Bone Tissue Engineering Strategies Research Articles

3D bioprinting of prevascularised implants for the repair of critically-sized bone defects

For 3D bioprinted tissues to be scaled-up to clinically relevant sizes, effective prevascularisation strategies are required to provide the necessary nutrients for normal metabolism and to remove associated waste by-products. The aim of this study was to develop a bioprinting strategy to engineer prevascularised tissues in vitro and to investigate the capacity of such constructs to enhance the vascularisation and regeneration of large bone defects in vivo. From a screen of different bioinks, a fibrin-based hydrogel was found to best support human umbilical vein endothelial cell (HUVEC) sprouting and the establishment of a microvessel network. When this bioink was combined with HUVECs and supporting human bone marrow stem/stromal cells (hBMSCs), these microvessel networks persisted in vitro. Furthermore, only bioprinted tissues containing both HUVECs and hBMSCs, that were first allowed to mature in vitro, supported robust blood vessel development in vivo. To assess the therapeutic utility of this bioprinting strategy, these bioinks were used to prevascularise 3D printed polycaprolactone (PCL) scaffolds, which were subsequently implanted into critically-sized femoral bone defects in rats. Micro-computed tomography (µCT) angiography revealed increased levels of vascularisation in vivo, which correlated with higher levels of new bone formation. Such prevascularised constructs could be used to enhance the vascularisation of a range of large tissue defects, forming the basis of multiple new bioprinted therapeutics. Statement of significanceThis paper demonstrates a versatile 3D bioprinting technique to improve the vascularisation of tissue engineered constructs and further demonstrates how this method can be incorporated into a bone tissue engineering strategy to improve vascularisation in a rat femoral defect model.

3D-Bioprinting Strategies Based on In Situ Bone-Healing Mechanism for Vascularized Bone Tissue Engineering.

Over the past decades, a number of bone tissue engineering (BTE) approaches have been developed to address substantial challenges in the management of critical size bone defects. Although the majority of BTE strategies developed in the laboratory have been limited due to lack of clinical relevance in translation, primary prerequisites for the construction of vascularized functional bone grafts have gained confidence owing to the accumulated knowledge of the osteogenic, osteoinductive, and osteoconductive properties of mesenchymal stem cells and bone-relevant biomaterials that reflect bone-healing mechanisms. In this review, we summarize the current knowledge of bone-healing mechanisms focusing on the details that should be embodied in the development of vascularized BTE, and discuss promising strategies based on 3D-bioprinting technologies that efficiently coalesce the abovementioned main features in bone-healing systems, which comprehensively interact during the bone regeneration processes.

Chaetocin Promotes Osteogenic Differentiation via Modulating Wnt/Beta-Catenin Signaling in Mesenchymal Stem Cells.

Mesenchymal stemXin cells (MSCs) are a great cell source for bone regeneration. Although combining MSCs with growth factors and scaffolds provides a useful clinical strategy for bone tissue engineering, the efficiency of MSC osteogenic differentiation remains to be improved. Epigenetic modification is related to the differentiation ability of MSCs during osteogenic induction. In this study, we evaluate the effect of Chaetocin, an inhibitor of lysine-specific histone methyltransferases, on the differentiation of MSCs. We found that MSCs treated with Chaetocin demonstrated increased osteogenic ability and reduced adipogenic ability. The expression of osteogenic markers (Runx2 and OPN) was induced in MSCs by Chaetocin during osteogenic induction. Moveover, treatment of Chaetocin in MSCs improves Wnt/β-catenin signaling pathways and its downstream targets. Finally, we showed increased bone formation of MSC and Wnt/β-catenin signaling activity by treatment of Chaetocin using in vivo bone formation assays. Our data uncovered a critical role of Chaetocin in MSC osteogenic differentiation and provide new insights into bone tissue regeneration and repair.

