Browsing by Subject "bone"
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Item Comparison of DNA Extraction Methods From Bone to be Used With the DNA IQ System on the Maxwell 16 for Human Identification(2008-08-01) Lopez, Kristen; John Planz; Arthur Eisenberg; Joseph WarrenLopez, Kristen M., Comparison of DNA Extraction Methods From Bone to be Used with the DNA IQ System on the Maxwell 16 for Human Identification . Masters of Science (Graduate School of Biomedical Sciences), August, 2008, 52 pp., 11 tables, 15 figures, bibliography, 24 titles. Extraction and purification of DNA from human bones is essential for correctly identifying the remains through DNA analysis. Current DNA extraction methods include a demineralization step, which extracts calcium and phosphate from the bone matrix, inactivation of DNAses, and the removal of Polymerase Chain Reaction (PCR) inhibitors. These methods often use harsh chemicals and may allow for residual DNA to be discarded in various wash steps. To assess the effectiveness of DNA extraction from bone samples, two extraction protocols were compared. The first method included a bone demineralization pretreatment solution of Sodium N-Laurylsarcosinate, 0.5 M EDTA, and Proteinase K (20 mg/ml). The second included a pretreatment using a Bone Incubation Buffer by Promega Corporation, with an addition of Proteinase K (18mg/ml). Various incubation times were included to assess the extraction at different time intervals. All extracted samples were purified with the DNA IQ Reference Sample Kit on the automated Maxwell 16 Instrument (Promega Corp.). Full and partial profiles were obtained from samples extracted with the Bone Incubation pretreatment, regardless of incubation time. Profiles were not observed with the standard demineralization pretreatment when amplified at 28 cycles, with partial profiles present in a few samples when amplified at 32 cycles.Item Development of an Osteoinductive Bone Graft(2010-12-01) Sule, Anupam A.; Slobodan Dan DimitrijevichBone is a unique tissue that serves multiple functions. One of its unique features is the ability to heal by formation of new bone, whereas most other tissues undergo the process of scar formation. When a large amount of bone is lost the only treatment available is the use of bone grafts. Multiple bone graft substitutes are being developed to address the shortage of autologous bone graft. 3-D models are being developed to further our understanding of the cellular processes taking place in vivo. In this study I examined the strategy of designing a 3-Dmodel of hard tissue and a potential bone graft substitute using collagen type I and several different porous scaffolds. Factors influencing collagen gel contraction by human mesenchymal stem cells (hMSC) during the process of osteogenic differentiation were studied and it was shown that collagen type I gels prepared in accordance with our patented technology contract far less than any other collagen gels reported in literature. The validity of MTT assay to track proliferation of hMSC in various 3-D matrices was established and allowed me to show that human mesenchymal stem cells (hMSC) proliferated, differentiated along an osteogenic lineage and mineralized the extracellular matrix (ECM). Higher cell seeding density and greater serum concentration in the culture medium, caused increased collagen type I gel contraction. Late passage cells and osteoblasts caused a greater collagen type I gel contraction than undifferentiated early passage hMSC. hMSC that had been transduced to constitutively express human telomerase reverse transcriptase (hTERT), and which had thereby acquired an extended in vitro life span (telomerized hMSC or TMSC), contracted the collagen gel lesser than hMSC. A Collagen type I Gel - Collagen type I foam Scaffold combination (CGCS) was investigated as a 3-D in vitro model to allow extrapolation of soft tissue results to those characteristic of hard tissue. Deep penetration of MSC into the CGCS with uniform distribution was achieved by the use of collagen type I gel, as the cell carrier. Collagen type I gel improved seeding efficiency and facilitated retention of cells that penetrated deep into the scaffold. Longterm survival, proliferation, viability and in situ osteogenic differentiation within the CGCS were demonstrated. A model that demonstrated migration of cells in and out of CGCS was assembled and tested. A need for the presence of fibrillar collagen gel for mineralization process to take place highlighted the benefit of adding collagen gel to the 3-D models. Porous Beta-tricalcium phosphate (-TCP) was used as the scaffold and impregnated with collagen gel to generate Collagen Gel Impregnated Porous Scaffolds (CGIPS). Highly efficient seeding of the cells throughout the porous scaffold was attained with collagen gel. hMSC proliferated in CGIPS without contracting the collagen gel. Cells could migrate into CGIPS and mineralized the ECM when cultured in vitro under osteogenic differentiation conditions. CGIPS allowed the application of pressure and hMSC responded to mechanical force by a change in proliferation. hMSC xenotransplanted into immunocompetent rats survived for a month and expressed markers of osteogenic differentiation. While cells alone improved vascularization of the implants, they did not improve mineralization. Presence of collagen gel alone allowed for faster invasion of cells into the implanted TCP and improved radiodensity but did not affect vascularization. A combination of cell and gel within the TCP (CGIPS) was necessary to improve all the measured varialbes (tissue invasion, vascularization, mineralization and radioopacity). Thus biocompatibility, greater vascularization and enhanced mineralization of CGIPS implants established the foundation to proceed with large animal bone defect model studies utilizing CGIPS in the future. I established that CGIPS could deliver small molecules into the surrounding milieu by a process of simple diffusion. A rapid intital burst followed by a slow sustained release was observed when collagen gel containing EphrinB2-Fc clusters was incorporated ointo CGIPS. The released EphrinB2-Fc was physiologically functional and increased hMSC proliferation and chemotaxis. CGIPS inhibited the growth of Methicillin resistant Staphylococcus aureus when vancomycin was incorporated into the CGIPS. Thus the potential of CGIPS to serve as a drug delivery device was demonstrated. This work has provided the scientific foundation for use of CGIPS as bone graft substitute and 3-D model of osteogenesis. In