FABRICATION AND CHARACTERIZATION OF BIOACTIVE, COMPOSITE ELECTROSPUN BONE TISSUE ENGINEERING SCAFFOLDS INTENDED FOR CLEFT PALATE REPAIR

Tissue Engineering is a scientific discipline that aims to regenerate tissues and organs that are diseased, lost or congenitally absent. It encompasses the use of suitable synthetic equivalents of native extracellular matrix that may or may not be supplemented with cells or relevant growth factors. Such scaffolds are designed to reside at the site of implantation for a variable period of time during which they induce the regeneration of native tissue. During this time, they also provide a template for new cells to attach, infiltrate, differentiate into appropriate phenotype and eventually restore function of the concerned tissue. Among the factors that affect the outcome are the composition of scaffold, methods of fabrication, bulk properties of the scaffold and topography and architecture at the cellular level. Bone is unique in the body in that it is one of the few tissues capable of complete regeneration even in adults, as seen during fracture healing. However, certain conditions (non-union of fractures, congenital and acquired bone deficiencies) exist in which the regenerative capacities of bone are exceeded and appropriate intervention becomes necessary. Current treatment options include autologous bone grafts harvested from iliac crest or de-cellularized allografts or synthetic substitutes made from metals, ceramics and polymers. However these options have serious limitations: while autografts are limited in supply, necessitate second surgery and show inadequate vascularization, allografts can transmit viral infections. Metals, ceramics and polymers are in essence structural replacements without performing any biological function. Other problems associated with these synthetic materials include adverse immune reactions, corrosion, stress-shielding and secondary fractures due to inadequate osseo-integration. Bone tissue engineering is a specialized field of research that provides an alternative strategy to repair bone defects by exploiting the advances in engineering and better understanding of bone biology. Scaffold-based tissue engineering approach is a promising field that involves implantation of a biomaterial that is specifically matched in terms of biological and material properties to the tissue it replaces. This study explores the feasibility of using electrospinning as a potential fabrication strategy for bone tissue engineering applications, more specifically intended for cleft palate repair. This model represents a congenital deformity that affects both hard and soft tissues and presents unique challenges and opportunities. Among the challenges are: the need for the implant allow growth of the most complex areas of the facial skeleton, integrate and grow with the patient through adolescence, the ability of the implant to not interfere with vital functions including breathing and feeding. Further the implant should provide a flexible matrix that can effectively support erupting teeth. In spite of these extreme demands, maxilla is a non load-bearing membranous bone, a favorable consideration from materials engineering perspective. The present study is organized into three independent sections. The first section investigates developing strategies intended to improve the material properties of electrospun bone scaffold. Bone is composed of a high volume fraction (50%) of inorganic hydroxyapatite nanocrystals that is closely associated with collagen. The dispersal of brittle mineral is critical in not only strengthening the bone in compression but also contributes to the osteoconductivity of the matrix. Since loading of mineral in a bone scaffold is a serious limitation, we attempted to achieve improved loading of bone mineral by dual mineralization approach. We first incorporated nanocrystalline hydroxyapatite (nHA) directly into the scaffold by adding it to the electrospinning polymer solution. The second step involves inducing biomimetic mineralization of electrospun scaffolds by incubating them in simulated body fluid (SBF) for 2 weeks. The hypothesis was that the nanocrystalline hydroxyapatite seeded during electrospinning would act as sites for nucleation and further crystal growth when incubated in solution supersaturated with respect to calcium and phosphate ions. We tested this approach in two synthetic, biocompatible polymers-polydioxanone and poly (lactide: glycolide) and four formulations of SBF with differential loading of nHA (0-50% by wt. of polymer). A modified Alizarin Red S (ARS) staining that specifically binds to calcium was developed that allowed us to quantify the mineral content of 3D scaffold with great accuracy. Results indicated a unique combination of factors: PDO scaffolds containing 50% nHA incubated in 1x revised-SBF incubated under static conditions gave maximum mineralization over a period of two weeks. We then sought to exploit these findings to engineer a stiffer scaffold by stacking multiple layers together and cold welding them under high pressure. Electrospun scaffolds (1, 2 or 4 layered stacks) were either compressed before or after mineralizing treatment with SBF. After two weeks, scaffolds were analyzed for total mineral content and stiffness by uniaxial tensile testing. Results indicated while compression of multiple layers significantly increases the stiffness of scaffolds, it also had lower levels of mineralization partly due to increased density of fibers and loss of surface area due to fiber welding. However this can be offset to a reasonable degree by increasing the number of stacks and hence this strategy can be successfully adopted to improve the mechanical properties of electrospun scaffolds. The second section introduces a novel infrared imaging technique to quantify and characterize the biological activity of biomaterials, based on cell adhesion. Cells attach to the surface by the formation of focal contacts where multiple proteins including vinculin and talin assemble to signal critical processes like cell survival, migration, proliferation and differentiation. After allowing MG-63 osteoblasts to adhere to 2D biomaterial surface coated with extracellular matrix proteins (collagen, gelatin, fibronectin) cells were fixed and probed with antibodies for vinculin and talin. Secondary antibodies, tagged with infrared-sensitive fluorescent dyes, were used to quantify the molecules of interest. In addition, the kinetics of focal contact formation in these different substrates was followed. Successful quantification of focal contacts were made and further research revealed phosphorylation of vinculin at pY-822 as one potential mechanism for recruitment of vinculin to focal contacts. Hence it could represent a subset of vinculin and might serve as a specific molecular marker for focal contacts. As an extension, we evaluated the possibility of using such an assay to quantify 3D electrospun tissue engineering scaffolds. We fabricated scaffolds of graded biological activity by electrospinning blends of polydioxanone and collagen in different ratios. Vinculin and talin expressed by MG-63 cultured on these scaffolds for 24 hours were quantified in a similar manner. Results indicate that while talin does not show a significant difference in expression among different scaffolds, vinculin showed a positive correlation with increasing biological activity of scaffolds. In conclusion, we have identified vinculin as a reliable marker of focal contacts in 3D scaffolds while phosphovinculin (pY-822) was more specific to focal contacts in coated 2D substrates. In both instances, infrared imaging proved to be reliable in study of focal contacts. The third section aims to make the bone scaffolds osteoinductive- a property of a material to induce new bone formation even when implanted in subcutaneous and intramuscular heterotopic sites. Bone morphogenetic proteins (BMP) are potent cytokines that can induce migration, proliferation and differentiation of stem cells along osteoblastic lineage. The therapeutic efficacy of BMPs in the treatment of severe bone defects has been identified and is currently FDA approved for specific orthopedic applications. BMPs are clinically administered in a buffer form that not only makes the treatment expensive but less effective. Suitable delivery systems for BMP delivery have been an intense area of investigation. We rationalized electrospinning as a strategy to incorporate BMP within the scaffold and that would enable controlled release when implanted. One of the drawbacks of using electrospinning to deliver bioactive molecules is the potential denaturing effect and eventual loss of activity of BMPs. The final section of this dissertation tries to develop sensitive and relevant assays that could answer intriguing questions about solvent-protein interaction. We chose to use the BMP-2/7 heterodimer as the osteoinductive molecule of choice because of its superior potency compared to homodimer counterparts. We characterized the detection and quantification of BMP-2/7 using a slot blot technique. Further, we used a novel cell line (C2C12 BRA) to test the retention of activity of BMP-2/7 that has been exposed to organic solvents. Results indicate significant loss of activity when BMPs are exposed to organic solvents but complete recovery was possible by diluting the solvent with an aqueous buffer

