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Regenerative biomaterials for musculoskeletal defects must address multi-scale mechanical challenges. Repairing craniomaxillofacial bone defects, which are often large and irregularly shaped, requires close conformal contact between implant and defect margins to aid healing. While mineralized collagen scaffolds can promote mesenchymal stem cell osteogenic differentiation in vitro and bone formation in vivo, their mechanical performance is insufficient for surgical translation. We report a generative design approach to create scaffold-mesh composites by embedding a macro-scale polymeric Voronoi mesh into the mineralized collagen scaffold. The mechanics of architected foam reinforced composites are defined by a rigorous predictive moduli equation. We show biphasic composites localize strain during loading. Further, planar and 3D mesh-scaffold composites can be rapidly shaped to aid conformal fitting. Voronoi-based composites overcome traditional porosity-mechanics relationship limits while enabling rapid shaping of regenerative implants to conformally fit complex defects unique for individual patients. STATEMENT OF SIGNIFICANCE: Biomaterial strategies for (craniomaxillofacial) bone regeneration are often limited by the size and complex geometry of the defects. Voronoi structures are open-cell foams with tunable mechanical properties which have primarily been used computationally. We describe generative design strategies to create Voronoi foams via 3D-printing then embed them into an osteogenic mineralized collagen scaffold to form a multi-scale composite biomaterial. Voronoi structures have predictable and tailorable moduli, permit stain localization to defined regions of the composite, and permit conformal fitting to effect margins to aid surgical practicality and improve host-biomaterial interactions. Multi-scale composites based on Voronoi foams represent an adaptable design approach to address significant challenges to large-scale bone repair., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Matrix-bound nanovesicles (MBV) are a distinct subtype of extracellular vesicles that are firmly embedded within biomaterials composed of extracellular matrix (ECM). MBV both store and transport a diverse, tissue specific portfolio of signaling molecules including proteins, miRNAs, and bioactive lipids. MBV function as a key mediator in ECM-mediated control of the local tissue microenvironment. One of the most important mechanisms by which MBV in ECM bioscaffolds support constructive tissue remodeling following injury is immunomodulation and, specifically, the promotion of an anti-inflammatory, pro-remodeling immune cell activation state. Recent in vivo studies have shown that isolated MBV have therapeutic efficacy in rodent models of both retinal damage and rheumatoid arthritis through the targeted immunomodulation of pro-inflammatory macrophages towards an anti-inflammatory activation state. While these results show the therapeutic potential of MBV administered independent of the rest of the ECM, the in vitro and in vivo safety and biodistribution profile of MBV remain uncharacterized. The purpose of the present study was to thoroughly characterize the pre-clinical safety profile of MBV through a combination of in vitro cytotoxicity and MBV uptake studies and in vivo toxicity, immunotoxicity, and imaging studies. The results showed that MBV isolated from porcine urinary bladder are well-tolerated and are not cytotoxic in cell culture, are non-toxic to the whole organism, and are not immunosuppressive compared to the potent immunosuppressive drug cyclophosphamide. Furthermore, this safety profile was sustained across a wide range of MBV doses. STATEMENT OF SIGNIFICANCE: Matrix-bound nanovesicles (MBV) are a distinct subtype of bioactive extracellular vesicles that are embedded within biomaterials composed of extracellular matrix (ECM). Recent studies have shown therapeutic efficacy of MBV in models of both retinal damage and rheumatoid arthritis through the targeted immunomodulation of pro-inflammatory macrophages towards an anti-inflammatory activation state. While these results show the therapeutic potential of MBV, the in vitro and in vivo biocompatibility and biodistribution profile of MBV remain uncharacterized. The results of the present study showed that MBV are a well-tolerated ECM-derived therapy that are not cytotoxic in cell culture, are non-toxic to the whole organism, and are not immunosuppressive. Collectively, these data highlight the translational feasibility of MBV therapeutics across a wide variety of clinical applications., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interest: This study was sponsored by ECM Therapeutics Inc., Warrendale PA. GSH and SFB are employees of and own stock in ECM Therapeutics Inc. The other authors have no financial conflicts or competing interests to disclose., (Copyright © 2022. Published by Elsevier Ltd.)

