Intramedullary implant stability affects patterns of fracture healing in mice with morphologically different bone phenotypes.

Almost all prior mouse fracture healing models have used needles or K-wires for fixation, unwittingly providing inadequate mechanical stability during the healing process. Our contention is that the reported outcomes have predominantly reflected this instability, rather than the impact of diverse biological conditions, pharmacologic interventions, exogenous growth factors, or genetic considerations. This important issue becomes obvious upon a critical review of the literature. Therefore, the primary aim of this study was to demonstrate the significance of mouse-specific implants designed to provide both axial and torsional stability (Screw and IM Nail) compared to conventional pins (Needle and K-wires), even when used in mice with differently sized marrow canals and diverse genetic backgrounds. B6 (large medullary canal), DBA, and C3H (smaller medullary canals) mice were employed, all of which have different bone morphologies. Closed femoral fractures were created and stabilized with intramedullary implants that provide different mechanical conditions during the healing process. The most important finding of this study was that appropriately designed mouse-specific implants, providing both axial and torsional stability, had the greatest influence on bone healing outcomes regardless of the different bone morphologies encountered. For instance, unstable implants in the B6 strain (largest medullary canal) resulted in significantly greater callus, with a fracture region mainly comprising trabecular bone along with the presence of cartilage 28 days after surgery. The DBA and C3H strains (with smaller medullary canals) instead formed significantly less callus, and only had a small amount of intracortical trabeculation remaining. Moreover, with more stable fracture fixation a higher BV/TV was observed and cortices were largely restored to their original dimensions and structure, indicating an accelerated healing and remodeling process. These observations reveal that the diaphyseal cortical thickness, influenced by the genetic background of each strain, played a pivotal role in determining the amount of bone formation in response to the fracture. These findings are highly important, indicating the rate and type of tissue formed is a direct result of mechanical instability, and this most likely would mask the true contribution of the tested genes, genetic backgrounds, or various therapeutic agents administered during the bone healing process.

Osteal macrophages support osteoclast-mediated resorption and contribute to bone pathology in a postmenopausal osteoporosis mouse model.

Osteal macrophages (osteomacs) support osteoblast function and promote bone anabolism, but their contribution to osteoporosis has not been explored. Although mouse ovariectomy (OVX) models have been repeatedly used, variation in strain, experimental design and assessment modalities have contributed to no single model being confirmed as comprehensively replicating the full gamut of osteoporosis pathological manifestations. We validated an OVX model in adult C3H/HeJ mice and demonstrated that it presents with human postmenopausal osteoporosis features with reduced bone volume in axial and appendicular bone and bone loss in both trabecular and cortical bone including increased cortical porosity. Bone loss was associated with increased osteoclasts on trabecular and endocortical bone and decreased osteoblasts on trabecular bone. Importantly, this OVX model was characterized by delayed fracture healing. Using this validated model, we demonstrated that osteomacs are increased post-OVX on both trabecular and endocortical bone. Dual F4/80 (pan-macrophage marker) and tartrate-resistant acid phosphatase (TRAP) staining revealed osteomacs frequently located near TRAP+ osteoclasts and contained TRAP+ intracellular vesicles. Using an in vivo inducible macrophage depletion model that does not simultaneously deplete osteoclasts, we observed that osteomac loss was associated with elevated extracellular TRAP in bone marrow interstitium and increased serum TRAP. Using in vitro high-resolution confocal imaging of mixed osteoclast-macrophage cultures on bone substrate, we observed macrophages juxtaposed to osteoclast basolateral functional secretory domains scavenging degraded bone byproducts. These data demonstrate a role for osteomacs in supporting osteoclastic bone resorption through phagocytosis and sequestration of resorption byproducts. Overall, our data expose a novel role for osteomacs in supporting osteoclast function and provide the first evidence of their involvement in osteoporosis pathogenesis. © 2021 American Society for Bone and Mineral Research (ASBMR).

Alkali-Treated Titanium Coated with a Polyurethane, Magnesium and Hydroxyapatite Composite for Bone Tissue Engineering.

The aim of this study was to form a functional layer on the surface of titanium (Ti) implants to enhance their bioactivity. Layers of polyurethane (PU), containing hydroxyapatite (HAp) nanoparticles (NPs) and magnesium (Mg) particles, were deposited on alkali-treated Ti surfaces using a cost-effective dip-coating approach. The coatings were assessed in terms of morphology, chemical composition, adhesion strength, interfacial bonding, and thermal properties. Additionally, cell response to the variably coated Ti substrates was investigated using MC3T3-E1 osteoblast-like cells, including assessment of cell adhesion, cell proliferation, and osteogenic activity through an alkaline phosphatase (ALP) assay. The results showed that the incorporation of HAp NPs enhanced the interfacial bonding between the coating and the alkali-treated Ti surface. Furthermore, the presence of Mg and HAp particles enhanced the surface charge properties as well as cell attachment, proliferation, and differentiation. Our results suggest that the deposition of a bioactive composite layer containing Mg and HAp particles on Ti implants may have the potential to induce bone formation.