Ensure Everyone Succeeds

Students in the post-baccalaureate certificate and graduate programs on the Health Science Campus (MS, PhD, and first professional programs including DDS, PharmD, and MD) are highly motivated to succeed in their chosen career paths. Needless to say, these courses are very heavy on didactics and challenge the best of the students. Typically, these programs have rigid curricular designs and offer little to no flexibility to students in choosing the courses. It is fast-paced, high in curricular hours and the students cannot afford to have mishaps without jeopardizing their careers. While this stringent curriculum is necessary for VCU to maintain its accreditation standards and global standing, it does take a toll on students’ performance and well-being. The Division of Academic Success (DAS) on the Health Science campus provides an excellent support net for these students in terms of identifying and supporting students needing accommodations, teaching time management and study habits, and test taking strategies. Conversations among team members, students and program directors representing different units in the Health Science Campus, as well as discussions with DAS staff, revealed lack of awareness of the comprehensive services provided by the DAS and no standard policy/ guideline across different schools as to when to engage the DAS to help students in need. To improve utilization, this project proposes that DAS focus on three key areas: education and training, promotion and marketing, and redesign and elevation of existing resources

Acute Fish Oil supplementation and Aspirin treatment modulates lipid profile in Platelet Rich Plasma: a randomized pilot trial