Regenerative repair of craniomaxillofacial bone injuries is challenging due to both the large size and irregular shape of many defects. Mineralized collagen scaffolds have previously been shown to be a promising biomaterial implant to accelerate craniofacial bone regeneration in vivo. Here we describe inclusion of a 3D-printed polymer or ceramic-based mesh into a mineralized collagen scaffold to improve mechanical and biological activity. Mineralized collagen scaffolds were reinforced with 3D-printed Fluffy-PLG (ultraporous polylactide-co-glycolide co-polymer) or Hyperelastic Bone (90wt% calcium phosphate in PLG) meshes. We show degradation byproducts and acidic release from the printed structures have limited negative impact on the viability of mesenchymal stem cells. Further, inclusion of a mesh formed from Hyperelastic Bone generates a reinforced composite with significantly improved mechanical performance (elastic modulus, push-out strength). Composites formed from the mineralized collagen scaffold and either Hyperelastic Bone or Fluffy-PLG reinforcement both supported human bone-marrow derived mesenchymal stem cell osteogenesis and new bone formation. This was observed by increased mineral formation in Fluffy-PLG composites and increased cell viability and upregulation of RUNX2, Osterix, and COL1A2 genes in both composites. Strikingly, composites reinforced with Hyperelastic Bone mesh elicited significantly increased secretion of osteoprotegerin, a soluble glycoprotein and endogenous inhibitor of osteoclast activity. These results suggest that architectured meshes can be integrated into collagen scaffolds to boost mechanical performance and actively instruct cell processes that aid osteogenicity; specifically, secretion of a factor crucial to inhibiting osteoclast-mediated bone resorption. Future work will focus on further adapting the polymer mesh architecture to confer improved shape-fitting capacity as well as to investigate the role of polymer reinforcement on MSC-osteoclast interactions as a means to increase regenerative potential., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier Ltd.)

Craniomaxillofacial bone defects can occur as a result of congenital, post-oncologic, and high-energy impact conditions. The scale and irregularity of such defects motivate new biomaterials to promote regeneration of the damaged bone. We have recently described a mineralized collagen scaffold capable of instructing stem cell osteogenic differentiation and new bone infill in the absence of traditional osteogenic supplements. Herein, we report the integration of a millimeter-scale reinforcing poly (lactic acid) frame fabricated via 3D-printing into the mineralized collagen scaffold with micron-scale porosity to form a multi-scale mineralized collagen-PLA composite. We describe modifications to the PLA frame design to increase the compressive strength (Young's Modulus, ultimate stress and strain) of the composite. A critical challenge beyond increasing the compressive strength of the collagen scaffold is addressing challenges inherent with the irregularity of clinical defects. As a result, we examined the potential for modifying the frame architecture to render the composite with increased compressive strength in one axis or radial compressibility and shape-fitting capacity in an orthogonal axis. A library of mineralized collagen-PLA composites was mechanically characterized via compression testing and push-out test to describe mechanical performance and shape-fitting capacity. We also report in vitro comparison of the bioactivity of porcine adipose derived stem cells in the mineralized collagen-PLA composite versus the mineralized collagen scaffold via metabolic activity, gene expression, and functional matrix synthesis. The results suggest that incorporation of the PLA reinforcing frame does not negatively influence the osteoinductive nature of the mineralized collagen scaffold. Together, these findings suggest a strategy to address often competing bioactivity, mechanical strength, and shape-fitting design requirements for biomaterials for craniomaxillofacial bone regeneration., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

A rapid and sensitive high-performance liquid chromatography assay method was developed to determine serum fluoxetine and norfluoxetine levels by single extraction of 0.1 ml of serum with sodium hydroxide. The mobile phase (55% acetonitrile-45% distilled water containing 10 mM aqueous triethylamine) was used to separate fluoxetine and norfluoxetine (25-1000 ng/ml, using clomipramine as the internal standard) by ultraviolet detection at 226 nm. The inter- and intra-day variabilities of fluoxetine and norfluoxetine were 13-18%, and the recoveries of both drugs exceeded 89%. This assay method was applied to a pharmacokinetic disposition study of fluoxetine in mice.

A rapid and highly sensitive HPLC assay method was developed to measure small amounts of imipramine and its major metabolite, desipramine. The assay involved simple extraction procedures using clomipramine as the internal standard. The mobile phase consisted of acetonitrile (60%) and 0.01 M triethylamine in distilled water (40%) with the pH adjusted to 3.0. Separations were achieved on a C18 column and the effluent measured for UV absorption at 260 nm. The chromatographic separation was excellent, with no interference from endogenous serum constituents. This assay was suitable for measuring drug concentrations in the range of 10-1000 ng/ml using a 0.1-ml serum sample. The method was applied to a drug disposition study in transgenic mice with increased plasma alpha 1-acid glycoprotein.