Biomimetic, biodegradable, and osteoinductive Microgels with open porous structure and excellent injectability for construction of microtissues for bone tissue engineering

Developing microtissues that could promote bone regeneration remains a challenge. This study develops a biomimetic, biodegradable and osteoinductive microgel called 40% strontium-substituted hydroxyapatite/Microgel (40% Sr-HA/Microgel) to construct microtissues that could facilitate bone tissue regeneration. The open porous structure (with a pore diameter of 33 μm and porosity of 88%) of the 40% Sr-HA/Microgels could promote cellular proliferation and realize cell–cell interactions. The 40% Sr-HA/Microgels could stably release Sr2+ and show excellent osteoinductivity. In addition, the 40% Sr-HA/Microgels show outstanding biodegradable and injectable performances. Therefore, the 40% Sr-HA/Microgels could support the formation of microtissues to boost bone tissue regeneration. This study proposes an advanced microgel that could meet the need to form microtissues for bone tissue regeneration, thereby offering an accelerated therapeutic strategy for bone tissue engineering.

Scaffold-free human mesenchymal stem cell construct geometry regulates long bone regeneration

Biomimetic bone tissue engineering strategies partially recapitulate development. We recently showed functional restoration of femoral defects using scaffold-free human mesenchymal stem cell (hMSC) condensates featuring localized morphogen presentation with delayed in vivo mechanical loading. Possible effects of construct geometry on healing outcome remain unclear. Here, we hypothesized that localized presentation of transforming growth factor (TGF)-β1 and bone morphogenetic protein (BMP)-2 to engineered hMSC tubes mimicking femoral diaphyses induces endochondral ossification, and that TGF-β1 + BMP-2-presenting hMSC tubes enhance defect healing with delayed in vivo loading vs. loosely packed hMSC sheets. Localized morphogen presentation stimulated chondrogenic priming/endochondral differentiation in vitro. Subcutaneously, hMSC tubes formed cartilage templates that underwent bony remodeling. Orthotopically, hMSC tubes stimulated more robust endochondral defect healing vs. hMSC sheets. Tissue resembling normal growth plate was observed with negligible ectopic bone. This study demonstrates interactions between hMSC condensation geometry, morphogen bioavailability, and mechanical cues to recapitulate development for biomimetic bone tissue engineering.

A novel magnesium ion-incorporating dual-crosslinked hydrogel to improve bone scaffold-mediated osteogenesis and angiogenesis

Osteogenesis is closely complemented by angiogenesis during the bone regeneration process. The development of functional hydrogel bone substitutes that mimic the extracellular matrix is a promising strategy for bone tissue engineering. However, the development of scaffold materials tailored to exhibit sufficient biomechanics, biodegradability, and favorable osteogenic and angiogenic activity continue to present a great challenge. Herein, we prepared a novel magnesium ion-incorporating dual-crosslinked hydrogel through the photocrosslinking of gelatin methacryloyl (GelMA), thiolated chitosan (TCS) and modified polyhedral oligomeric silsesquioxane (POSS) nanoparticles, and active Mg2+ ions were then introduced into system via coordination bonds of MgS, which can be tailored to possess superior mechanical strength, a stable network structure and more suitable pore size and degradation properties. The fabricated GelMA/TCS/POSS-Mg hydrogels effectively promoted cell adhesion, spreading, and proliferation, demonstrating that the introduction of POSS and Mg2+ not only stimulates the osteogenic differentiation of BMSCs but also promotes angiogenesis both in vitro and in vivo, thereby facilitating subsequent bone regeneration in calvarial defects of rats. Taken together, the results of this study indicate that the GelMA/TCS/POSS-Mg hydrogel has promising potential for repairing bone defects by promoting cell adhesion, osteogenesis and vascularization.

Three-dimensionally printed polycaprolactone/multicomponent bioactive glass scaffolds for potential application in bone tissue engineering