Aims: Platelet rich plasma (PRP) has been used in tissue repair to treat numerous inflammatory pathophysiologies. Recent studies have elucidated that the bioactive lipid fraction of PRP significantly contributes towards the resolution of inflammation. There has been great interest in how therapeutics could modulate the PRP lipidome to formulate a more pro-resolving matrix. Many of the pathways used to produce either pro-resolving or pro-inflammatory lipids are shared between ω-3 and ω-6 polyunsaturated fatty acids (PUFA). Here, we explored the separate and interacting effects acute exogenous ω-3 PUFA supplementation and aspirin had on the lipidome of PRP within 6 hours. Methods:PRP from 45 patients was obtained at baseline and after 6-hours from either control or those receiving one 1400 mg fish oil tablet, Bayer low-dose aspirin (81mg), or combinational therapy. Lipids were acquired by liquid chromatography mass spectrometry. Spearman rank correlation analysis visually assessed what effects treatments had on the relative abundance of PUFA derivatives. The control group was referenced for lipid selection across groups; lipids were selected on the basis that they significantly (p Results: Fish oil ω-3 PUFA supplementation and aspirin had separate and interacting effects on oxylipin and neutral lipid correlations. Strongly correlated (rho \u3e 0.65) ω-6 PUFA metabolites were reversed or reduced in magnitude following either treatment. A total of 24 lipid species were significantly modulated in the fish oil treatment group, with notable (p4), and lipoxin A4 (LXA4). Conclusion: We can confirm that fish oil supplementation and aspirin do exert modulatory effects on the lipid fraction of PRP within a short period of time (6-hours). The PUFAs composing fish oil impacted a wide range of the lipidome – possibly though a mechanism of ω-3/ω-6 enzymatic competition. Our results support that ω-3 PUFA supplementation may improve the efficacy of PRP for short-term use

Mesenchymal Stem Cells Derived from Healthy and Diseased Human Gingiva Support Osteogenesis on Electrospun Polycaprolactone Scaffolds

Periodontitis is a chronic inflammatory disease affecting almost half of the adult US population. Gingiva is an integral part of the periodontium and has recently been identified as a source of adult gingiva-derived mesenchymal stem cells (GMSCs). Given the prevalence of periodontitis, the purpose of this study is to evaluate differences between GMSCs derived from healthy and diseased gingival tissues and explore their potential in bone engineering. Primary clonal cell lines were established from harvested healthy and diseased gingival and characterized for expression of known stem-cell markers and multi-lineage differentiation potential. Finally, they were cultured on electrospun polycaprolactone (PCL) scaffolds and evaluated for attachment, proliferation, and differentiation. Flow cytometry demonstrated cells isolated from healthy and diseased gingiva met the criteria defining mesenchymal stem cells (MSCs). However, GMSCs from diseased tissue showed decreased colony-forming unit efficiency, decreased alkaline phosphatase activity, weaker osteoblast mineralization, and greater propensity to differentiate into adipocytes than their healthy counterparts. When cultured on electrospun PCL scaffolds, GMSCs from both sources showed robust attachment and proliferation over a 7-day period; they exhibited high mineralization as well as strong expression of alkaline phosphatase. Our results show preservation of ‘stemness’ and osteogenic potential of GMSC even in the presence of disease, opening up the possibility of using routinely discarded, diseased gingival tissue as an alternate source of adult MSCs

Incorporation of Fibrin Matrix into Electrospun Membranes for Periodontal Wound Healing

Guided tissue regeneration (GTR) aims to regenerate the lost attachment apparatus caused by periodontal disease through the use of a membrane. The goal of this study is to create and characterize a novel hybrid membrane that contains biologically active fibrin matrix within a synthetic polycaprolactone (PCL) electrospun membrane. Three-dimensional fibrin matrices and fibrin-incorporated electrospun membrane were created from fresh frozen plasma by centrifugation in glass vials under three different conditions: 400 g for 12 min, 1450 g for 15 min and 3000 g for 60 min. Half the membranes were crosslinked with 1% genipin. Degradation against trypsin indicated biologic stability while uniaxial tensile testing characterized mechanical properties. Continuous data was analyzed by ANOVA to detect differences between groups (p = 0.05). Fibrin-incorporated electrospun membranes showed statistically significant increase in mechanical properties (elastic modulus, strain at break and energy to break) compared to fibrin matrices. While crosslinking had marginal effects on mechanical properties, it did significantly increase biologic stability against trypsin (p < 0.0001). Lastly, membranes generated at 400 g and 1450 g were superior in mechanical properties and biologic stability compared to those generated at 3000 g. Fibrin-incorporated, crosslinked electrospun PCL membranes generated at lower centrifugation forces offers a novel strategy to generate a potentially superior membrane for GTR procedures

Peracetic Acid: A Practical Alternative to Formalin for Disinfection of Extracted Human Teeth

