ABSTRAKT:Isolering och karaktärisering av mesenkymala stamceller från periodontalligamentet hos friskatänderSYFTE: Att isolera och odla celler från periodontalligamentet samt karaktärisera dem sommesenkymala stamceller.MATERIAL OCH METOD: Friska premolarer gjordes tillgängliga vid ortodontiskaextraktioner. Den mellersta 1/3 av periodontalligamentet skrapades varpå en enzymatiskmetod användes för isolering av individuella celler. Resulterande celler odlades understandardiserade metoder. Karaktärisering av celler skedde genom flödescymetri med 2 olikapaneler av cellyta markörer; en för etablerat positiva uttryck och en för kända negativauttryck hos mesenkymala stamceller. Möjlighet av celler att differentieras in vitro tilladipocyter och osteocyter testades genom tillförsel av specifika substanser till odlingsmediet.RESULTAT: Celler från 11 av 13 tänder isolerades och odlades framgångsrikt adherenta tillodlingsytan i upp till 8 generationer. Celluttryck av de positiva markörerna CD73, CD90 samtCD44 bekräftades genom flödescymetri. Inget uttryck observerades för den negativa panelenCD45, CD34, CD11b, CD19 eller HLA class II. Uttrycket av CD105 kunde inte fastställas pgaofullständigt data. Försök till differentiering av celler till adipocyter och osteocyter visade påfenotypiska förändringar efter 21 dagar.SLUTSATS: Den här studien har bidragit till framgångsrik isolering och delvis karaktäriseringav mesenkymala stamceller från periodontalligamentet hos friska tänder. En icke-invasivmetod av detta slag, resulterande i tillgång till denna cellpopulation utgör ett lovande verktygför framtida studier med goda möjligheter till ytterligare kunskap applicerbart till kliniskasituationer inom tandvården. / ABSTRACT:Isolation and Characterization of Mesenchymal Stem Cells from the Periodontal Ligament ofHealthy TeethAIM: To isolate and culture viable cells from the periodontal ligament and confirming theiridentity as mesenchymal stem cells.METHODS AND MATERIALS: Healthy premolars were collected at the time oforthodontic extractions. The middle 1/3 of the periodontal ligament was scraped andsubsequent cell isolation was performed using an enzymatic method; yielding single cellisolates. Cells were cultured and maintained under standard culture conditions. Cellcharacterization was performed by flow cytometry using two sets of cell surface markers; oneknown to be present and one known to be absent in mesenchymal stem cells. Ability of thecells for in vitro differentiation into adipogenic and osteogenic lineages was tested usingspecifically formulated media supplements.RESULTS: Cells were successfully isolated from 11 of 13 teeth and were maintained asadherent cultures for up to 8 generations. Cellular expression of positive markers; CD73, CD90and CD44 were confirmed by flow cytometry. For the negative marker panel, expression ofCD45, CD34, CD11b, CD19 and HLA class II were not detectable. The expression of CD105was inconclusive. As determined by phenotypic changes, cells appeared to have undergoneadipogenic and osteocytic differentiation at 21 days.CONCLUSION: This study has resulted in successful isolation and partial characterization ofmesenchymal stem cells from the periodontal ligament of healthy teeth. Non-invasive accessto these cells, provides an excellent tool for future studies, potentially leading to beneficialknowledge transferable to the dental clinical situation.

The purpose of this dissertation was to study the effects of graft pretension in anterior cruciate ligament (ACL) reconstruction through a series of experiments. First, an in vitro study of 5 human knees was conducted to determine if intact joint kinematics could be restored when using the ideal graft - the intrinsic ACL. The ACL tibial insertion site was freed, and pretensions of 0, 10, 20, 30, and 40 N were applied to the ligament using a custom designed load cell connection. Kinematics during a simulated active extension were compared to those of the intact knee. Intact knee kinematics were not restored. Pretensions that best restored tibial anterior/posterior translation and internal/external rotation ranged from 0-40 N. Furthermore, the pretensions that best restored these kinematic variables were widely disparate in two specimens. Second, the in vitro kinematics during a simulated active extension of human and porcine knees were compared and contrasted both prior to and following transection of the ACL. The ACL limited: (1) tibial anterior translation in both species, (2) tibial internal rotation in humans, and (3) tibial external rotation in pigs. Differences in kinematic patterns for tibial internal/external rotation and abduction/adduction between the species was explained by requirements for biped and quadruped stances. Third, the mechanical characteristics of porcine patellar tendon (PT) were investigated by uniaxial tensile testing at two strain rates. Patella-PT-tibia complexes from freshly sacrificed skeletally immature and mature animals were loaded to failure at elongation rates of 20 and 200 mm/min. Both strain rate and skeletal maturity significantly affected failure mode, tangent modulus, and ultimate stress of the tendons, and hence are important considerations in the mechanical evaluation of porcine PT. Fourth, ACL reconstructions were performed using pretensions of 10 or 20 N in an in vivo porcine model with a specially designed load cell/telemetry system to monitor graft load. Graft pretension was seen to increase during fixation with interference screws. Following sacrifice at 4 weeks, tissues were mechanically, histologically, and biochemically analyzed. A pretension of 20 N resulted in a tissue more similar to the intrinsic ACL. / Ph. D.

