in the case of total hip arthroplasty, new solutions should be developed to compensate for the double negative factors, peri-implant osteolysis, and osteoporotic bone loss, affecting the quality of implant outcome. The goal of this study was then to establish a proof of concept for orthopedic implant used as Zoledronate delivery in osteoporotic rats, and in particular, to verify if this approach could increase the initial implant stability. Twenty-five female 6-month-old Wistar rats were ovariectomized 6 weeks before the implantation to induce osteoporosis. The animals were randomly separated in five groups representing the different Zoledronate concentrations in the HA coating: 0, 0.2, 2.1, 8.5, and 16 microg/implant. Histomorphometric measures and peri-implant bone volume fraction were assessed and mechanical stability tests were performed. Bone volume fraction and biomechanical results clearly illustrate the positive effect of Zoledronate coated implants in the osteoporotic rats. A remarkable result was to show the existence of a window of Zoledronate content (0.2 to 8.5 microg/implant) in which the mechanical fixation of the implant increased. We were able to establish the proof of concept for orthopedic implants used as a drug delivery system in osteoporotic rats. The local bisphosphonate delivery from a calcium phosphate coating allowed increase of the mechanical fixation of an orthopedic implant. This study shows that orthopedic implants containing bisphosphonates could be beneficial for osteoporotic patients in need of a total joint replacement.

In vivo bone regeneration with injectable calcium phosphate biomaterial: a three-dimensional micro-computed tomographic, biomechanical and SEM study.

This in vivo study investigated the efficiency of an injectable calcium phosphate bone substitute (IBS) for bone regenerative procedures through non-destructive three-dimensional (3D) micro-tomographic (microCT) imaging, biomechanical testing with a non-destructive micro-indentation technique and 2D scanning electron microscopy (SEM) analysis. The injectable biomaterial was obtained by mixing a biphasic calcium phosphate (BCP) ceramic mineral phase and a cellulosic polymer. The BCP particles were 200-500 microm or 80-200 microm in diameter. The injectable material was implanted for 6 weeks into critical-sized bone defects at the distal end of rabbit femurs. Extensive new bone apposition was noted with both 2D and 3D techniques. Micro-CT showed that newly formed bone was in perfect continuity with the trabecular host bone structure and demonstrated the high interconnectivity of the restored bone network. For both IBS formulations, SEM and microCT gave very close measurements. The only detected significant difference concerned the amount of newly formed bone obtained with IBS 80-200 that appeared significantly higher with microCT analysis than with SEM (p=0.00007). Student t-tests did not show any significant difference in the amount of newly formed bone and remaining ceramic obtained from microCT analysis or SEM. Regression analysis showed satisfactory correlation between both the amount of newly formed bone and remaining ceramic obtained from microCT or SEM. For IBS 200-500, the newly formed bone rate inside the defect was 28.0+/-5.2% with SEM and yield strength of the samples was 18.8+/-5.4 MPa. For IBS 80-200, the newly formed bone rate inside the defect was 31.7+/-5.1% with SEM and yield strength of the samples was 26.8+/-4.5 MPa. Yield strength appeared well correlated with the amount of newly formed bone, specially observed with microCT. This study showed the ability of non-destructive techniques to investigate biological and mechanical aspects of bone replacement with injectable biomaterials.

3D Bioprinting of complex channels-Effects of material, orientation, geometry, and cell embedding.

Creating filled or hollow channels within 3D tissues has become increasingly important in tissue engineering. Channels can serve as vasculature enhancing medium perfusion or as conduits for nerve regeneration. The 3D biofabrication seems to be a promising method to generate these structures within 3D constructs layer-by-layer. In this study, geometry and interface of bioprinted channels were investigated with micro-computed tomography and fluorescent imaging. In filament printing, size and shape of printed channels are influenced by their orientation, which was analyzed by printing horizontally and vertically aligned channels, and by the ink, which was evaluated by comparing channels printed with an alginate-gelatin hydrogel or with an emulsion. The influence of geometry and cell-embedding in the hydrogel on feature size and shape was investigated by printing more complex channels. The generation of hollow channels, induced through leaching of a support phase, was monitored over time. Horizontally aligned channels provided 16× smaller cross-sectional areas than channels in vertical orientation. The smallest feature size of hydrogel filaments was twice as large compared to emulsion filaments. Feature size and shape depended on the geometry but did not alter when living cells were embedded. With that knowledge, channels can be consciously tailored to the particular needs.