Workflow for highly porous resorbable custom 3D printed scaffolds using medical grade polymer for large volume alveolar bone regeneration.

OBJECTIVES: This study investigates the design, workflow, and manufacture of highly porous, resorbable additively manufactured, 3-dimensional (3D) custom scaffolds for the regeneration of large volume alveolar bone defects. MATERIALS AND METHODS: Computed tomography (CT) scans of 5 posterior mandibular vertical bone defects were obtained. Surface masks (3D surface contours) of the recipient site were first isolated using a contrast threshold, transformed into 3D objects, and used to guide the formation of custom implant template models. To determine model accuracy and fit, the gap and overlap between the patient geometry models and the idealized template 3D models were quantified. Models were 3D printed from medical grade polycaprolactone (PCL) into porous scaffolds. For scaffold dimensional quantification, scaffolds were scanned using a micro-computed tomography (µCT) scanner. RESULTS: The design and printing processes each achieved dimensional errors of <200 µm on average. The average gap between the template implant model and the scanned scaffold model was found to be 74 ± 14 µm. The printed scaffold was confirmed as having a porosity of 83.91%, a mean polymer or filament thickness of 200 ± 46 µm, and a mean pore size of 590 ± 243 µm. CONCLUSION: The approach described in this study is straightforward, adaptable to a range of patient geometries, and results in the formation of reproducible, dimensionally accurate custom implants. These highly porous 3D structures manufactured from resorbable medical grade material represent a potentially transformative technology toward the clinical implementation of scaffold-guided bone regeneration procedures.

Genetic variation in mice affects closed femoral fracture pattern outcomes.

The purpose of this study was to determine whether differences in structural and material properties of bone between different mouse strains influence the fracture patterns produced under experimental fracture conditions. Femurs of C57BL/6 (B6), C3H/HeJ (C3H), and DBA/2 (DBA) strains were evaluated using micro-computed tomography (µCT), measurements derived from radiographic images and mechanical testing to determine differences in the geometry and mechanical properties. A fracture device was used to create femoral fractures on freshly sacrificed animals using a range of kinetic energies (∼20-80mJ) which were classified as transverse, oblique, or comminuted. B6 femurs had the lowest bone volume/total volume (BV/TV) and bone mineral density (BMD), thinnest cortex, and had the most variable fracture patterns, with 77.5% transverse, 15% oblique, and 7.5% comminuted fractures. In contrast, C3H had the highest BV/TV, BMD, and thickest cortices, resulting in 97.5% transverse, 2.5% oblique, and 0% comminuted fractures. DBA had an intermediate BV/TV and thickness of cortices, with BMD similar to C3H, resulting in 92.9% transverse, 7.1% oblique, and 0% comminuted fractures. A binomial logistic regression confirmed that bone morphometry was the single strongest predictor of the resulting fracture pattern. This study demonstrated that the reproducibility of closed transverse femoral fractures was most influenced by the structural and material properties of the bone characteristics in each strain, rather than the kinetic energy or body weight of the mice. This was evidenced through geometric analysis of X-ray and µCT data, and further supported by the bone mineral density measurements from each strain, derived from µCT. Furthermore, this study also demonstrated that the use of lower kinetic energies was more than sufficient to reproducibly create transverse fractures, and to avoid severe tissue trauma. The creation of reproducible fracture patterns is important as this often dictates the outcomes of fracture healing, and those studies that do not control this potential variability could lead to a false interpretation of the results.

Release of lithium from 3D printed polycaprolactone scaffolds regulates macrophage and osteoclast response.

The immunomodulatory effects of lithium have been reported across a range of models and contexts. Lithium appears to have a positive effect on osteogenesis in vivo, while in vitro outcomes throughout the literature are varied. Tissue engineering approaches have rarely targeted local lithium delivery within a regenerative setting. We hypothesized that part of the positive effects of lithium in vivo may be due to an immunomodulatory effect manifesting in a local environment. To achieve a sustained lithium release from scaffold constructs, we blended lithium carbonate, a soluble salt of lithium, with the biomaterial polymer polycaprolactone (PCL). We printed constructs of PCL alone, and with 5% (5Li) and 10% (10Li) lithium carbonate. Mechanical testing revealed that mechanical properties were largely retained with lithium carbonate incorporation, and we measured a consistent release of the ion over a 7 day period. The efficacy of our construct system was then assessed using a primary mouse macrophage culture, and a differentiated osteoclast culture. We found that the lithium released from constructs had a great effect on macrophage polarization, resulting in pronounced upregulation of immunomodulatory (M2) genes, and a decrease in pro-inflammatory (M1) genes. This was reflected in cytokine expression, and illustrated through immunofluorescent staining. Osteoclast activity was greatly suppressed by the lithium incorporation, with a marked effect on gene expression and actin ring formation. Our work demonstrated an effective system for local lithium delivery, confirmed the pronounced effects that lithium has on macrophage and osteoclast response, and sets the stage for further innovations in ion release for targeted tissue engineering.

A Multifunctional Zinc Oxide/Poly(Lactic Acid) Nanocomposite Layer Coated on Magnesium Alloys for Controlled Degradation and Antibacterial Function.