Partial or complete tears of the anterior cruciate ligament (ACL) can greatly afflict quality of life and often require surgical reconstruction with autograft or allograft tissue to restore native knee biomechanical function. However, limitations exist with these treatments that include donor site pain and weakness found with autografts, and longer "ligamentization" and integration times due to the devitalization of allograft tissue. Alternatively, a tissue engineering approach has been proposed for the fabrication of patient-specific grafts that can more rapidly and completely heal after ACL reconstruction. Electrospun micro-fiber networks have been widely utilized as biomaterial scaffolds to support the growth and differentiation of mesenchymal stem cells toward many tissue lineages including ligament. However, these micro-fiber networks do not possess suitable sizes and shapes for a ligament application and cannot support cell infiltration. The objective of this work was to develop techniques to 1) rapidly cellularize micro-fiber networks, 2) assemble micro-fiber networks into cylindrical composites, 3) provide cues to mesenchymal stem cells (MSCs) to guide their differentiation toward a ligament phenotype. The cellularization of micro-fiber networks was performed utilizing a co-electrospinning/electrospraying technique. Cells deposited within a cell culture medium solution remained where they were deposited and did not proliferate. The inclusion of space-filling hydrogel network such as collagen was necessary to reduce the density of the micro-fiber network to facilitate spreading. However, it became apparent that the incorporation of significant collagen phase was necessary for long-term MSC survival within the micro-fiber network. Next, two approaches were developed to fabricate large cylindrical, composites. The first approach utilized a co-electrospinning/electrospraying technique to generate micro-fiber/collagen composites that were subsequently rolled into cylinders. These cylindrical composites exhibited greater diameters and water weight percentages as collagen content increased. However, the high micro-fiber content of these composites was inhibitory to cell survival. In the second approach, thin layers (~5-10 fibers) of aligned electrospun PEUR fibers were encapsulated within a collagen gel and subsequently rolled the composites into cylinders. These sparse-fiber composites were nearly 98% by weight water and confocal imaging revealed the presence of sparse fiber layers (~5 fibers thick) separated by approximately 200 μm thick collagen layers. We hypothesize that the proliferation and migration of MSCs within these micro-fiber/collagen composites may not be restricted by the presence of a dense, non-manipulatable electrospun fiber network present in traditionally rolled fiber composites. Simple model platforms were then developed to study the influence of sparse micro-fibers on MSCs differentiation within a collagen hydrogel. MSCs in the presence of the softest (5.6 MPa) micro-fibers elongated and oriented to the underlying network and exhibited greater expression of scleraxis, and α-smooth muscle actin compared to the stiffest (31 MPa) fibers. Additionally, preliminary results revealed that the incorporation of fibroblast growth factor-2 and growth and differentiation factor-5 onto micro-fibers through chemical conjugation enhanced expression of the ligamentous markers collagen I, scleraxis, and tenomodulin. In conclusion, micro-fiber/collagen composite materials must possess sufficient space to support the infiltration and differentiation of MSCs. The strategies described in this document could be combined to fabricate large, micro-fiber/collagen composites that can support cell infiltration and provide relevant cues to guide the formation of an engineered ligament tissue. / Ph. D.

Significant cost and debilitation results from connective tissue injury and disease every year. Prolotherapy is an effective medical treatment used to increase joint stability. However, most associated studies are retrospective or case studies, rather than comprehensive laboratory investigation originating with the cellular response to exposure to the proliferant solutions. As a parallel consideration, nanoparticles are being investigated for use in drug delivery and heat shock treatment of cancerous tissue due to their unique structural and thermal properties. The phenomenal strength and stiffness of carbon nanoparticles have been used for commercial purposes in composite materials, but investigation of biomedical applications is still fairly nascent. In an attempt to develop a non-surgical approach to supporting and healing damaged ligaments and tendons resulting from injury or disease by combining prolotherapy and the use of nanoparticles, the author presents studies investigating the cellular response to proliferative therapy solution as well as tendon and ligament tissue's mechanical and cellular response to exposure to nanoparticles. In the prolotherapy solution cell studies, the results suggested that there is an optimal dosage of the proliferant for in vitro studies, different responses between cell types, and a dosage-dependent response in cell viability and collagen production to the solution P2G in preosteoblasts. In the nanoparticle studies, cell populations tolerated nanoparticles at the levels tested, tendon mechanical properties were increased (stiffness significantly so), and bright field and transmission electron microscopic histological images were taken of connective tissue and carbon nanohorn interactions. / Master of Science

