Biomaterials Research and Development at Missouri S&T

Advances in Biomaterials and Medical Devices PanelBiomaterials are critically important to future developments in the life sciences. They provide a backbone for biomedical research in academia and industry, and are a key component of reconstructive medical and dental research as well as patient and animal care. The worldwide demand for biomaterials for use as biological substitutes to restore and improve tissue function, currently estimated at $40 billion, is expected to grow substantially (10-20% per year) with future advances and an aging population. A major area of emphasis for future biomedical research is the development of new, more advanced biomaterials to replace hard and soft tissues, lost because of age-related attrition or degeneration, disease, or trauma. Although existing biomaterials and reconstructive procedures have improved the quality of life for many patients, the clinical performance and longevity of prosthetic replacements is considerably lower than that of the original tissue. The promising field of tissue engineering has the potential for overcoming many of the limitations of existing materials and procedures. The success of tissue engineering will, to a large extent, depend on biomaterials and the understanding of their biological interactions with molecules, cells, tissues, and organs. This presentation will provide an overview of the biomaterials research and development efforts in the Center for Bone and Tissue Repair and Regeneration (CBTRR) at Missouri S&T. Areas such as bioactive glass for hard (bone) and soft tissue repair, hydroxyapatite and bioactive glass microspheres for drug delivery, orthopaedic biomaterials, and advanced techniques for fabricating biomaterials with the requisite anatomical shape and structure, will be covered. Ongoing research collaborations in biomaterials and tissue engineering with the University of Missouri campuses at Kansas City and Columbia will be described. Potential economic and health benefits that could result from expansion of these collaborative research efforts among the University of Missouri campuses will be discussed

Antibiotic Elution and Mechanical Strength of PMMA Bone Cement Loaded With Borate Bioactive Glass

A grant from the One-University Open Access Fund at the University of Kansas was used to defray the author's publication fees in this Open Access journal. The Open Access Fund, administered by librarians from the KU, KU Law, and KUMC libraries, is made possible by contributions from the offices of KU Provost, KU Vice Chancellor for Research & Graduate Studies, and KUMC Vice Chancellor for Research. For more information about the Open Access Fund, please see http://library.kumc.edu/authors-fund.xml.Introduction: Local delivery of antibiotics using bone cement as the delivery vehicle is an established method of managing implant-associated orthopedic infections. Various fillers have been added to cement to increase antibiotic elution, but they often do so at the expense of strength. This study evaluated the effect of adding a borate bioactive glass, previously shown to promote bone formation, on vancomycin elution from PMMA bone cement. Methods: Five cement composites were made: three loaded with borate bioactive glass along with 0, 1, and 5 grams of vancomycin and two without any glass but with 1 and 5 grams vancomycin to serve as controls. The specimens were soaked in PBS. Eluate of vancomycin was collected every 24 hours and analyzed by HPLC. Orthopedic-relevant mechanical properties of each composite were tested over time. Results: The addition of borate bioactive glass provided an increase in vancomycin release at Day 1 and an increase in sustained vancomycin release throughout the treatment period. An 87.6% and 21.1% increase in cumulative vancomycin release was seen for both 1g and 5g loading groups, respectively. Compressive strength of all composites remained above the weight-bearing threshold of 70 MPa throughout the duration of the study with the glass-containing composites showing comparable strength to their respective controls. Conclusion: The incorporation of borate bioactive glass into commercial PMMA bone cement can significantly increase the elution of vancomycin. The mechanical strength of the cement-glass composites remained above 70 MPa even after soaking for 8 weeks, suggesting their suitability for orthopedic weight-bearing applications.Marc A. and Elinor J. Asher Orthopedic Research Endowmen

Selective Laser Sintering and Freeze Extrusion Fabrication of Bioglass Bone Scaffolds [abstract]

