A Multifunctional Therapeutic Strategy Using P7C3 as A Countermeasure Against Bone Loss and Fragility in An Ovariectomized Rat Model of Postmenopausal Osteoporosis.

By 2060, an estimated one in four Americans will be elderly. Consequently, the prevalence of osteoporosis and fragility fractures will also increase. Presently, no available intervention definitively prevents or manages osteoporosis. This study explores whether Pool 7 Compound 3 (P7C3) reduces progressive bone loss and fragility following the onset of ovariectomy (OVX)-induced osteoporosis. Results confirm OVX-induced weakened, osteoporotic bone together with a significant gain in adipogenic body weight. Treatment with P7C3 significantly reduced osteoclastic activity, bone marrow adiposity, whole-body weight gain, and preserved bone area, architecture, and mechanical strength. Analyses reveal significantly upregulated platelet derived growth factor-BB and leukemia inhibitory factor, with downregulation of interleukin-1 R6, and receptor activator of nuclear factor kappa-B (RANK). Together, proteomic data suggest the targeting of several key regulators of inflammation, bone, and adipose turnover, via transforming growth factor-beta/SMAD, and Wingless-related integration site/be-catenin signaling pathways. To the best of the knowledge, this is first evidence of an intervention that drives against bone loss via RANK. Metatranscriptomic analyses of the gut microbiota show P7C3 increased Porphyromonadaceae bacterium, Candidatus Melainabacteria, and Ruminococcaceae bacterium abundance, potentially contributing to the favorable inflammatory, and adipo-osteogenic metabolic regulation observed. The results reveal an undiscovered, and multifunctional therapeutic strategy to prevent the pathological progression of OVX-induced bone loss.

Suitability of Gelatin Methacrylate and Hydroxyapatite Hydrogels for 3D-Bioprinted Bone Tissue.

BACKGROUND: Complex bone defects are challenging to treat. Autografting is the gold standard for regenerating bone defects; however, its limitations include donor-site morbidity and increased surgical complexity. Advancements in 3D bioprinting (3DBP) offer a promising alternative for viable bone grafts. In this experiment, gels composed of varying levels of gelatin methacrylate (GelMA) and hydroxyapatite (HA) and gelatin concentrations are explored. The objective was to increase the hydroxyapatite content and find the upper limit before the printability was compromised and determine its effect on the mechanical properties and cell viability. METHODS: Design of Experiments (DoE) was used to design 13 hydrogel bioinks of various GelMA/HA concentrations. These bioinks were assessed in terms of their pipettability and equilibrium modulus. An optimal bioink was designed using the DoE data to produce the greatest stiffness while still being pipettable. Three bioinks, one with the DoE-designed maximal stiffness, one with the experimentally defined maximal stiffness, and a literature-based control, were then printed using a 3D bioprinter and assessed for print fidelity. The resulting hydrogels were combined with human bone-marrow-derived mesenchymal stromal cells (hMSCs) and evaluated for cell viability. RESULTS: The DoE ANOVA analysis indicated that the augmented three-level factorial design model used was a good fit (p < 0.0001). Using the model, DoE correctly predicted that a composite hydrogel consisting of 12.3% GelMA, 15.7% HA, and 2% gelatin would produce the maximum equilibrium modulus while still being pipettable. The hydrogel with the most optimal print fidelity was 10% GelMA, 2% HA, and 5% gelatin. There were no significant differences in the cell viability within the hydrogels from day 2 to day 7 (p > 0.05). There was, however, a significantly lower cell viability in the gel composed of 12.3% GelMA, 15.7% HA, and 2% gelatin compared to the other gels with a lower HA concentration (p < 0.05), showing that a higher HA content or print pressure may be cytotoxic within hydrogels. CONCLUSIONS: Extrusion-based 3DBP offers significant advantages for bone-tissue implants due to its high customizability. This study demonstrates that it is possible to create printable bone-like grafts from GelMA and HA with an increased HA content, favorable mechanical properties (145 kPa), and a greater than 80% cell viability.

Optimizing Bioink Composition for Human Chondrocyte Expression of Lubricin.

