Thomas J. Kean

The Achilles tendon is essential for energy efficient walking through its ability to store and return elastic energy. Although the Achilles tendon has traditionally been evaluated as a single functional unit, it is composed of distinct subtendons from the gastrocnemius and soleus, each with unique mechanical and neuromuscular characteristics. Consequently, it remains unclear how individual subtendons contribute to walking mechanics across different gait speeds. Thus, this study aimed to quantify the contributions of the individual Achilles subtendons to tendon mechanics and walking function. Eight healthy participants underwent passive ankle dorsiflexion testing using ultrasound, motion capture, and a load cell, followed by gait analysis on an instrumented treadmill at self-selected slow, normal, and fast speeds. Lower extremity kinematics, as well as gastrocnemius Achilles subtendon force, energy, and induced ankle moment were analyzed. The gastrocnemius subtendon exhibited increased force, energy storage, induced ankle moment, and relative contribution to overall ankle moment with increasing walking speed. These findings demonstrate that the Achilles subtendons do not contribute uniformly to walking mechanics and instead exhibit speed dependent functional specialization. This noninvasive approach for quantifying Achilles tendon properties and individual subtendon contributions can be further utilized to evaluate surgical outcomes, improve treatment planning for various musculoskeletal conditions, and enhance the accuracy of musculoskeletal modeling.

Background: Hyaluronic acid (HA) filler-induced vascular occlusion is a serious complication in aesthetic medicine, yet the microvascular behavior of HA gels under physiologically relevant flow conditions remains poorly characterized. Objectives: To evaluate the embolic fragmentation, dispersal, and occlusive behavior of 5 commercially available HA fillers within a physiologically calibrated microvascular perfusion model. Methods: Five HA fillers were tested using a modified PULSAR system incorporating a branched microtubular adapter (200-1000 µm channels) with physiologic arterial flow parameters. Products were injected through 22 and 27 G microcannulas and assessed for occlusion patterns, fragment morphology, and particle size. Flow dynamics were recorded through videography, and fragment characteristics were analyzed using imaging software. Statistical comparisons were conducted across products and cannula gauges. Results: HA gels fragmented extensively into microparticles (mean area = 0.140 mm2; interquartile range, 0.024-0.254 mm2) generating high rates of occlusion predominantly in channels ≤300 µm (P < .0001). A 22 G injection produced larger particles and higher occlusion rates than 27 G (31% vs 17%, P = .025), most notably with large-particle, high-elasticity products. Fragment morphology varied with rheology: solid gels fractured into ovoid embolic particles, whereas soft, high-tan δ gels formed filamentous, nonocclusive strands. Across all products, particle size was lower in the microvascular simulation compared with previous macrovascular experiments, indicating vessel-caliber-dependent fragmentation. Conclusions: HA fillers behave as deformable embolic particles that disperse distally under physiologic microtubular conditions. These findings support a concurrent microembolic mechanism underlying filler-induced ischemia. Product rheology, cannula gauge, and vascular anatomy are important determinants of embolic particle behavior.

Two new diacid monomers containing either fluorene or sulfone moieties polymerize via step-growth polymerization in the presence of their diol counterparts or alkyl diols (ethylene glycol and 1,6-hexanediol), forming eight new cardo structure polyesters. The polymers exhibit high optical transparency in the thin-film form with refractive indices ranging from 1.56 to 1.69, tunable glass transition temperatures from ca. 40 to 116 °C with no melting temperature, and resistance to thermal degradation in a nitrogenous atmosphere, reaching 350-398 °C before observing 10% weight loss. Their molecular weights, M _w , range from ca. 17 to 77 kDa, with an average polydispersity, Đ, of 1.5, and average purified yields of 82%. The polymers absorb light primarily in the UV region from ca. 228 to 320 nm with no absorption from 320 to 800 nm. A critical finding is that some of the fluorene and sulfone monomers induce negative bioactivity and even cell death in T47D-KBLuc cells, a breast cancer cell line, at high concentrations. This report details the project inspiration, monomer synthesis, polymerization steps, structural confirmation, material characterization, and the impact of the new monomer's estrogenic and antiestrogenic bioactivity.