Random shifts in blood cell genotypic composition are commonly observed in allophenic mice. This phenomenon was studied in 16 mosaic mice produced from very closely related strains, and no such changes were observed in the mosaic composition of erythrocytes, platelets, and lymphocytes over a period of 14 weeks. Furthermore, the mosaic distribution of a large group (66) of these mosaic mice was markedly biased in favor of those animals containing major contributions of both strains. This contrasts with what is normally found in collections of allophenic mice, in which the mosaic distribution curves are usually much flatter. While most allophenic mice have been produced from inbred strains with many genetic differences our results were obtained with congenic strains. This suggests that both properties, the unstable mosaic composition of blood cells and the flat mosaic distribution curves, are caused by specific genetic differences between cells of the two strains and are not inherent properties of allophenic mice. We propose that genetic differences cause these phenomena by inhibiting the mixing of cells of the two strains. Such might occur for example if, throughout development, cells of the same H-2 haplotype had greater affinity for each other than for ones of disparate H-2 haplotypes.

Mice of strain DBA/2J were found to produce red cells considerably more resistant to osmotic lysis than cells from C57BL/6J or the F1 hybrid between the two strains. Such strain-specific differences in osmotic fragility could be the result of genetically determined humoral or other systemic differences that indirectly influence red cell properties. Alternatively, this phenotypic variation might be an inherent property of the erythrocyte themselves and be directly controlled by their genotype. Analysis of red cells from allophenic (mosaic) mice of the strain composition C57BL/6J in equilibrium DBA/2J demonstrated that the latter possibility is the case. In such mice, erythrocytes of the DBA/2J genotype are relatively more resistant to osmotic lysis than are those of the C57BL/6J genotype; partial lysis of allophenic blood at intermediate salt concentrations results in marked enrichment for DBA/2J cells among the survivors. Future experiments designed to determine the mechanism underlying this difference can now focus on the properties of the red blood cells per se with the certainty that this property is inherent to the genotype of each cell.

Cholesterol is a major risk factor for coronary heart disease. Of 117 employees seen consecutively in the Tennessee State Employee Health Service voluntary screening program, 86 (74%) had cholesterol levels above the reference range reported by a commercial clinical laboratory. This was three times greater than the calculated expected number of 29 (25%, 17 at moderate risk, 12 at high risk for coronary disease). Age and sex adjustment using Lipid Research Clinic guidelines reduced the number with elevated cholesterol to 55 (47%, 24 at moderate risk, 31 at high risk). Split sample cholesterol assays run independently by the commercial laboratory and a university laboratory showed excellent correlation (r = 0.99, commercial laboratory = 1.0 (university laboratory) + 10.8), but a systematic difference of 12.4 mg/dL (SD = 6.4 mg/dL, paired-t = 9.63, p less than 0.00001) between the two laboratories. Further adjustment for this difference reduced the number with elevated cholesterol to 41 (36%, 26 at moderate risk, 15 at high risk). This experience illustrates how small systematic laboratory errors in cholesterol determination can greatly exaggerate the number of persons reported to have clinically important cholesterol elevations. Clinical laboratories should report age and sex adjusted cholesterol reference ranges and provide clients periodic quality assurance reports that their measurements of cholesterol levels are accurate.

C57BL/6 mice exhibit impaired lens development as evidenced by competition studies with allophenic (chimeric) mice. Strain-specific differences in electrophoretic forms of glucose phosphate isomerase were exploited to determine the strain composition of various ocular and nonocular tissues in allophenic mice constructed from C57BL/6 and DBA/2 strains. As expected, there was variation from one animal to the next in overall mosaic composition, as well as variation among most ocular and nonocular tissues within individual animals. In contrast, when individual lenses from the same animals were assayed there was a marked and consistent deficiency of the C57BL/6 component. Control experiments indicated that these results are not the consequence of peculiarities of marker enzyme expression in the lens. Since lens formation occurs early in embryogenesis any anomaly at this stage could influence the normal induction of later ocular structures. We believe that the impaired lens-forming capacity of C57BL/6 mice may be the underlying cause of the 5-10% frequency of overt ocular defects in this strain.