The surface zone of articular cartilage is the first area impacted by cartilage defects, commonly resulting in osteoarthritis. Chondrocytes in the surface zone of articular cartilage synthesize and secrete lubricin, a proteoglycan that functions as a lubricant protecting the deeper layers from shear stress. Notably, 3D bioprinting is a tissue engineering technique that uses cells encapsulated in biomaterials to fabricate 3D constructs. Gelatin methacrylate (GelMA) is a frequently used biomaterial for 3D bioprinting cartilage. Oxidized methacrylated alginate (OMA) is a chemically modified alginate designed for its tunable degradation rate and mechanical properties. To determine an optimal combination of GelMA and OMA for lubricin expression, we used our novel high-throughput human articular chondrocyte reporter system. Primary human chondrocytes were transduced with PRG4 (lubricin) promoter-driven Gaussia luciferase, allowing for temporal assessment of lubricin expression. A lubricin expression-driven Design of Experiment screen and subsequent validation identified 14% GelMA/2% OMA for further study. Therefore, DoE optimized 14% GelMA/2% OMA, 14% GelMA control, and 16% GelMA (total solid content control) were 3D bioprinted. The combination of lubricin protein expression and shape retention over the 22 days in culture, successfully determined the 14% GelMA/2%OMA to be the optimal formulation for lubricin secretion. This strategy allows for rapid analysis of the role(s) of biomaterial composition, stiffness or other cell manipulations on lubricin expression by chondrocytes, which may improve therapeutic strategies for cartilage regeneration.

Micronutrient optimization for tissue engineered articular cartilage production of type II collagen.

Tissue Engineering of cartilage has been hampered by the inability of engineered tissue to express native levels of type II collagen in vitro. Inadequate levels of type II collagen are, in part, due to a failure to recapitulate the physiological environment in culture. In this study, we engineered primary rabbit chondrocytes to express a secreted reporter, Gaussia Luciferase, driven by the type II collagen promoter, and applied a Design of Experiments approach to assess chondrogenic differentiation in micronutrient-supplemented medium. Using a Response Surface Model, 240 combinations of micronutrients absent in standard chondrogenic differentiation medium, were screened and assessed for type II collagen promoter-driven Gaussia luciferase expression. While the target of this study was to establish a combination of all micronutrients, alpha-linolenic acid, copper, cobalt, chromium, manganese, molybdenum, vitamins A, E, D and B7 were all found to have a significant effect on type II collagen promoter activity. Five conditions containing all micronutrients predicted to produce the greatest luciferase expression were selected for further study. Validation of these conditions in 3D aggregates identified an optimal condition for type II collagen promoter activity. Engineered cartilage grown in this condition, showed a 170% increase in type II collagen expression (Day 22 Luminescence) and in Young's tensile modulus compared to engineered cartilage in basal media alone.Collagen cross-linking analysis confirmed formation of type II-type II collagen and type II-type IX collagen cross-linked heteropolymeric fibrils, characteristic of mature native cartilage. Combining a Design of Experiments approach and secreted reporter cells in 3D aggregate culture enabled a high-throughput platform that can be used to identify more optimal physiological culture parameters for chondrogenesis.

An osteogenic magnesium alloy with improved corrosion resistance, antibacterial, and mechanical properties for orthopedic applications.

The aim of this study was to develop a novel biodegradable magnesium (Mg) alloy for bone implant applications. We used scandium (Sc; 2 wt %) and strontium (Sr; 2 wt %) as alloying elements due to their high biocompatibility, antibacterial efficacy, osteogenesis, and protective effects against corrosion. In the present work, we also examined the effect of a heat treatment process on the properties of the Mg-Sc-Sr alloy. Alloys were manufactured using a metal casting process followed by heat treatment. The microstructure, corrosion, mechanical properties, antibacterial activity, and osteogenic activity of the alloy were assessed in vitro. The results showed that the incorporation of Sc and Sr elements controlled the corrosion, reduced the hydrogen generation, and enhanced mechanical properties. Furthermore, alloying with Sc and Sr demonstrated a significantly enhanced antibacterial activity and decreased biofilm formation compared to control Mg. Also, culturing Mg-Sc-Sr alloy with human bone marrow-derived mesenchymal stromal cells showed a high degree of biocompatibility (>90% live cells) and a significant increase in osteoblastic differentiation in vitro shown by Alizarin red staining and alkaline phosphatase activity. Based on these results, the Mg-Sc-Sr alloy heat-treated at 400°C displayed optimal mechanical properties, corrosion rate, antibacterial efficacy, and osteoinductivity. These characteristics make the Mg-Sc-Sr alloy a promising candidate for biodegradable orthopedic implants in the fixation of bone fractures such as bone plate-screws or intramedullary nails.