Background: Despite the recently increased incidence and improved awareness of filler-induced ischemic injuries, the arterioembolic behavior of filler products has not been well described. Objectives: To evaluate the embolic behavior of varying dilutions of calcium hydroxylapatite-carboxymethylcellulose (CaHA-CMC) gel mixtures within an artificial macrovascular perfusion model of the proximal facial artery with correlation against published instances of ischemic injuries in the literature. Methods: CaHA-CMC gel mixtures were assessed through the Pulsatile Unit for the Laboratory Simulation of Arterioembolic Restrictions (PULSAR) system at different flow rates. The occlusive behavior, embolic particle size distributions, and morphological attributes were evaluated through direct photographic and videographic captures followed by digital image processing. The PubMed database was systematically queried for all published instances of CaHA-CMC-associated ischemic injuries. Results: Undiluted CaHA-CMC demonstrated highly cohesive behavior upon PULSAR inoculation, with a tendency toward proximal occlusion. Gel fragmentation resulted in a polydisperse embolic mixture averaging 0.151 ± 0.61 mm² (interquartile range: 0.006-0.022 mm²) in size. Product dilution had a profound effect on embolic behavior, with a significant reduction in average particle size (0.018 ± 0.03 mm²; interquartile range: 0.005-0.018 mm²; P < .0001) and complete elimination of proximally occlusive capacity for hyperdiluted mixtures compared with undiluted product (P = .002). Confirmed hyperdiluted CaHA-CMC-associated ischemic injuries represented only 3% of published reports, with a predominantly self-limited clinical course. Conclusions: Embolized CaHA-CMC gels produce polydisperse particle mixtures with a preponderance of microparticles. Hyperdilution profoundly reduced the proximally occlusive potential of the product.

Background/Objectives: Osteoarthritis (OA) is a highly prevalent, degenerative joint disease capable of causing severe pain and impaired mobility. Current treatments mitigate symptoms but do not cure the disease. The development of a disease-modifying osteoarthritis drug (DMOAD) could improve patient outcomes by slowing, halting, or reversing joint damage. Many DMOADs have progressed to clinical trials, but very few have made a significant impact, and none have been approved for clinical use. The purpose of this review is to present an update on the current status of DMOADs with a particular focus on results published since 2010. Methods: A comprehensive search was conducted within PubMed and ClinicalTrials.gov for novel DMOADs enrolled in phase II and III clinical trials between 1 January 2010 and 1 July 2024. Results: Eleven DMOAD candidates are reviewed and critically analyzed for their potential benefit in OA treatment—Lorecivivint (SM04690), TissueGene-C, Cindunistat (SD-6010), Sprifermin, UBX0101, TPX-100, GLPG1972/S201086, Lutikizumab (ABT-981), SAR113945, MIV-711, and LNA043—and relevant challenges to their development are discussed. Conclusions: Six DMOADs have demonstrated statistically significant evidence of a structural or symptomatic benefit without major safety concerns in phase II and III randomized controlled trials post-2010.

Our aim was to develop a novel approach to identify disease‐modifying drugs for osteoarthritis (OA), focusing on stimulating type II collagen anabolism in chondrocytes. As ELISA or western blot are destructive, laborious and time consuming, primary human chondrocytes expressing Gaussia luciferase under the control of the type II collagen promoter were developed and used. We cultured them in 3D cartilage aggregates under physioxia, to temporally screen a natural product library over 3‐weeks. Hit compounds were analyzed for their potential targets in silico, first by structure, then by RNA‐Seq. Two hit compounds were then further analyzed using biochemical assays, dose‐response curves, and histological analyses to confirm their effects on type II collagen expression and chondrogenesis. Aromoline shows promise as a potential disease modifying compound, demonstrating an increase in type II collagen expression within cartilage sourced from chondrocytes of three distinct donors. Aromoline is a bisbenzylisoquinoline alkaloid that has been studied for its antiproliferative, anti‐inflammatory, and antimicrobial properties, and we are the first to explore its effects on chondrocytes and chondrogenesis. In silico analysis revealed the dopamine receptor D4 (DRD4) as a potential target, confirmed by type II collagen upregulation after aromoline treatment and with DRD4‐specific agonist ABT‐724. This novel approach combining in silico and in vitro methods provides a platform for drug discovery in a challenging and under‐researched area. In conclusion, a novel drug (aromoline) and target receptor (DRD4) were identified as stimulating type II collagen, with the future goal of treating or preventing OA.

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.

Background: Complex bone defects are challenging to treat. Autografting is the gold standard for regenerating bone defects, however limitations include donor site morbidity and increased surgical complexity. Advancements in 3D bioprinting (3DBP) offer a promising alternative for viable bone grafts. Methods: Design of Experiments (DoE) was used to design 12 hydrogel bioinks of various Gelatin meth-acrylate/hydroxyapatite (GelMA/HA) concentrations. These bioinks were assessed for pipettability and equilibrium modulus. An optimal bioink was designed using the DoE data to produce the greatest stiffness while still being pipettable. Two bioinks, the DoE designed maximal stiff-ness and the experimentally defined maximal stiffness vs. 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: DoE ANOVA analysis indicated that the augmented 3 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 cell viability within 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 lower HA concentration (p < 0.05), showing that higher HA 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 it is possible to create printable bone-like grafts from GelMA and HA with increased HA levels, favorable mechanical properties (145kPa) and >80% cell viability.

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.

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.