Extracted human teeth provide the closest approximation to teeth in situ and play important roles in dental education and materials research. Since extracted teeth are potentially infectious, the Centers for Disease Control recommend their sterilization by autoclaving or disinfection by formalin immersion to ensure safe handling. However, autoclaving is not recommended for teeth with amalgam fillings and formalin is hazardous. The goal of the present study was to investigate the potential of peracetic acid (PA) as an alternative method to achieve reliable disinfection of freshly extracted teeth. A total of 80 extracted teeth were collected for this study. Whole teeth were incubated in one of four solutions for defined periods of time: sterile water (2 weeks), formalin (2 weeks), PA 1000 ppm (2 weeks), and PA 2000 ppm (1 week). After sectioning, the crown and root fragments were transferred into separate tubes containing brain–heart infusion broth and incubated at 37 °C under anaerobic conditions for 72 h. Absence of broth turbidity was used to assess effectiveness of disinfection. No turbidity was observed in any of the formalin-treated or peracetic acid-treated samples, signifying complete disinfection. Our results indicate that PA can effectively disinfect extracted human teeth, providing a reliable alternative to formalin and autoclaving

Science of nanofibrous scaffold fabrication: Strategies for next generation tissue-engineering scaffolds

Native extracellular matrix (ECM) provides structural support to the multicellular organism on a macroscopic scale and establishes a unique microenvironment (niche) to tissue- and organ-specific cell types. Both these functions are critical for optimal function of the organism. These natural ECMs comprise predominantly fibrillar proteins, collagen and elastin and are synthesized as monomers but undergo hierarchical organization into well-defined nanoscaled structural units. The interaction between the cells and ECM is dynamic, reciprocal and essential for tissue development, maintenance of function, repair and regeneration processes. Tissue-engineering scaffolds are synthetic, biomimetic ECM analogues that have great promise in regenerative medicine. Ongoing efforts in mimicking the native ECM in terms of composition and dimension have resulted in three strategies that permit the generation of scaffolds in nanometer dimensions. Although excellent reviews regarding the applications of these strategies in tissue engineering are available, a comprehensive review of the science behind these fabrication techniques does not exist. This review intends to fill this critical gap in the existing knowledge in the fast-expanding field of nanofibrous scaffolds. A thorough understanding of the fabrication processes would enable us to better exploit available technologies to produce superior tissue-engineering scaffolds

Extracellular matrix regenerated: Tissue engineering via electrospun biomimetic nanofibers

While electrospinning had seen intermittent use in the textile industry from the early twentieth century, it took the explosion of the field of tissue engineering, and its pursuit of biomimetic extracellular matrix (ECM) structures, to create an electrospinning renaissance. Over the past decade, a growing number of researchers in the tissue engineering community have embraced electrospinning as a polymer processing technique that effectively and routinely produces non-woven structures of nanoscale fibers (sizes of 80 nm to 1.5 μm). These nanofibers are of physiological significance as they closely resemble the structure and size scale of the native ECM (fiber diameters of 50 to 500 nm). Attempts to replicate the many roles of native ECM have led to the electrospinning of a wide array of polymers, both synthetic (poly(glycolic acid), poly(lactic acid), polydioxanone, polycaprolactone, etc.) and natural (collagen, fibrinogen, elastin, etc.) in origin, for a multitude of different tissue applications. With various compositions, fiber dimensions and fiber orientations, the biological, chemical and mechanical properties of the electrospun materials can be tailored. In this review we highlight the role of electrospinning in the engineering of different tissues and applications (skin/wound healing, cartilage, bone, vascular tissue, urological tissues, nerve, and ligament), and discuss its potential role in future work. © 2007 Society of Chemical Industry

Evaluation of biological activity of bone morphogenetic proteins on exposure to commonly used electrospinning solvents

Bone tissue engineering is one of the emerging strategies for developing functionally viable bone substitutes. The recent trend in bone tissue engineering is to combine the benefits of a three-dimensional nanofibrous scaffold with biologically active molecules and responsive stem cells. Electrospinning is the most versatile of the scaffold fabrication strategies and may involve the use of an organic solvent at one stage or another. In spite of all distinct advantages of electrospinning, valid concerns about potentially denaturing interactions between the organic solvent and the biomolecules exist. Efforts are ongoing to incorporate osteoinductive molecules, such as bone morphogenetic proteins (BMPs), during the electrospinning process. The challenge lies in ensuring that the biological activity of these incorporated molecules survives the process. This study was specifically designed to investigate the effects of exposure to commonly used organic solvents on heterodimeric BMP-2/7 using slot-blot assay quantified by infrared imaging and on embryonic myoblasts stably transfected with BMP-specific response element linked to a luciferase reporter-C2C12BRA. Overall, the biological activity of these molecules significantly decreased when exposed to organic solvents but can be restored to their original values by increasing the polarity of the solvent. It was found that an aqueous buffer can effectively overcome the deleterious effects of organic solvents on BMPs, thus generating osteoinductive bone scaffolds. © SAGE Publications 2011