Biomaterial composition and stiffness as decisive properties of 3D bioprinted constructs for type II collagen stimulation.

Gelatin methacrylate (GelMA) and hyaluronic acid methacrylate (HAMA) are frequently used biomaterials for 3D bioprinting, with individual well-established material characteristics. To identify an ideal combination of GelMA and HAMA for chondrogenesis, a novel, primary human chondrocyte COL2A1-Gaussia luciferase reporter system (HuCol2gLuc) was developed. With this non-destructive, high-throughput temporal assay, Gaussia luciferase is secreted from the cells and used as a proxy for measuring type II collagen production. GelMA:HAMA ratios were screened using the reporter system before proceeding to 3D bioprinting. This method is efficient, saving on time and materials, resulting in a streamlined process of biomaterial optimization. The screen revealed that the addition of HAMA to GelMA improved chondrogenesis over GelMA (15%) alone. Storage moduli were measured using dynamic mechanical analysis of the same GelMA:HAMA ratios and established an initial threshold for chondrogenesis of ∼30kPa. To determine if biomaterial storage moduli impact cell mobility, human primary chondrocytes transduced with green fluorescent protein (GFP) were 3D bioprinted in either 1:1 or 2:1 ratios with storage moduli of 32kPa and 57.9kPa, respectively. We found that reduced cell mobility, in the stiffer biomaterial, had higher type II collagen expression, than the softer material with more cell mobility. Finally, after 3D bioprinting with HuCol2gLuc cells we successfully identified an optimal combination (2:1) of GelMA:HAMA and photo-crosslinking time (38s) for chondrogenesis. STATEMENT OF SIGNIFICANCE: One challenge of 3D bioprinting is identifying ideal biomaterials that stimulate articular cartilage development. To identify an optimal combination of gelatin methacrylate and hyaluronic acid methacrylate for chondrogenesis we developed a primary human chondrocyte type II collagen Gaussia luciferase reporter cell (HuCol2gLuc). This non-destructive, high-throughput assay uses a secreted Gaussia luciferase as a proxy for temporal type II collagen production. This reporter system streamlines the biomaterial optimization process before 3D bioprinting. We also used it to determine the level of stiffness required for chondrogenesis. And for the first time, we quantified chondrocyte mobility in a 3D bioprinted construct. Together these results indicate that a biomaterial with a higher storage modulus and less cell mobility, improves chondrogenesis.

Systematic review on the application of 3D-bioprinting technology in orthoregeneration: current achievements and open challenges.

BACKGROUND: Joint degeneration and large or complex bone defects are a significant source of morbidity and diminished quality of life worldwide. There is an unmet need for a functional implant with near-native biomechanical properties. The potential for their generation using 3D bioprinting (3DBP)-based tissue engineering methods was assessed. We systematically reviewed the current state of 3DBP in orthoregeneration. METHODS: This review was performed using PubMed and Web of Science. Primary research articles reporting 3DBP of cartilage, bone, vasculature, and their osteochondral and vascular bone composites were considered. Full text English articles were analyzed. RESULTS: Over 1300 studies were retrieved, after removing duplicates, 1046 studies remained. After inclusion and exclusion criteria were applied, 114 articles were analyzed fully. Bioink material types and combinations were tallied. Cell types and testing methods were also analyzed. Nearly all papers determined the effect of 3DBP on cell survival. Bioink material physical characterization using gelation and rheology, and construct biomechanics were performed. In vitro testing methods assessed biochemistry, markers of extracellular matrix production and/or cell differentiation into respective lineages. In vivo proof-of-concept studies included full-thickness bone and joint defects as well as subcutaneous implantation in rodents followed by histological and µCT analyses to demonstrate implant growth and integration into surrounding native tissues. CONCLUSIONS: Despite its relative infancy, 3DBP is making an impact in joint and bone engineering. Several groups have demonstrated preclinical efficacy of mechanically robust constructs which integrate into articular joint defects in small animals. However, notable obstacles remain. Notably, researchers encountered pitfalls in scaling up constructs and establishing implant function and viability in long term animal models. Further, to translate from the laboratory to the clinic, standardized quality control metrics such as construct stiffness and graft integration metrics should be established with investigator consensus. While there is much work to be done, 3DBP implants have great potential to treat degenerative joint diseases and provide benefit to patients globally.