Biomedical Tissue Engineering, Biomaterials, and Medical Devices Poster SessionBioactive glasses are promising materials for bone scaffolds due to their ability to assist in tissue regeneration. When implanted in vivo, bioactive glasses can convert to hydroxyapatite, the main mineral constituent of human bone and form a strong bond with the surrounding tissues, providing an advantage over polymer scaffold materials. Bone scaffold fabrication using additive manufacturing (solid freeform fabrication) methods provides control over design and fabrication of pores in the scaffold. 13-93 bioglass (manufactured by Mo-Sci Corporation), a third-generation bioactive and resorbable material designed to accelerate the body's natural ability to heal itself, was used in the research described herein to fabricate bone scaffolds using two different additive manufacturing methods - Selective Laser Sintering and Freeze Extrusion Fabrication. Selective Laser Sintering (SLS) is a process where a laser light is controlled to selectively sinter the particles in a powder bed layer-by-layer to fabricate a 3D part based on a CAD model. The SLS machine used in this research was a DTM Sinterstation 2000. 13-93 bioglass mixed with stearic acid (as the polymer binder) by ball milling was used as the powder feedstock for the SLS machine. The fabricated green scaffolds underwent binder burnout to remove the stearic acid binder and then sintered at temperatures between 6500C and 7000C. After sintering, the scaffolds were mechanically tested, achieving a maximum compressive strength of 16 MPa for scaffolds with 60% apparent porosity. Bioactivity results showed the ability of the SLS scaffolds to support the growth of osteoblastic cells. Scanning electron microsocopy analysis and MTT formazan formation measurements provided evidence that the bioglass scaffolds fabricated by the SLS process offer a surface capable of supporting robust cell growth. Freeze Extrusion Fabrication (FEF) is a process where an aqueous-based glass paste is extruded and deposited layer-by-layer to fabricate a 3D part in a sub-freezing temperature environment. The FEF system, developed at Missouri S&T, consists of a 3-axis positioning system, a ram extruder for paste extrusion, and position and force sensors for measurement and control. Bioglass slurry was prepared by ball milling 13-93 bioglass particles along with a dispersant (surfynol) and a binder (aquazol). Further, a lubricant (PEG-400) was added to the paste to aid in extrusion. The bioglass slurry was then heated to obtain bioglass paste. Scaffolds with varying pore sizes from 300μm to 800μm were successfully fabricated using the FEF process. Post processing of green scaffolds, including binder burnout and sintering, is currently being performed. Scaffolds produced by the FEF process will be evaluated and compared with the scaffolds obtained using the SLS process

Bioreactor for microalgal cultivation systems: strategy and development

Microalgae are important natural resources that can provide food, medicine, energy and various bioproducts for nutraceutical, cosmeceutical and aquaculture industries. Their production rates are superior compared to those of terrestrial crops. However, microalgae biomass production on a large scale is still a challenging problem in terms of economic and ecological viability. Microalgal cultivation system should be designed to maximize production with the least cost. Energy efficient approaches of using light, dynamic mixing to maximize use of carbon dioxide (CO2) and nutrients and selection of highly productive species are the main considerations in designing an efficient photobioreactor. In general, optimized culture conditions and biological responses are the two overarching attributes to be considered for photobioreactor design strategies. Thus, fundamental aspects of microalgae growth, such as availability of suitable light, CO2 and nutrients to each growing cell, suitable environmental parameters (including temperature and pH) and efficient removal of oxygen which otherwise would negatively impact the algal growth, should be integrated into the photobioreactor design and function. Innovations should be strategized to fully exploit the wastewaters, flue-gas, waves or solar energy to drive large outdoor microalgae cultivation systems. Cultured species should be carefully selected to match the most suitable growth parameters in different reactor systems. Factors that would decrease production such as photoinhibition, self-shading and phosphate flocculation should be nullified using appropriate technical approaches such as flashing light innovation, selective light spectrum, light-CO2 synergy and mixing dynamics. Use of predictive mathematical modelling and adoption of new technologies in novel photobioreactor design will not only increase the photosynthetic and growth rates but will also enhance the quality of microalgae composition. Optimizing the use of natural resources and industrial wastes that would otherwise harm the environment should be given emphasis in strategizing the photobioreactor mass production. To date, more research and innovation are needed since scalability and economics of microalgae cultivation using photobioreactors remain the challenges to be overcome for large-scale microalgae production

The evolving SARS-CoV-2 epidemic in Africa: Insights from rapidly expanding genomic surveillance

INTRODUCTION Investment in Africa over the past year with regard to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequencing has led to a massive increase in the number of sequences, which, to date, exceeds 100,000 sequences generated to track the pandemic on the continent. These sequences have profoundly affected how public health officials in Africa have navigated the COVID-19 pandemic. RATIONALE We demonstrate how the first 100,000 SARS-CoV-2 sequences from Africa have helped monitor the epidemic on the continent, how genomic surveillance expanded over the course of the pandemic, and how we adapted our sequencing methods to deal with an evolving virus. Finally, we also examine how viral lineages have spread across the continent in a phylogeographic framework to gain insights into the underlying temporal and spatial transmission dynamics for several variants of concern (VOCs). RESULTS Our results indicate that the number of countries in Africa that can sequence the virus within their own borders is growing and that this is coupled with a shorter turnaround time from the time of sampling to sequence submission. Ongoing evolution necessitated the continual updating of primer sets, and, as a result, eight primer sets were designed in tandem with viral evolution and used to ensure effective sequencing of the virus. The pandemic unfolded through multiple waves of infection that were each driven by distinct genetic lineages, with B.1-like ancestral strains associated with the first pandemic wave of infections in 2020. Successive waves on the continent were fueled by different VOCs, with Alpha and Beta cocirculating in distinct spatial patterns during the second wave and Delta and Omicron affecting the whole continent during the third and fourth waves, respectively. Phylogeographic reconstruction points toward distinct differences in viral importation and exportation patterns associated with the Alpha, Beta, Delta, and Omicron variants and subvariants, when considering both Africa versus the rest of the world and viral dissemination within the continent. Our epidemiological and phylogenetic inferences therefore underscore the heterogeneous nature of the pandemic on the continent and highlight key insights and challenges, for instance, recognizing the limitations of low testing proportions. We also highlight the early warning capacity that genomic surveillance in Africa has had for the rest of the world with the detection of new lineages and variants, the most recent being the characterization of various Omicron subvariants. CONCLUSION Sustained investment for diagnostics and genomic surveillance in Africa is needed as the virus continues to evolve. This is important not only to help combat SARS-CoV-2 on the continent but also because it can be used as a platform to help address the many emerging and reemerging infectious disease threats in Africa. In particular, capacity building for local sequencing within countries or within the continent should be prioritized because this is generally associated with shorter turnaround times, providing the most benefit to local public health authorities tasked with pandemic response and mitigation and allowing for the fastest reaction to localized outbreaks. These investments are crucial for pandemic preparedness and response and will serve the health of the continent well into the 21st century