AbstractOver the last years, three-dimensional (3D) printing has been successfully applied to produce suitable substitutes for treating bone defects. In this work, 3D printed composite scaffolds of polycaprolactone (PCL) and strontium (Sr)- and cobalt (Co)-doped multi-component melt-derived bioactive glasses (BGs) were prepared for bone tissue engineering strategies. For this purpose, 30% of as-prepared BG particles (size <38 μm) were incorporated into PCL, and then the obtained composite mix was introduced into a 3D printing machine to fabricate layer-by-layer porous structures with the size of 12 × 12 × 2 mm3.The scaffolds were fully characterized through a series of physico-chemical and biological assays. Adding the BGs to PCL led to an improvement in the compressive strength of the fabricated scaffolds and increased their hydrophilicity. Furthermore, the PCL/BG scaffolds showed apatite-forming ability (i.e., bioactivity behavior) after being immersed in simulated body fluid (SBF). The in vitro cellular examinations revealed the cytocompatibility of the scaffolds and confirmed them as suitable substrates for the adhesion and proliferation of MG-63 osteosarcoma cells. In conclusion, 3D printed composite scaffolds made of PCL and Sr- and Co-doped BGs might be potentially-beneficial bone replacements, and the achieved results motivate further research on these materials.

Regenerative Approaches for the Treatment of Large Bone Defects.

A variety of engineered materials have gained acceptance in orthopedic practice as substitutes for autologous bone grafts, although the regenerative efficacy of these engineered grafts is still limited compared with that of transplanted native tissues. For bone defects greater than 4-5 cm, however, common bone grafting procedures are insufficient and more complicated surgical interventions are required to repair and regenerate the damaged or missing bone. In this review, we describe current grafting materials and surgical techniques for the reconstruction of large bone defects, followed by tissue engineering (TE) efforts to develop improved therapies. Particular emphasis is placed on graft vascularization, because for both autologous bone and engineered alternatives, achieving adequate vascular development within the regenerating bone tissues remains a significant challenge in the context of large bone defects. To this end, TE and surgical strategies to induce development of a vasculature within bone grafts are discussed. Impact statement This review aims to present an accessible and thorough overview of current orthopedic surgical techniques as well as bone tissue engineering and vascularization strategies that might one day offer improvements to clinical therapies for the repair of large bone defects. We consider the lessons that clinical orthopedic reconstructive practices can contribute to the push toward engineered bone.

A nano approach towards the creation of a biointerface as stimulator of osteogenic differentiation

There is a great need for tissue engineering constructs with the ability to modulate stem cell behavior. The initial adhesion, growth and differentiation of stem cell are a key strategy in bone tissue engineering and it can be controlled through biomaterial-cell interface. Here we engineered a polycaprolactone/gelatin/bioactive glass (PCL/GT/BG) nanocomposite scaffold coated with Fibronectin (FN) as a potential candidate to aid the bone regeneration process by giving cells a temporary template to grow into. For this purpose, initially BG nanoparticles (nBG) of 70 ± 15 nm were synthesized, characterized and then impregnated into PCL/GT matrix to create a nanocomposite fibrous mesh. An optimized structure was selected based on fiber uniformity, diameter, and the mechanical properties. Cell adhesion, growth, and the expression of osteogenic-related genes as a result of FN tethering, through specific surface interactions, was evaluated. Furthermore, the potential of optimized nanofiberous structure as a drug delivery vehicle for the local release of therapeutic agents was studied by using amoxicillin as a model drug. The release profile revealed that around 70% of drug was released in an hour for non-crosslinked fibers (burst release) followed by a gradual release up to 72 h. The release profile was steadier for crosslinked fibers. The scaffold also showed an antibacterial effect against ubiquitous gram-positive Staphylococcus aureus. The current study provides an insight for future researchers who aim to create nanocomposite materials as multifunctional scaffolds for bone tissue engineering applications.

The Bone-Forming Properties of Periosteum-Derived Cells Differ Between Harvest Sites.

