Triply periodic minimal surface (TPMS) microarchitectures combine low weight with high mechanical strength, and, in particular, G-gyroid-based microarchitectures represent a promising option for bone substitutes. Most studies on G-gyroid-based bone substitutes have reported only in silico or in vitro results, whereas in vivo data remain scarce and are generally limited to single G-gyroid microarchitectures. To identify the optimal G-gyroid microarchitecture for bone substitute applications, we compared three different G-gyroid designs with varying surface-to-surface distances to determine the most suitable architecture for osteoconduction and vertical bone augmentation. From a mechanical perspective, constructs with a wall-to-wall distance of 0.50 mm (gyroid05) exhibited higher compressive strength than those with distances of 0.80 mm (gyroid08) and 1.10 mm (gyroid11). In vivo assessment in a rabbit calvarial defect model demonstrated that defect bridging was improved by 44% with gyroid05 and by 40% with gyroid11 compared with gyroid08. In contrast, evaluation in a rabbit calvarial vertical bone augmentation model showed that bone height gain increased by 39% and 32% with gyroid08 and gyroid11, respectively, relative to gyroid05. Overall, the gyroid11 design demonstrated superior in vivo performance in defect bridging and bone augmentation, indicating that it may represent the most promising universal G-gyroid microarchitecture for bone substitutes.

Extracellular vesicles (EVs) derived from osteoblasts are emerging as key regulators of bone formation, yet functional differences between vesicles from distinct extracellular compartments remain unclear. In this study, we compared media-derived extracellular vesicles (MEs) and matrix-bound extracellular vesicle-enriched fractions (MBEs) isolated from mineralized osteoblasts (MOBs) and evaluated their effects on human bone marrow-derived mesenchymal stromal cells (hBMSCs). Both preparations, characterized by nanoparticle tracking analysis and transmission electron microscopy, displayed similar size distributions (30-200 nm) and concentrations. Moreover, both preparations showed enrichment of the EV-associated marker CD63, with no detectable GAPDH and only minimal Grp94 signals in a subset of samples. mRNA profiling revealed that MBE-enriched fractions were selectively enriched in RUNX2, whereas other osteogenesis-related transcripts were comparable between them. Functional analyses demonstrated that both EV populations promoted osteogenic differentiation of hBMSCs, although with distinct biological profiles. MBE-enriched fractions were associated with higher alkaline phosphatase activity under control conditions, whereas MEs induced greater osteocalcin expression and showed a numerical tendency toward increased matrix mineralization, particularly under osteogenic conditions. These findings suggest that extracellular vesicles associated with different extracellular compartments exhibit distinct osteogenic activities rather than a uniform biological effect. Although the matrix-bound preparation likely contains extracellular matrix-associated components co-isolated during the extraction procedure, the present study highlights the importance of extracellular compartmentalization in shaping EV-associated bioactivity and provides a foundation for future studies aimed at optimizing EV-based strategies for bone regeneration.

In regenerative medicine, addressing the complex challenge of bone tissue regeneration demands innovative strategies. Exosomes, nanoscale vesicles rich in bioactive molecules, have shown great promise in tissue repair. This study focuses on exosomes derived from mineralized osteoblasts (MOBs), which play a pivotal role in bone formation. We investigated the therapeutic potential of exosomes isolated from osteoblasts cultured in osteogenic medium for 21 days, delivered via 3D-printed gyroid scaffolds composed of hydroxyapatite (HA) and tricalcium phosphate (TCP). The exosomes were characterized through nanoparticle tracking analysis to determine size, morphology, and concentration, while proteomics revealed their cargo contents. In vitro, rabbit bone marrow mesenchymal stromal cells (rBMSCs) were cultured as monolayers and within ceramic scaffolds, where MOB-derived exosomes were shown to promote osteogenic differentiation. In vivo, their osteoconductive and bone augmentation capabilities were evaluated in two rabbit calvarial models, while the osteoinductive potential was further tested in a heterotopic mouse model. Neo-bone formation was assessed using µCT and histological analysis. Our findings demonstrated that MOB-derived exosomes upregulated bone-related gene expression and promoted mineralization in rBMSCs, even in the absence of osteogenic medium. Proteomics confirmed the presence of bone-associated proteins in these exosomes. In rabbit models, however, exosomes did not significantly enhance bone formation. In contrast, in the heterotopic mouse model, exosomes functionalized onto ceramic scaffolds exhibited strong osteoinductive activity. This study highlights the potential of MOB-derived exosomes to enhance 3D-printed ceramic scaffolds for bone regeneration, offering a promising avenue for bone healing without the need for additional growth factors or stem cells. STATEMENT OF SIGNIFICANCE: This study presents a novel strategy for bone regeneration by integrating exosomes derived from mineralized osteoblasts (MOBs) into 3D-printed ceramic scaffolds. While exosomes are increasingly studied for regenerative applications, the unique osteoinductive potential of MOB-derived exosomes has remained underexplored. Our findings demonstrate that these exosomes promote osteogenic differentiation and mineralization of stem cells without added growth factors. In vivo, their efficacy varied by anatomical site and species, highlighting key considerations for clinical translation. This work introduces a cell-free, bioactive scaffold platform that leverages naturally secreted vesicles for bone repair, offering potential alternatives to cell-based therapies. It will be of broad interest to researchers in tissue engineering, biomaterials, and translational regenerative medicine.