Synoviocyte-Derived Extracellular Matrix and bFGF Speed Human Chondrocyte Proliferation While Maintaining Differentiation Potential.

Improving the ability of human chondrocytes to proliferate, while maintaining their differentiation potential, has presented a great challenge in cartilage tissue engineering. In this study, human chondrocytes were cultured under four unique growth conditions at physiologic oxygen tension: tissue culture plastic (TCP) only, synoviocyte matrix (SCM)-coated flasks only, SCM-coated flasks with bFGF media supplement, and TCP with bFGF media supplement. The results indicated that, compared to standard TCP, all test conditions showed significantly increased cell expansion rates and an increase in both glycosaminoglycan (GAG) and collagen content during redifferentiation culture. Specifically, the combined SCM + bFGF growth condition showed an additive effect, with an increase of approximately 36% more cells per passage (5-7 days) when compared to the SCM alone. In conclusion, the results of this study demonstrate that bFGF and SCM can be used as supplements to enhance the growth of human chondrocytes both as individual enhancers and as a combined additive.

Transforming Capillary Alginate Gel (Capgel) into New 3D-Printing Biomaterial Inks.

Three-dimensional (3D) printing has great potential for creating tissues and organs to meet shortfalls in transplant supply, and biomaterial inks are key components of many such approaches. There is a need for biomaterial inks that facilitate integration, infiltration, and vascularization of targeted 3D-printed structures. This study is therefore focused on creating new biomaterial inks from self-assembled capillary alginate gel (Capgel), which possesses a unique microstructure of uniform tubular channels with tunable diameters and densities. First, extrusions of Capgel through needles (0.1-0.8 mm inner diameter) were investigated. It was found that Capgel ink extrudes as slurries of fractured and entangled particles, each retaining capillary microstructures, and that extruded line widths W and particle sizes A were both functions of needle inner diameter D, specifically power-law relationships of W~D0.42 and A~D1.52, respectively. Next, various structures were successfully 3D-printed with Capgel ink, thus demonstrating that this biomaterial ink is stackable and self-supporting. To increase ink self-adherence, Capgel was coated with poly-L-lysine (PLL) to create a cationic "skin" prior to extrusion. It was hypothesized that, during extrusion of Capgel-PLL, the sheared particles fracture and thereby expose cryptic sites of negatively-charged biomaterial capable of forming new polyelectrolyte bonds with areas of the positively-charged PLL skin on neighboring entangled particles. This novel approach resulted in continuous, self-adherent extrusions that remained intact in solution. Human lung fibroblasts (HLFs) were then cultured on this ink to investigate biocompatibility. HLFs readily colonized Capgel-PLL ink and were strongly oriented by the capillary microstructures. This is the first description of successful 3D-printing with Capgel biomaterial ink as well as the first demonstration of the concept and formulation of a self-adherent Capgel-PLL biomaterial ink.

Physioxia Stimulates Extracellular Matrix Deposition and Increases Mechanical Properties of Human Chondrocyte-Derived Tissue-Engineered Cartilage.

Cartilage tissue has been recalcitrant to tissue engineering approaches. In this study, human chondrocytes were formed into self-assembled cartilage sheets, cultured in physiologic (5%) and atmospheric (20%) oxygen conditions and underwent biochemical, histological and biomechanical analysis at 1- and 2-months. The results indicated that sheets formed at physiological oxygen tension were thicker, contained greater amounts of glycosaminoglycans (GAGs) and type II collagen, and had greater compressive and tensile properties than those cultured in atmospheric oxygen. In all cases, cartilage sheets stained throughout for extracellular matrix components. Type II-IX-XI collagen heteropolymer formed in the neo-cartilage and fibrils were stabilized by trivalent pyridinoline cross-links. Collagen cross-links were not significantly affected by oxygen tension but increased with time in culture. Physiological oxygen tension and longer culture periods both served to increase extracellular matrix components. The foremost correlation was found between compressive stiffness and the GAG to collagen ratio.