Osteoregenerative Potential of 3D-Printed Poly ε-Caprolactone Tissue Scaffolds In Vitro Using Minimally Manipulative Expansion of Primary Human Bone Marrow Stem Cells.

The repair of orthopedic and maxillofacial defects in modern medicine currently relies heavily on the use of autograft, allograft, void fillers, or other structural material composites. This study examines the in vitro osteo regenerative potential of polycaprolactone (PCL) tissue scaffolding, fabricated via a three-dimensional (3D) additive manufacturing technology, i.e., a pneumatic micro extrusion (PME) process. The objectives of this study were: (i) To examine the innate osteoinductive and osteoconductive potential of 3D-printed PCL tissue scaffolding and (ii) To perform a direct in vitro comparison of 3D-printed PCL scaffolding with allograft Allowash® cancellous bone cubes with regards to cell-scaffold interactions and biocompatibility with three primary human bone marrow (hBM) stem cell lines. This study specifically examined cell survival, cell integration, intra-scaffold cell proliferation, and differentiation of progenitor cells to investigate the potential of 3D-printed PCL scaffolds as an alternative to allograft bone material for the repair of orthopedic injuries. We found that mechanically robust PCL bone scaffolds can be fabricated via the PME process and the resulting material did not elicit detectable cytotoxicity. When the widely used osteogenic model SAOS-2 was cultured in PCL extract medium, no detectable effect was observed on cell viability or proliferation with multiple test groups showing viability ranges of 92.2% to 100% relative to a control group with a standard deviation of ±10%. In addition, we found that the honeycomb infill pattern of the 3D-printed PCL scaffold allowed for superior mesenchymal stem-cell integration, proliferation, and biomass increase. When healthy and active primary hBM cell lines, having documented in vitro growth rates with doubling times of 23.9, 24.67, and 30.94 h, were cultured directly into 3D-printed PCL scaffolds, impressive biomass increase values were observed. It was found that the PCL scaffolding material allowed for biomass increase values of 17.17%, 17.14%, and 18.18%, compared to values of 4.29% for allograph material cultured under identical parameters. It was also found that the honeycomb scaffold infill pattern was superior to the cubic and rectangular matrix structures, and provided a superior microenvironment for osteogenic and hematopoietic progenitor cell activity and auto-differentiation of primary hBM stem cells. Histological and immunohistochemical studies performed in this work confirmed the regenerative potential of PCL matrices in the orthopedic setting by displaying the integration, self-organization, and auto-differentiation of hBM progenitor cells within the matrix. Differentiation products including mineralization, self-organizing "proto-osteon" structures, and in vitro erythropoiesis were observed in conjunction with the documented expression of expected bone marrow differentiative markers including CD-99 (>70%), CD-71 (>60%), and CD-61 (>5%). All of the studies were conducted without the addition of any exogenous chemical or hormonal stimulation and exclusively utilized the abiotic and inert material polycaprolactone; setting this work apart from the vast majority of contemporary investigations into synthetic bone scaffold fabrication In summary, this study demonstrates the unique clinical potential of 3D-printed PCL scaffolds for stem cell expansion and incorporation into advanced microstructures created via PME manufacturing to generate a physiologically inert temporary bony defect graft with significant autograft features for enhanced end-stage healing.

Minimally Manipulative Method for the Expansion of Human Bone Marrow Mesenchymal Stem Cells to Treat Osseous Defects.

Lack of standardization of clinically compliant culture protocols of mesenchymal stem cells for re-implantation in humans have hindered clinical progress in the field of tissue regeneration to repair maxillofacial and orthopedic defects. The goal of this study was to establish a clinically relevant osteogenic protocol for collection and expansion of autologous stem cells to be used at Marshall University for re-implantation and repair of maxillofacial and orthopedic conditions. Human bone marrow (hBM) samples were collected from patients undergoing intramedullary nail fixation for closed femoral fractures. hBM mesenchymal cells were expanded by growing them first in Petri dishes for two weeks, followed by a week of culture using Perfecta 3D Hanging Drop Plates®. Various scaffold materials were tested and analyzed for cellular integration, vitality, and differentiation capacity of harvested hBM-MSCs including: 60/40 blend of hydroxyapatite biomatrix; Acellular bone composite discs; Allowash®, cancellous bone cubes; PLGA (poly lactic-co-glycolic acid); and Woven chitin derived fiber. We found that the 3D spheroid culture allowed production of hBM mesenchymal cells that retained osteoblast differentiation capacity over a monolayer culture of hBM-MSCs without the need to use chemical or hormonal modulation. We also observed that hydroxyapatite and Allowash cancellous bone scaffolds allowed better cell integration and viability properties as compared to other materials tested in this study. In conclusion, the multimodal culture methodology we developed creates actively differentiating stem-cell spheroids that can then be readily utilized in clinical practices to improve the regeneration of tissues of the head and the body.