Additive manufacturing has emerged as a transformative tool in biomedical engineering, offering precise control over scaffold design for bone tissue engineering and regenerative medicine. While much attention has been focused on optimizing pore-based scaffold architectures, filament-based microarchitectures remain relatively understudied, despite the fact that the majority of 3D-printers generate filament-based structures. Here, we investigated the influence of filament characteristics on bone regeneration outcomes using a lithography-based additive manufacturing approach. Three distinct filament-based scaffolds (Fil050, Fil083, and Fil125) identical in macroporosity and transparency, crafted from tri-calcium phosphate (TCP) with varying filament thicknesses and distance, were evaluated in a rabbit model of bone augmentation and non-critical calvarial defect. Additionally, two scaffold types differing in filament directionality (Fil and FilG) were compared to elucidate optimal design parameters. Distance of bone ingrowth and percentage of regenerated area within scaffolds were measured by histomorphometric analysis. Our findings reveal filaments of 0.50 mm as the most effective filament-based scaffold, demonstrating superior bone ingrowth and bony regenerated area compared to larger size filament (i.e., 0.83 mm and 1.25 mm scaffolds). Optimized directionality of filaments can overcome the reduced performance of larger filaments. This study advances our understanding of microarchitecture’s role in bone tissue engineering and holds significant implications for clinical practice, paving the way for the development of highly tailored, patient-specific bone substitutes with enhanced efficacy.

Triply periodic minimal surface microarchitectures (TPMS) were developed by mathematicians and evolved in all kingdoms of living organisms. Renowned for their lightweight yet robust attributes, TPMS structures find application in diverse fields, such as the construction of satellites, aircrafts, and electric vehicles. Moreover, these microarchitectures, despite their intricate geometric patterns, demonstrate potential for application as bone substitutes, despite the inherent gothic style of natural bone microarchitecture. Here, we produced three TPMS microarchitectures, D-diamond, G-gyroid, and P-primitive, by 3D printing from hydroxyapatite. We explored their mechanical characterization and, further, implanted them to study their bone augmentation and osteoconduction potential. In terms of strength, the D-diamond and G-gyroid performed significantly better than the P-primitive. In a calvarial defect model and a calvarial bone augmentation model, where osteoconduction is determined as the extent of bony bridging of the defect and bone augmentation as the maximal vertical bone ingrowth, the G-gyroid performed significantly better than the P-primitive. No significant difference in performance was observed between the G-gyroid and D-diamond. Since, in real life, the treatment of bone deficiencies in patients comprises elements of defect bridging and bone augmentation, ceramic scaffolds with D-diamond and G-gyroid microarchitectures appear as the best choice for a TPMS-based scaffold in bone tissue engineering.