this research study, a number of challenges were solved and questions answered, and the applications of the proposed strategy formulated. However, as is frequently the case many more avenues of future research have been exposed and a variety of new questions posed to be pursued and answered in future.studies.Item Effect of Detergent Selection on Quantity of DNA Obtained and on STR Profile Developed from Bone-Derived DNA(2016-05-01) Proctor, Frances N.; Joseph E. Warren; Bobby L. LaRue; Raghu R. KrishnamoorthyBone is sometimes the only source of DNA in cases of unidentified persons, missing persons, and mass fatality incidents, but bone characteristically provides lower quantities of DNA and lower quality short tandem repeat (STR) profiles than that of other sample types. Bone composition is very different from that of blood and soft tissue, therefore using a method designed for these other sample types is less effective for bone. A bone-specific extraction method is needed in order to improve these results. This study investigated the effects of detergent selection on both the quantity of DNA obtained and on the STR profiles produced from bone-derived DNA, as a part of developing a bone-specific DNA extraction protocol.Item Testing Minimum Ultraviolet Light Exposures to Effectively Remove Contaminating DNA for Use in Forensics(2005-08-01) Kanaly, Angela Catherine; John Planz; Joseph Warren; Arthur EisenbergThis study introduces a solar lamp UV light source, for the purpose of removing contaminating DNA in direct relation to forensic testing. The study attempts to demonstrate what level of decontamination occurs from sun lamp exposure at given time intervals of exposure, set at distances from the lamp, and for different types of biological samples. A FS-40 solar lamp was used to irradiate samples of amplified DNA and cellular samples at distances of 5 cm, 10 cm, and 60 cm from the source, with varied exposure times of 15 min, 30 min, 3 hrs, 6 hrs, 12 hrs and 24 hrs. Common forensic DNA typing concerns include contamination by previously amplified DNA products or from transfer of cellular material onto testing materials. Samples exposed included dried PCR products amplified by AmpFlSTR COfiler kit, dried whole blood, and dried saliva. An organic extraction of the blood and saliva samples isolated any remaining genomic DNA. Control blood and saliva samples were quantitated for accurate DNA concentration. All samples were then amplified by AmpFlSTR COfiler kit and analyzed on an ABI 310 Genetic Analyzer, along with an unexposed control PCR product, blood, and saliva samples, reagent blank run alongside each PCR product, blood, and saliva series, and positive and negative PCR controls. Fragment analysis data was analyzed by GeneScan and Genotyper software to obtain any detectable genetic profile from the samples. This experimental design mimics a true forensic casework scenario by following a routine chain of procedures used widely throughout the field. The current standard in forensic DNA testing measures short tandem repeats (STRs), which vary significantly in length between individuals. There are thirteen loci used by the Combined DNA Index System (CODIS), the national DNA index managed by the FBI Laboratory. All thirteen loci are typed in a typical DNA test, with the AmpFlSTR COfiler kit amplifying seven of these loci. For the purposes of this study, successfully decontaminated PCR products, blood, and saliva samples would show no detectable genotype at any of the seven loci. Other DNA testing, such as mitochondrial DNA analysis from hair, bone or teeth, or very low copy number DNA from a small number of cells, require extreme caution to avoid contamination, as these tests have increased sensitivity over standard STR testing. The level of decontamination detected through UV exposure in this study would not provide sufficient information for application to the more sensitive techniques.Item The Effect of Diet on Craniofacial Growth in Osteogenesis Imperfecta Mouse Model(2018-05) Ladd, Summer H.; Menegaz, Rachel A.; Maddux, Scott D.; Reeves, Rustin E.; Borejdo, JulianOsteogenesis imperfecta (OI, or "brittle bone disease") is a rare disorder that is caused by genetic point mutations (COL1A1/COL1A2) that affect type 1 collagen. In OI type III (severe) patients, limb bones are more susceptible to skeletal fractures and the bones of the craniofacial region are underdeveloped. Some OI type III patients also suffer from dental malocclusions or fractures (dentinogenesis imperfecta, DI). The goals of this project are 1) to describe the facial phenotype in an OI mouse model, to see if this model can be used to test potential behavioral and pharmaceutical interventions; and 2) to determine if diet and masticatory loading affect the development of the craniofacial region in the OI model. The homozygous OI murine (OIM-/-), a mouse strain with a nonlethal recessively inherited mutation of the COL1A2 gene, is a potential model for the human OI type III. OIM-/- and wild type (WT) littermates were raised from weaning (21 days) to adulthood (16 weeks). Digital 3D craniofacial landmarks were taken from in-vivo micro CT scans, and Kuskal-Wallis ANOVAs, along with Mann-Whitney tests, were used to compare centroid size and interlandmark distances among treatment groups. This practicum focuses on the Week 10 mice, with 3 treatment groups: OIMxM, WTxM, and WTxP. We acknowledge that the sample is incomplete due to factors beyond our immediate control, such as OIM-/- survivability. Adolescent OIM-/- mice (week 10) were found to have on average smaller cranial and mandibular centroid sizes compared to WT mice regardless of diet. Week 10 OIM-/- mice also show several morphological similarities to the OI type III human phenotype, such as shortened cranial vault height, shortened jaw length, and altered dental spacing secondary to a shortened tooth row. We conclude that the OIM mouse model shows potential for future investigations of the growth mechanisms underlying the craniofacial presentation of OI. Furthermore, preliminary results suggest that masticatory loading during the early growth period can be used to stimulate craniofacial bone growth and improve bone quality in the OIM mouse model. Future studies will continue to improve sample size by treatment and age groups. The significance of this project is that it will give a better understanding of the role of type 1 collagen and the biomechanical mechanics of craniofacial development, which are important in the search for a new treatment method in OI.