In the present work, magnesium (Mg) AZ31 alloy was coated with a multifunctional membrane layer composed of ZnO nanoparticles (NPs) embedded in a poly(lactic acid) (PLA) matrix. We aimed to produce a stable coating that would be used to control the degradation rate of the Mg alloy and promote a local antibacterial activity. ZnO NPs were dispersed at 5 and 10 wt % in a PLA solution and dip-coated onto the AZ31 substrate. Surface topography, chemical composition, thickness, electrochemical corrosion performance, mass variation, antibacterial activity, adhesion performance, and cytotoxicity of an uncoated control and coated alloys were investigated. The results indicated that the incorporation of ZnO NPs at various concentrations affords a dramatic control over surface topography and degradation rates under in vitro and in vivo environmental conditions when compared to the uncoated Mg alloy control. In addition, the results confirmed that the coated layer exerts antibacterial properties and supports cell growth, indicating this system may have utility for bone tissue engineering applications.

Electrospun biphasic tubular scaffold with enhanced mechanical properties for vascular tissue engineering.

Polymer scaffolds produced through an electrospinning process are frequently explored as tissue substitutes for regenerative medicine. Despite offering desirable surface area to volume ratios and tailorable pore sizes, their poor structural mechanical properties limit their applicability in load-bearing regions. In this study, we present a simple strategy to improve the mechanical properties of a vascular graft scaffold. We achieved the formation of biphasic tubular scaffolds by electrospinning polyurethane (PU) onto an airbrushed tube made of polycaprolactone (PCL). After preparation, the scaffold was subsequently thermally-crosslinked (60°C) to strengthen the bonding between the two materials. The tensile strength and tensile elastic (Young's) modulus of the biphasic scaffolds were significantly enhanced from 4.5±1.72 and 45±15MPa (PU-only) up to 67.5±2.4 and 1039±81.8MPa (PCL/PU; p<0.05). Additionally, suture retention force, burst pressure, and compliance were all improved. The cytotoxicity of the fabricated samples was investigated using an MTT assay after 7days of cell culture and found to be negligible (~100% viability). In conclusion, we have demonstrated the preparation and characterization of a stable and mechanically robust vascular graft scaffold using a novel combination of well-established fabrication techniques. This study could also be extended to the fabrication of other biphasic scaffolds to better enhance the mechanical properties of the electrospun fibers mat without deteriorating its architecture structure.

A Hydrogel Model Incorporating 3D-Plotted Hydroxyapatite for Osteochondral Tissue Engineering.

The concept of biphasic or multi-layered compound scaffolds has been explored within numerous studies in the context of cartilage and osteochondral regeneration. To date, no system has been identified that stands out in terms of superior chondrogenesis, osteogenesis or the formation of a zone of calcified cartilage (ZCC). Herein we present a 3D plotted scaffold, comprising an alginate and hydroxyapatite paste, cast within a photocrosslinkable hydrogel made of gelatin methacrylamide (GelMA), or GelMA with hyaluronic acid methacrylate (HAMA). We hypothesized that this combination of 3D plotting and hydrogel crosslinking would form a high fidelity, cell supporting structure that would allow localization of hydroxyapatite to the deepest regions of the structure whilst taking advantage of hydrogel photocrosslinking. We assessed this preliminary design in terms of chondrogenesis in culture with human articular chondrocytes, and verified whether the inclusion of hydroxyapatite in the form presented had any influence on the formation of the ZCC. Whilst the inclusion of HAMA resulted in a better chondrogenic outcome, the effect of HAP was limited. We overall demonstrated that formation of such compound structures is possible, providing a foundation for future work. The development of cohesive biphasic systems is highly relevant for current and future cartilage tissue engineering.

Effects of scaffold architecture on mechanical characteristics and osteoblast response to static and perfusion bioreactor cultures.

Tissue engineering focuses on the repair and regeneration of tissues through the use of biodegradable scaffold systems that structurally support regions of injury while recruiting and/or stimulating cell populations to rebuild the target tissue. Within bone tissue engineering, the effects of scaffold architecture on cellular response have not been conclusively characterized in a controlled-density environment. We present a theoretical and practical assessment of the effects of polycaprolactone (PCL) scaffold architectural modifications on mechanical and flow characteristics as well as MC3T3-E1 preosteoblast cellular response in an in vitro static plate and custom-designed perfusion bioreactor model. Four scaffold architectures were contrasted, which varied in inter-layer lay-down angle and offset between layers, while maintaining a structural porosity of 60 ± 5%. We established that as layer angle was decreased (90° vs. 60°) and offset was introduced (0 vs. 0.5 between layers), structural stiffness, yield stress, strength, pore size, and permeability decreased, while computational fluid dynamics-modeled wall shear stress was increased. Most significant effects were noted with layer offset. Seeding efficiencies in static culture were also dramatically increased due to offset (∼ 45% to ∼ 86%), with static culture exhibiting a much higher seeding efficiency than perfusion culture. Scaffold architecture had minimal effect on cell response in static culture. However, architecture influenced osteogenic differentiation in perfusion culture, likely by modifying the microfluidic environment.