The functionalization of bone substitutes with exosomes appears to be a promising technique to enhance bone tissue formation. This study investigates the potential of exosomes derived from bone marrow mesenchymal stromal cells (BMSCs) to improve bone healing and bone augmentation when incorporated into wide open-porous 3D-printed ceramic Gyroid scaffolds. We demonstrated the multipotent characteristics of BMSCs and characterized the extracted exosomes using nanoparticle tracking analysis and proteomic profiling. Through cell culture experimentation, we demonstrated that BMSC-derived exosomes possess the ability to attract cells and significantly facilitate their differentiation into the osteogenic lineage. Furthermore, we observed that scaffold architecture influences exosome release kinetics, with Gyroid scaffolds exhibiting slower release rates compared to Lattice scaffolds. Nevertheless, in vivo implantation did not show increased bone ingrowth in scaffolds loaded with exosomes, suggesting that the scaffold microarchitecture and material were already optimized for osteoconduction and bone augmentation. These findings highlight the lack of understanding about the optimal delivery of exosomes for osteoconduction and bone augmentation by advanced ceramic scaffolds.

Beta-tricalcium phosphate (β-TCP) is a biocompatible ceramic material widely used in the field of oral regeneration. Due to its excellent biological and mechanical properties, it is increasingly utilized for alveolar ridge augmentation or guided bone regeneration (GBR). With recent advances in computer-aided design and manufacturing (CAD/CAM), β-TCP can now be used in the form of digitally designed patient-specific scaffolds for customized bone regeneration (CBR) of advanced defects in a two-stage implant therapy concept. In this case report following the CARE case report guidelines, we present a novel application of a patient-specific β-TCP scaffold in pre-implant mandibular alveolar ridge augmentation. A 63-year-old female patient with significant horizontal bone loss in the posterior mandible was treated with a custom β-TCP scaffold in the context of a two-stage backward-planned implant therapy. Cone-beam computed tomography nine months after augmentation showed successful integration of the scaffold into the surrounding bone, allowing implant placement. Follow-up until two years after initial surgery showed excellent oral and peri-implant health. This case highlights the potential of patient-specific β-TCP scaffolds for alveolar ridge augmentation and their advantage over traditional techniques, including avoidance of xeno-, allo-, and autografts. The results provide encouraging evidence for their use in clinical practice. Patient-specific β-TCP scaffolds may be a promising alternative for clinicians seeking to provide their patients with safe, predictable, and effective alveolar ridge augmentation results in customized bone regeneration procedures.

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.

The first set of data refers to Insulin-like Growth Factor-1 (IGF-1) protein incorporation via emulsion electrospinning into a DegraPolⓇ random fiber mesh and its characterization. Specifically, the fiber thickness was assessed and compared to pure DegraPolⓇ fibers without IGF-1 (control). Furthermore, the mechanical properties of these meshes were assessed and data on ultimate tensile stress, Young's modulus and ultimate fracture strain are presented for ring specimen and rectangular pieces taken from electrospun tubes in the transverse direction as well as rectangular pieces taken in the axial direction of the electrospun tube. Moreover, the static and the dynamic water contact angles were determined.The second set of data represents morphological aspects, such as the cytoskeletal aspect ratio (i.e. length of the cell divided by its width) for rabbit Achilles tenocytes stimulated in vitro with 1, 10, and 100 ng/mL IGF-1 supplementation compared to the corresponding cell culture without IGF-1 (control). Furthermore, qPCR was performed and collagen I, ki67 and tenomodulin gene expression data are presented for rabbit Achilles tenocytes in vitro with 0.1, 1 and 10 ng/mL IGF-1 supplementation, respectively, as well as with a supplementation of released IGF-1 from the DegraPol mesh (concentration was 1 ng/mL).

Biomimetic nanocrystalline apatites analogous to bone mineral can be prepared using soft chemistry. Due to their high similarity to bone apatite, as opposed to stoichiometric hydroxyapatite for example, they now represent an appealing class of compounds to produce bioactive ceramics for which drug delivery and ion exchange abilities have been described extensively. However, immersion in aqueous media of dried non-carbonated biomimetic apatite crystals may generate an acidification event, which is often disregarded and not been clarified to-date. Yet, this acidification process could limit their further development if it is not understood and overcome if necessary. This may, for example, alter biological test outcomes, during their evaluation as bone repair materials, due to potentially deleterious effects of the acidic environment on cells, especially in in vitro static conditions. In this study, we explore the origins of this acidification phenomenon based on complementary experimental data and we point out the central role of the hydrated ionic layer present on apatite nanocrystals. We then propose a practical strategy to circumvent this acidification effect using an adequate post-precipitation equilibration step that was optimized. Using this enutralization protocol, we then showed the possibility of performing (micro)biological assessments on such compounds and provide an illustration with the examples of post-equilibrated Cu2+- and Ag+-doped nanocrystalline apatites. We demonstrate their non-cytotoxicity to osteoblast cells and their antibacterial features as tested versus five major pathogens involved in bone infections, therefore pointing to their relevance in the field of antibacterial bone substitutes. The preliminary in vivo implantation of a relevant sample in a rat’s calvarial defect confirmed its biocompatibility and the absence of adverse reaction. Understanding and eliminating this technical barrier should help promoting biomimetic apatites as a genuine new class of biomaterial-producing compounds for bone regeneration applications, e.g., with antibacterial features, far from being solely considered as “laboratory curiosities”.