The development of alternatives for autologous bone grafts is a major focus of bone tissue engineering. To produce living bone-forming implants, skeletal stem and progenitor cells (SSPCs) are envisioned as key ingredients. SSPCs can be obtained from different tissues including bone marrow, adipose tissue, dental pulp, and periosteum. Human periosteum-derived cells (hPDCs) exhibit progenitor cell characteristics and have well-documented in vivo bone formation potency. Here, we have characterized and compared hPDCs derived from tibia with craniofacial hPDCs, from maxilla and mandible, respectively, each representing a potential source for cell-based tissue engineered implants for craniofacial applications. Maxilla and mandible-derived hPDCs display similar growth curves as tibial hPDCs, with equal trilineage differentiation potential toward chondrogenic, osteogenic, and adipogenic cells. These craniofacial hPDCs are positive for SSPC-markers CD73, CD164, and Podoplanin (PDPN), and negative for CD146, hematopoietic and endothelial lineage markers. Bulk RNA-sequencing identified genes that are differentially expressed between the three sources of hPDC. In particular, differential expression was found for genes of the HOX and DLX family, for SOX9 and genes involved in skeletal system development. The in vivo bone formation, 8 weeks after ectopic implantation in nude mice, was observed in constructs seeded with tibial and mandibular hPDCs. Taken together, we provide evidence that hPDCs show different profiles and properties according to their anatomical origin, and that craniofacial hPDCs are potential sources for cell-based bone tissue engineering strategies. The mandible-derived hPDCs display - both in vitro and in vivo - chondrogenic and osteogenic differentiation potential, which supports their future testing for use in craniofacial bone regeneration applications.

3-D macro/microporous-nanofibrous bacterial cellulose scaffolds seeded with BMP-2 preconditioned mesenchymal stem cells exhibit remarkable potential for bone tissue engineering

Bone repair using BMP-2 is a promising therapeutic approach in clinical practices, however, high dosages required to be effective pose issues of cost and safety. The present study explores the potential of low dose BMP-2 treatment via tissue engineering approach, which amalgamates 3-D macro/microporous-nanofibrous bacterial cellulose (mNBC) scaffolds and low dose BMP-2 primed murine mesenchymal stem cells (C3H10T1/2 cells). Initial studies on cell-scaffold interaction using unprimed C3H10T1/2 cells confirmed that scaffolds provided a propitious environment for cell adhesion, growth, and infiltration, owing to its ECM-mimicking nano-micro-macro architecture. Osteogenic studies were conducted by preconditioning the cells with 50 ng/mL BMP-2 for 15 min, followed by culturing on mNBC scaffolds for up to three weeks. The results showed an early onset and significantly enhanced bone matrix secretion and maturation in the scaffolds seeded with BMP-2 primed cells compared to the unprimed ones. Moreover, mNBC scaffolds alone were able to facilitate the mineralization of cells to some extent. These findings suggest that, with the aid of ‘osteoinduction’ from low dose BMP-2 priming of stem cells and ‘osteoconduction’ from nano-macro/micro topography of mNBC scaffolds, a cost-effective bone tissue engineering strategy can be designed for quick and excellent in vivo osseointegration.

Bone Defect Reconstruction Via Endochondral Ossification: A Developmental Engineering Strategy

Bone tissue engineering (BTE) is a growing area of research in extensive bone defect reconstruction. Traditional BTE strategies induce direct bone-like matrix formation by mimicking the embryological process of intramembranous ossification. However, clinical translation of traditional tissue-engineered bone grafts is hampered by their limited in vivo vascularization and poor bone regeneration after implantation. To overcome this hurdle, an emerging engineering strategy advocates to fabricate cartilaginous constructs for reconstruction of large bone defects, which was inspired by recapitulating the developmental processes of endochondral ossification (ECO). Such developmentally engineered constructs can act as a transient biomimetic template to facilitate bone regeneration in a critical-size defect. Accordingly, various studies following the principles of developmental engineering, including ectopic bone formation and orthotopic bone regeneration studies, have recently shown their unique abilities to survive under hypoxic conditions as well as induce neovascularization and ossification. This review introduces the concept and mechanism of the developmental BTE, highlights the routes of endochondral graft engineering, discusses states of large bone defect reconstruction via the ECO-based strategies, and offers perspectives on the challenges and future directions of translating current knowledge from bench to bedside.

Patterned Piezoelectric Scaffolds for Osteogenic Differentiation.

The morphological clues of scaffolds can determine cell behavior and, therefore, the patterning of electroactive polymers can be a suitable strategy for bone tissue engineering. In this way, this work reports on the influence of poly(vinylidene fluoride-co-trifluoroethylene) (P(VDF-TrFE)) electroactive micropatterned scaffolds on the proliferation and differentiation of bone cells. For that, micropatterned P(VDF-TrFE) scaffolds were produced by lithography in the form of arrays of lines and hexagons and then tested for cell proliferation and differentiation of pre-osteoblast cell line. Results show that more anisotropic surface microstructures promote bone differentiation without the need of further biochemical stimulation. Thus, the combination of specific patterns with the inherent electroactivity of materials provides a promising platform for bone regeneration.