Detection and confirmation of Clostridium botulinum in water used for cooling at a plant producing low-acid canned foods.

Our laboratory tested water samples used for cooling low-acid canned foods at a canning facility under investigation by the U.S. Food and Drug Administration. We used an enzyme-linked immunosorbent assay with digoxigenin-labeled antibodies (DIG-ELISA) and real-time PCR as screening methods and confirmed the presence of neurotoxin-producing Clostridium botulinum in the samples by mouse bioassay.

Evaluating the use of fatty acid profiles to identify Francisella tularensis.

Rapid capillary gas chromatography (GC) with flame-ionization detection was used to determine the cellular fatty acid profiles of Francisella tularensis. Two subspecies of F. tularensis, the live vaccine strain (LVS) derived from holarctica and a novicida strain Utah 112 (U112), were used to compare the extracted fatty acid methyl esters (FAMEs). A data set for the 2 subspecies was prepared using fatty acid profiles of bacteria grown on 2 types of media, Mueller-Hinton and cysteine heart agar supplemented with 5% rabbit blood (CHAB), and harvested at various time intervals (Day 1 through Day 4) with replicates prepared on different days. A total of 204 samples were analyzed. The results showed that these fatty acid quantitative profiles were unique for each of the subspecies and could be used as a fingerprint for the organism. It was determined by this rapid method that approximately 88% of the fatty acids in both the LVS and U112 strains included 6 saturated fatty acids: 10:0, 12:0, 14:0, 16:0, 18:0, and 20:0; and 4 hydroxy fatty acids 10:0 2OH, 16:0 3OH, 17:0 3OH, and 18:0 3OH. Data analysis and determination of clustering were performed by principal component analysis (PCA) and soft independent modeling of class analogy (SIMCA). Both PCA and SIMCA showed clear separation of the LVS and U112 strain and would be useful for prediction of unknowns. It was determined that the incubation time can be reduced from 48 to 24 h, and results are highly predictive for the identification of F. tularensis. In summary, analysis of FAMEs from F. tularensis subspecies LVS and U112 grown on CHAB or Mueller-Hinton media, and using a rapid GC method can provide a sensitive procedure for identification of these organisms.

Translocation of YopE and YopN into eukaryotic cells by Yersinia pestis yopN, tyeA, sycN, yscB and lcrG deletion mutants measured using a phosphorylatable peptide tag and phosphospecific antibodies.

Yersinia pestis, the causative agent of plague, exports a set of virulence proteins called Yops upon contact with eukaryotic cells. A subset of these Yops is translocated directly into the cytosol of host cells. In this study, a novel protein tag-based reporter system is used to measure the translocation of Yops into cultured eukaryotic cells. The reporter system uses a small bipartite phosphorylatable peptide tag, termed the Elk tag. Translocation of an Elk-tagged protein into eukaryotic cells results in host cell protein kinase-dependent phosphorylation of the tag at a specific serine residue, which can subsequently be detected with phosphospecific antibodies. The YopN, TyeA, SycN, YscB and LcrG proteins function to prevent Yop secretion before host cell contact. The role of these proteins was investigated in the translocation of Elk-tagged YopE (YopE129-Elk) and YopN (YopN293-Elk) into HeLa cells. Y. pestis yopN, tyeA, sycN and yscB deletion mutants showed reduced levels of YopE129-Elk phosphorylation compared with the parent strain, indicating that these mutants translocate reduced amounts of YopE. We also demonstrate that YopN293-Elk is translocated into HeLa cells and that this process is more efficient in a Yersinia yop polymutant strain lacking the six translocated effector Yops. Y. pestis sycN and yscB mutants translocated reduced amounts of YopN293-Elk; however, tyeA and lcrG mutants translocated higher amounts of YopN293-Elk compared with the parent strain. These data suggest that TyeA and LcrG function to suppress the secretion of YopN before host cell contact, whereas SycN and YscB facilitate YopN secretion and subsequent translocation.