Tendon injuries can result in two major drawbacks. Adhesions to the surrounding tissue may limit the range of motion, while fibrovascular scar formation can lead to poor biomechanical outcomes. Prosthetic devices may help to mitigate those problems. Emulsion electrospinning was used to develop a novel three-layer tube based on the polymer DegraPol (DP), with incorporated insulin-like growth factor-1 (IGF-1) in the middle layer. Scanning electron microscopy was utilized to assess the fiber diameter in IGF-1 containing pure DP meshes. Further characterization was performed with Fourier Transformed Infrared Spectroscopy, Differential Scanning Calorimetry, and water contact angle, as well as through the assessment of mechanical properties and release kinetics from ELISA, and the bioactivity of IGF-1 by qPCR of collagen I, ki67, and tenomodulin in rabbit Achilles tenocytes. The IGF-1-containing tubes exhibited a sustained release of the growth factor up to 4 days and showed bioactivity by significantly upregulated ki67 and tenomodulin gene expression. Moreover, they proved to be mechanically superior to pure DP tubes (significantly higher fracture strain, failure stress, and elastic modulus). The novel three-layer tubes intended to be applied over conventionally sutured tendons after a rupture may help accelerate the healing process. The release of IGF-1 stimulates proliferation and matrix synthesis of cells at the repair site. In addition, adhesion formation to surrounding tissue can be reduced due to the physical barrier.

Revolutionary fabrication technologies such as three-dimensional (3D) printing to develop dental structures are expected to replace traditional methods due to their ability to establish constructs with the required mechanical properties and detailed structures. Three-dimensional printing, as an additive manufacturing approach, has the potential to rapidly fabricate complex dental prostheses by employing a bottom-up strategy in a layer-by-layer fashion. This new technology allows dentists to extend their degree of freedom in selecting, creating, and performing the required treatments. Three-dimensional printing has been narrowly employed in the fabrication of various kinds of prostheses and implants. There is still an on-demand production procedure that offers a reasonable method with superior efficiency to engineer multifaceted dental constructs. This review article aims to cover the most recent applications of 3D printing techniques in the manufacturing of dental prosthetics. More specifically, after describing various 3D printing techniques and their advantages/disadvantages, the applications of 3D printing in dental prostheses are elaborated in various examples in the literature. Different 3D printing techniques have the capability to use different materials, including thermoplastic polymers, ceramics, and metals with distinctive suitability for dental applications, which are discussed in this article. The relevant limitations and challenges that currently limit the efficacy of 3D printing in this field are also reviewed. This review article has employed five major scientific databases, including Google Scholar, PubMed, ScienceDirect, Web of Science, and Scopus, with appropriate keywords to find the most relevant literature in the subject of dental prostheses 3D printing.

Autologous bone remains the gold standard bone substitute in clinical practice. Therefore, the microarchitecture of newly developed synthetic bone substitutes, which reflects the spatial distribution of materials in the scaffold, aims to recapitulate the natural bone microarchitecture. However, the natural bone microarchitecture is optimized to obtain a mechanically stable, lightweight structure adapted to the biomechanical loading situation. In the context of synthetic bone substitutes, the application of a Triply Periodic Minimum Surface (TPMS) algorithm can yield stable lightweight microarchitectures that, despite their demanding architectural complexity, can be produced by additive manufacturing. In this study, we applied the TPMS derivative Adaptive Density Minimal Surfaces (ADMS) algorithm to produce scaffolds from hydroxyapatite (HA) using a lithography-based layer-by-layer methodology and compared them with an established highly osteoconductive lattice microarchitecture. We characterized them for compression strength, osteoconductivity, and bone regeneration. The in vivo results, based on a rabbit calvaria defect model, showed that bony ingrowth into ADMS constructs as a measure of osteoconduction depended on minimal constriction as it limited the maximum apparent pore diameter in these scaffolds to 1.53 mm. Osteoconduction decreased significantly at a diameter of 1.76 mm. The most suitable ADMS microarchitecture was as osteoconductive as a highly osteoconductive orthogonal lattice microarchitecture in noncritical- and critical-size calvarial defects. However, the compression strength and microarchitectural integrity in vivo were significantly higher for scaffolds with their microarchitecture based on the ADMS algorithm when compared with high-connectivity lattice microarchitectures. Therefore, bone substitutes with high osteoconductivity can be designed with the advantages of the ADMS-based microarchitectures. As TPMS and ADMS microarchitectures are true lightweight structures optimized for high mechanical stability with a minimal amount of material, such microarchitectures appear most suitable for bone substitutes used in clinical settings to treat bone defects in weight-bearing and non-weight-bearing sites.