Rational design of hydrogels to enhance osteogenic potential.

Bone tissue engineering (BTE) encompasses the field of biomaterials, cells, and bioactive molecules to successfully guide the growth and repair of bone tissue. Current BTE strategies rely on delivering osteogenic molecules or cells via scaffolding materials. However, growth factor- and stem cell-based treatments have several limitations, such as source restriction, low stability, difficulties in predicting long-term efficacy, and high costs, among others. These issues have promoted the development of material-based therapy with properties of accessibility, high stability, tunable efficacy, and low-cost production. Hydrogels are widely used in BTE applications because of their unique hydrophilic nature and tunable physicochemical properties to mimic the native bone environment. However, current hydrogel materials are not ideal candidates due to minimal osteogenic capability on their own. Therefore, recent studies of BTE hydrogels attempt to counterbalance these issues by modifying their biophysical properties. In this article, we review recent progress in the design of hydrogels to instruct osteogenic potential, and present strategies developed to precisely control its bone healing properties.

18F- based Quantification of the Osteogenic Potential of hMSCs.

In bone tissue engineering, there is a constant need to design new methods for promoting in vitro osteogenic differentiation. Consequently, there is a strong demand for fast, effective and reliable methods to track and quantify osteogenesis in vitro. In this study, we used the radiopharmacon fluorine-18 (18F) to evaluate the amount of hydroxylapatite produced by mesenchymal stem cells (MSCs) in a monolayer cell culture in vitro. The hydroxylapatite bound tracer was evaluated using µ-positron emission tomography (µ-PET) scanning and activimeter analysis. It was therefore possible to determine the amount of synthesized mineral and thus to conclude the osteogenic potential of the cells. A Student’s t-test revealed a highly significant difference regarding tracer uptake between the osteogenic group and the corresponding control group (µ-PET p = 0.043; activimeter analysis p = 0.012). This tracer uptake showed a highly significant correlation with the gold standard of quantitative Alizarin Red staining (ARS) (r2 = 0.86) as well as with the absolute calcium content detected by inductively coupled plasma mass spectrometry (r2 = 0.81). The results showed that 18F labeling is a novel method to prove and quantify hydroxyapatite content in MSC monolayer cultures. The mineral layer remains intact for further analysis. This non-destructive in vitro method can be used to rapidly investigate bone tissue engineering strategies in terms of hydroxylapatite production, and could therefore accelerate the process of implementing new strategies in clinical practice.

Computational Fluid Dynamics in 3D-Printed Scaffolds with Different Strand-Orientation in Perfusion Bioreactors

Bone tissue engineering strategies use fluid flow dynamics inside 3D-scaffolds in perfusion, bioreactors mechanically stimulate cells in these scaffolds. Fluid flow dynamics depends on the bioreactor’s inlet flow rate and 3D-scaffold architecture. We aimed to employ a computational evaluation to assess fluid dynamics in 3D-printed scaffolds with different angular orientations between strands in each layer inside a perfusion bioreactor at different inlet flow rates. 3D-printed cubic scaffolds (0.6×0.6×0.6 cm; total volume 216×10-3 cm3) containing strands (diameter 100 µm) with regular internal structure and different angular orientation (30°, 45°, 60°, and 90° between strands in each layer) were used for modeling. The finite element method showed that the perfusion bioreactor’s inlet flow rate (0.02, 0.1, 0.5 mL/min) was linearly related to average fluid velocity, average fluid shear stress, and average wall shear stress inside 3D-printed scaffolds with different angular orientation (30°, 45°, 60°, 90°) between strands in each layer. At all inlet flow rates, strands at 30° angular orientation increased average fluid velocity (1.2-1.5-fold), average fluid shear stress (6-10-fold), and average wall shear stress (1.4-2-fold) compared to strands at 45°, 60°, and 90° angular orientation providing similar results. In conclusion, significant local changes in fluid dynamics inside 3D-printed scaffolds result from varying the degree of angular orientation between strands in each layer, and the perfusion bioreactor’s inlet flow rate. By decreasing the angular orientation between strands in each layer and increasing the inlet flow rate of a perfusion bioreactor, the magnitude and distribution of fluid velocity, fluid shear stress, and wall shear stress inside the scaffold increased. The average fluid velocity, average fluid shear stress, and average wall shear stress inside the scaffold within the bioreactor increased linearly with the inlet flow rate. This might have important implications for bone tissue engineering strategies using cells, scaffolds, and bioreactors.