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Modulating notochordal differentiation of human induced pluripotent stem cells using natural nucleus pulposus tissue matrix.

Human induced pluripotent stem cells (hiPSCs) can differentiate into notochordal cell (NC)-like cells when cultured in the presence of natural porcine nucleus pulposus (NP) tissue matrix. The method promises massive production of high-quality, functional cells to treat degenerative intervertebral discs (IVDs). Based on our previous work, we further examined the effect of cell-NP matrix contact and culture medium on the differentiation, and further assessed the functional differentiation ability of the generated NC-like. The study showed that direct contact between hiPSCs and NP matrix can promote the differentiation yield, whilst both the contact and non-contact cultures can generate functional NC-like cells. The generated NC-like cells are highly homogenous regarding the expression of notochordal marker genes. A culture medium containing a cocktail of growth factors (FGF, EGF, VEGF and IGF-1) also supported the notochordal differentiation in the presence of NP matrix. The NC-like cells showed excellent functional differentiation ability to generate NP-like tissue which was rich in aggrecan and collagen type II; and particularly, the proteoglycan to collagen content ratio was as high as 12.5-17.5 which represents a phenotype close to NP rather than hyaline cartilage. Collectively, the present study confirmed the effectiveness and flexibility of using natural NP tissue matrix to direct notochordal differentiation of hiPSCs, and the potential of using the generated NC-like cells for treating IVD degeneration

Workflow chart for the experiments.

<p>Workflow chart for the experiments.</p

Functional differentiation of NCs to generate NP-like tissue induced by TGF-beta 3.

<p>The cultures were analyzed after 2 and 4 weeks. (A): The transcript of Sox9 and collagen type II (COL2A1) gene was remarkably up-regulated in both cells (NCs derived from contact or non-contact cultures) and at both time points. The transcript of aggrecan (ACAN) attenuated after 2 and 4 weeks comparing to the undifferentiated control (0-day), however, it is noteworthy that the ACNA transcript was maintained at significant levels in all the cultures (Ct∼30). The data are reported in relative mRNA expression which was analyzed by 2^−ΔΔCt method using day 0 control as reference. Three biological samples were pooled and measured so that each result represents the average of three replicates. The NC-like cells derived from both contact and non-contact cultures generated spherical balls with diameter of approximately 1 mm after 4 weeks (B). Extracellular biochemistry of the pellet cultures was analyzed after 4 weeks. Histological sections were cut at 5 um. (D): Safranin O staining of proteoglycans in the extracellular matrix. (E) represents immunohistochemical staining of aggrecan and collagen type II. Negative control (primary antibody omitted) was shown in (C). Magnification: (C, D and E): 400x. (F): Quantification of cell population (dsDNA content), glycosaminoglycans (GAGs) content, hydroxyproline content, and GAGs/hydroxyproline ratio of the NP differentiation cultures. The outcomes were compared between the cells derived from contact and non-contact cultures. Noticeably, the GAGs/hydroxyproline was as high as 12.6–17.4, which was remarkably higher than that of native hyaline cartilage (∼2), and much closer to that of native nucleus pulposus tissue (∼27). *: p<0.05, n = 3.</p

Influence of culture media on the notochordal differentiation.

<p>Two media was investigated: one contained 10% fetal bovine serum (FBS medium) and another contained a cocktail of growth factors (EGM2-MV medium, which is a commercial product of Lonza). (A) and (B) represents the cells differentiated for 5 days in FBS medium and EGM2-MV medium, respectively. Arrow heads indicate the floating NP tissue (out of focus in the image). Frequently the cells (arrows) in B displayed vacuole morphology which is also observable in primary culture of notochordal cells. (C): The transcript levels of three typical notochordal genes of the two cultures. The data are reported in relative mRNA expression which was analyzed by 2^−ΔΔCt method using hiPSCs as reference. Three biological samples were pooled and measured so that each result represents the average of three replicates.</p