The early phase of bone healing is a complex and poorly understood process. With additive manufacturing, we can generate a specific and customizable library of bone substitutes to explore this phase. In this study, we produced tricalcium phosphate-based scaffolds with microarchitectures composed of filaments of 0.50 mm in diameter, named Fil050G, and 1.25 mm named Fil125G, respectively. The implants were removed after only 10 days in vivo followed by RNA sequencing (RNAseq) and histological analysis. RNAseq results revealed upregulation of adaptive immune response, regulation of cell adhesion, and cell migration-related genes in both of our two constructs. However, significant overexpression of genes linked to angiogenesis, regulation of cell differentiation, ossification, and bone development was observed solely in Fil050G scaffolds. Moreover, quantitative immunohistochemistry of structures positive for laminin revealed a significantly higher number of blood vessels in Fil050G samples. Furthermore, µCT detected a higher amount of mineralized tissue in Fil050G samples suggesting a superior osteoconductive potential. Hence, different filament diameters and distances in bone substitutes significantly influence angiogenesis and regulation of cell differentiation involved in the early phase of bone regeneration, which precedes osteoconductivity and bony bridging seen in later phases and as consequence, impacts the overall clinical outcome.

Triply periodic minimal surfaces (TPMSs) are found to be promising microarchitectures for bone substitutes owing to their low weight and superior mechanical characteristics. However, existing studies on their application are incomplete because they focus solely on biomechanical or in vitro aspects. Hardly any in vivo studies where different TPMS microarchitectures are compared have been reported. Therefore, we produced hydroxyapatite-based scaffolds with three types of TPMS microarchitectures, namely Diamond, Gyroid, and Primitive, and compared them with an established Lattice microarchitecture by mechanical testing, 3D-cell culture, and in vivo implantation. Common to all four microarchitectures was the minimal constriction of a sphere of 0.8 mm in diameter, which earlier was found superior in Lattice microarchitectures. Scanning by μCT revealed the precision and reproducibility of our printing method. The mechanical analysis showed significantly higher compression strength for Gyroid and Diamond samples compared with Primitive and Lattice. After in vitro culture with human bone marrow stromal cells in control or osteogenic medium, no differences between these microarchitectures were observed. However, from the TPMS microarchitectures, Diamond- and Gyroid-based scaffolds showed the highest bone ingrowth and bone-to-implant contact in vivo. Therefore, Diamond and Gyroid designs appear to be the most promising TPMS-type microarchitectures for scaffolds produced for bone tissue engineering and regenerative medicine. Impact Statement Extensive bone defects require the application of bone grafts. To match the existing requirements, scaffolds based on triply periodic minimal surface (TPMS)–based microarchitectures could be used as bone substitutes. This work is dedicated to the investigation of mechanical and osteoconductive properties of TPMS-based scaffolds to determine the influencing factors on differences in their behavior and choose the most promising design to be used in bone tissue engineering.

Healing large bone defects remains challenging in orthopedic surgery and is often associated with poor outcomes and complications. A major issue with bioengineered constructs is achieving a continuous interface between host bone and graft to enhance biological processes and mechanical stability. In this study, we have developed a new bioengineering strategy to produce oriented biocompatible 3D PLGA/aCaP nanocomposites with enhanced osseointegration. Decellularized scaffolds -containing only extracellular matrix- or scaffolds seeded with adipose-derived mesenchymal stromal cells were tested in a mouse model for critical size bone defects. In parallel to micro-CT analysis, SAXS tensor tomography and 2D scanning SAXS were employed to determine the 3D arrangement and nanostructure within the critical-sized bone. Both newly developed scaffold types, seeded with cells or decellularized, showed high osseointegration, higher bone quality, increased alignment of collagen fibers and optimal alignment and size of hydroxyapatite minerals.