Study on the bone morphogenetic protein 2 loaded synergistic hierarchical porous silk/carbon nanocage scaffold for the repair of bone defect

Bone morphogenic protein-2 (BMP2) as an outstanding growth factor has a dominant role in the new bone formation, which still leaves much room for efficiency improvements to its clinical application owning to the potential side effects with high dosage. In this study, carbon nanocages (CNCs) were firstly considered as a synergistic factor for BMP2. CNC could improve the osteogenesis of BMP2 (0.8 μg/mL) at a concentration of 20–80 μg/mL in a dose-dependent manner. Moreover, a composite hierarchical porous scaffold composed of silk fibroin (SF) and CNC (SF/CNC) was developed for the controlled delivery of BMP2, which provided delivery of BMP2 with an initial burst release of 23.9% and a gradual release over the subsequent 6 days to about 47.7%. According to the ALP staining and quantification, CNC and SF/CNC scaffold (without BMP2) were evidenced to promote osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) dose-dependently. After loading BMP2, BMP2/SF/CNC scaffold could significantly increase the osteogenic differentiation of BMSCs in vitro and promote new bone formation in vivo. BMP2/SF/CNC scaffold developed in this study could efficiently prolong the local retention of BMP2 and its synergic interaction duration with CNC, which provides a new strategy for bone tissue engineering.

The sialylation profile of IgG determines the efficiency of antibody directed osteogenic differentiation of iMSCs by modulating local immune responses and osteoclastogenesis

Antibody-mediated osseous regeneration (AMOR) has been proved as a promising strategy for osteogenic differentiation of induced pluripotent stem cells derived MSCs (iMSCs). The key characteristic of antibody that determines the AMOR potential is largely unknown. The glycosylation profile of immunoglobulin G (IgG) represents a key checkpoint that determines its effector functions. Herein, we modified the sialylation profile of BMP2 antibodies to investigate the effects of glycosylation on antibody-mediated osteogenic differentiation of iMSCs. We found that over-sialylated BMP2 antibodies stimulated the highest amount of new bone while those non- or low-sialylated led to bone porosity and collapse. The immune response aroused by BMP2 immune complexes (BMP2-ICs) was intensified by desialylation, which contributed to an environment that favored osteoclastogenesis while inhibited osteoblastogenesis. In vitro study further demonstrated that the osteogenic potential of BMP2-ICs was not significantly affected by the degree of sialylation. On the other hand, BMP2-ICs could stimulate osteoclastogenesis by binding FcγRs on preosteoclasts directly, which was significantly intensified by desialylation and attenuated by over-sialylation. Bone defects implanted with alginate microbeads loaded with iMSCs and over-sialylated antibodies showed more bone formation than those sites with non- or low sialylated antibodies. Taken together, our study demonstrated that sialylation profile is one of the traits that decide the AMOR potential of BMP2 antibodies. Enhancement of sialylation may be a promising strategy to optimize antibody for iMSCs application in bone tissue engineering. Statement of significanceAntibody-mediated osseous regeneration (AMOR) is a promising strategy for bone tissue engineering that takes advantage of the specific reactivity of antibodies to sequester endogenous BMP2 and present it to osteoprogenitor cells. We previously demonstrated that BMP2 immune complex can drive iPSCs derived MSCs to osteogenic lineage. In this study, we analyze the effects of glycosylation profile on antibody directed osteogenic differentiation of iMSCs because glycosylation profile represents a key checkpoint that determines the effector functions of antibodies, and it is susceptible to variations in different clones. The results showed that sialylation profile is one of the traits that decides the AMOR potential of BMP2 antibody, and the enhancement of sialylation maybe a promising strategy to optimize antibodies for AMOR.