Additive manufacturing enables the realization of the macro- and microarchitecture of bone substitutes. The macroarchitecture is determined by the bone defect and its shape makes the implant patient specific. The preset distribution of the 3D-printed material in the macroarchitecture defines the microarchitecture. At the lower scale, the nanoarchitecture of 3D-printed scaffolds is dependent on the post-processing methodology such as the sintering temperature. However, the role of microarchitecture and nanoarchitecture of scaffolds for osteoconduction is still elusive. To address these aspects in more detail, we produced lithography-based osteoconductive scaffolds from hydroxyapatite (HA) of identical macro- and microarchitecture and varied their nanoarchitecture, such as microporosity, by increasing the maximum sintering temperatures from 1100 to 1400 °C. The different scaffold types were characterized for microporosity, compression strength, and nanoarchitecture. The in vivo results, based on a rabbit calvarial defect model showed that bony ingrowth, as a measure of osteoconduction, was independent from scaffold's microporosity. The same applies to in vitro osteoclastic resorbability, since on all tested scaffold types, osteoclasts formed on their surfaces and resorption pits upon exposure to mature osteoclasts were visible. Thus, for wide-open porous HA-based scaffolds, a low degree of microporosity and high mechanical strength yield optimal osteoconduction and creeping substitution. Based on our study, non-unions, the major complication during demanding bone regeneration procedures, could be prevented.

INTRODUCTION:An oesophageal tube feeder was used to administer milk to six clinically healthy calves every 12 hours for a total of three feedings. The calves were seven to nine days of age, and each feeding consisted of a volume of milk that was 6 % of the body weight of the calves. The reticulum and abomasum were scanned ultrasonographically before, during and after drenching. Blood samples for D-lactate determination and venous blood gas analysis were collected at -12, 0 (first drenching), 6, 12 (second drenching), 18, 24 (third drenching), 30 and 36 hours. Ruminal fluid was collected for measurement of pH and D-lactate concentration at -12 and 36 hours. Based on simultaneous ultrasonographic examinations of the reticulum and abomasum, some of the force-fed milk flowed into the reticulum and some directly into the abomasum; both organs contained milk immediately after drenching. Drenching led to ruminal acidosis evidenced by a significant decrease in median pH from 7,0 (6,0 - 7,0) to 4,5 (3,0 - 5,0) and a 93-fold increase in median D-lactate concentration from 0,39 (0,14 - 1,33) to 36,3 (17,9 - 53,3) mmol/l but it did not cause metabolic acidosis. Based on these findings, we concluded that milk administered to calves via an oesophageal tube feeder flows into the reticulum and abomasum simultaneously and three force feedings 12 hours apart cause acute ruminal acidosis, which was not accompanied by metabolic acidosis in the short term.

63Additive manufacturing can be applied to produce personalized bone substitutes. At present, the major three-dimensional (3D) printing methodology relies on filament extrusion. In bioprinting, the extruded filament consists mainly of hydrogels, in which growth factors and cells are embedded. In this study, we used a lithography-based 3D printing methodology to mimic filament-based microarchitectures by varying the filament dimension and the distance between the filaments. In the first set of scaffolds, all filaments were aligned toward bone ingrowth direction. In a second set of scaffolds, which were derived from the identical microarchitecture but tilted by 90°, only 50% of the filaments were in line with the bone ingrowth direction. Testing of all tricalcium phosphate-based constructs for osteoconduction and bone regeneration was performed in a rabbit calvarial defect model. The results revealed that if all filaments are in line with the direction of bone ingrowth, filament size and distance (0.40-1.25 mm) had no significant influence on defect bridging. However, with 50% of filaments aligned, osteoconductivity declined significantly with an increase in filament dimension and distance. Therefore, for filament-based 3D- or bio-printed bone substitutes, the distance between the filaments should be 0.40 to 0.50 mm irrespective of the direction of bone ingrowth or up to 0.83 mm if perfectly aligned to it.