Inlet flow rate of perfusion bioreactors affects fluid flow dynamics, but not oxygen concentration in 3D-printed scaffolds for bone tissue engineering: Computational analysis and experimental validation

Fluid flow dynamics and oxygen-concentration in 3D-printed scaffolds within perfusion bioreactors are sensitive to controllable bioreactor parameters such as inlet flow rate. Here we aimed to determine fluid flow dynamics, oxygen-concentration, and cell proliferation and distribution in 3D-printed scaffolds as a result of different inlet flow rates of perfusion bioreactors using experiments and finite element modeling. Pre-osteoblasts were treated with 1 h pulsating fluid flow with low (0.8 Pa; PFFlow) or high peak shear stress (6.5 Pa; PFFhigh), and nitric oxide (NO) production was measured to validate shear stress sensitivity. Computational analysis was performed to determine fluid flow between 3D-scaffold-strands at three inlet flow rates (0.02, 0.1, 0.5 ml/min) during 5 days. MC3T3-E1 pre-osteoblast proliferation, matrix production, and oxygen-consumption in response to fluid flow in 3D-printed scaffolds inside a perfusion bioreactor were experimentally assessed. PFFhigh more strongly stimulated NO production by pre-osteoblasts than PFFlow. 3D-simulation demonstrated that dependent on inlet flow rate, fluid velocity reached a maximum (50–1200 μm/s) between scaffold-strands, and fluid shear stress (0.5–4 mPa) and wall shear stress (0.5–20 mPa) on scaffold-strands surfaces. At all inlet flow rates, gauge fluid pressure and oxygen-concentration were similar. The simulated cell proliferation and distribution, and oxygen-concentration data were in good agreement with the experimental results. In conclusion, varying a perfusion bioreactor's inlet flow rate locally affects fluid velocity, fluid shear stress, and wall shear stress inside 3D-printed scaffolds, but not gauge fluid pressure, and oxygen-concentration, which seems crucial for optimized bone tissue engineering strategies using bioreactors, scaffolds, and cells.

Interspecies Comparison of Alveolar Bone Biology, Part I: Morphology and Physiology of Pristine Bone.

Few interspecies comparisons of alveolar bone have been documented, and this knowledge gap raises questions about which animal models most accurately represent human dental conditions or responses to surgical interventions. The objective of this study was to employ state-of-the-art quantitative metrics to directly assess and compare the structural and functional characteristics of alveolar bone among humans, mini pigs, rats, and mice. The same anatomic location (i.e., the posterior maxillae) was analyzed in all species via micro-computed tomographic imaging, followed by quantitative analyses, coupled with histology and immunohistochemistry. Bone remodeling was evaluated with alkaline phosphatase activity and tartrate-resistant acid phosphatase staining to identify osteoblast and osteoclast activities. In vivo fluorochrome labeling was used as a means to assess mineral apposition rates. Collectively, these analyses demonstrated that bone volume differed among the species, while bone mineral density was equal. All species showed a similar density of alveolar osteocytes, with a highly conserved pattern of collagen organization. Collagen maturation was equal among mouse, rat, and mini pig. Bone remodeling was a shared feature among the species, with morphologically indistinguishable hemiosteonal appearances, osteocytic perilacunar remodeling, and similar mineral apposition rates in alveolar bone. Our analyses demonstrated equivalencies among the 4 species in a plurality of the biological features of alveolar bone. Despite contradictory results from older studies, we found no evidence for the superiority of pig models over rodent models in representing human bone biology. Animal models are extensively used to evaluate bone tissue engineering strategies, yet there are few state-of-the-art studies that rigorously compare and quantify the factors influencing selection of a given animal model. Consequently, there is an urgent need to assess preclinical animal models for their predictive value to dental research. Our article addresses this knowledge gap and, in doing so, provides a foundation for more effective standardization among animal models commonly used in dentistry.