Grafts currently used for the repair of anterior cruciate ligament (ACL) ruptures integrate poorly with bone due to a significant mismatch in properties between graft and bone. Specifically, conventional grafts (e.g., hamstring tendon) are unable to recapitulate intricate gradients in mechano-chemical properties and extracellular matrix (ECM) architecture found at natural bone-ligament (B-L) transitions, and thus result in stress-concentrations at the graft-bone interface leading to graft failure. In contrast, tissue-engineered scaffolds possessing gradients in properties can potentially guide the establishment of phenotypic gradients in bone marrow stromal cells (BMSCs), and thus aid the regeneration of B-L transitions in the long-term. Towards the eventual goal of regenerating complex tissue transitions, this project employs three complementary strategies to fabricate graded scaffolds. The three strategies involve the presentation of gradients in 1) mineral content, 2) scaffold architecture and 3) growth factor (GF) concentration within scaffolds to control BMSC morphology and phenotype. The first strategy involved co-electrospinning two polymers (one doped with hydroxyapatite) from offset spinnerets onto a rotating drum to produce scaffolds possessing a gradient in mineral content. Post-electrospinning, these graded scaffolds were treated with a simulated body fluid to further enhance the gradient. Analysis of mRNA expression of osteoblastic makers by BMSCs and the deposition of bone-specific ECM proteins indicated that the scaffolds could guide the formation of an osteoblastic phenotypic gradient. The second strategy involved electrospinning two polymer solutions onto a custom-designed dual-drum collector to fabricate scaffolds possessing region-wise differences in fiber alignment, diameter and chemistry. Specifically, electrospinning onto the dual-drum collector resulted in the deposition of aligned fibers from one polymer solution in the gap region between the drums, randomly oriented fibers from the other polymer solution on one of the drums and a mixture of fibers from both polymer solutions in the overlap region in between. The topographical cues within these scaffolds were shown to result in region-dependent BMSC morphology and orientation. Although the long-term goal of the third strategy was to create a co-electrospun scaffold possessing a gradient in GF concentration, a new technique to protect GF activity within electrospun scaffolds via the use of gelatin microspheres was first validated. Preliminary results from these studies indicate that microspheres can protect and deliver a model protein (lysozyme) in active conformation from electrospun scaffolds. These results further suggest that gradients of GF concentration can be achieved in the long-term by protecting GFs within microspheres and co-electrospinning as described in the first strategy. In conclusion, the results from this project suggest that graded scaffolds can help guide the formation of gradients in cell morphology, orientation and phenotype, and thus potentially promote the regeneration of B-L transitions in the long-term. The three strategies described in this project can be employed in concert to create scaffolds intended for the regeneration of complex tissue transitions. / Ph. D.

Objective: To evaluate the relative position of the femur and tibia in cranial cruciate ligament (CCL) intact stifles, CCL deficient stifles, and stifles following a novel extracapsular procedure (lateral extracapsular suture system or LESSa) under load at specific joint angles. Study Design: In vitro biomechanical study. Methods: Twenty pelvic limbs from 11 dogs were used to evaluate the relative position of the femur and tibia between 3 stifle conditions (CCL intact, CCL deficient, and LESSa treated) at a load of 30 % and stifle angles of 125", 135", and 145" using electromagnetic tracking sensors. Results: Cranial cruciate ligament deficient stifles had significantly greater (p <0.0001) cranial displacement and internal rotation of the tibia relative to the femur than CCL intact stifles or LESSa treated stifles at all stifle angles. Cranial displacement of the tibia relative to the femur for CCL intact and LESSa treated were not significantly different from one another at stifle angles of 125", but were significantly different at stifle angles of 135" (p = 0.0182) and 145" (p = 0.0012). There was no significant difference in internal rotation of the tibia relative to the femur between CCL intact and LESSa treated stifles at any of the stifle angles. Conclusion: LESSa effectively decreases cranial tibial displacement and eliminates internal rotation of the tibia relative / Master of Science

Objectives: To compare optical values in the osteotomy gap created after a Tibial Tuberosity Advancement (TTA) treated with autogenous cancellous bone graft (ACBG), extracorporeal shock wave therapy (ESWT), a combination of ACBG and ESWT, and absence of both ACBG and ESWT using densitometry. Methods: Dogs presenting for surgical repair of a cranial cruciate ligament rupture were randomly assigned to one of four groups; TTA with ACBG (TTA-G), TTA with ACBG and ESWT (TTA-GS), TTA with ESWT (TTA-S), and TTA with no additional therapy (TTA-O). Mediolateral radiographs at 0, 4 and 8 weeks after surgery were evaluated to compare healing of the osteotomy gap via densitometry. An analysis of variance (ANOVA) statistical analysis was used to compare the densitometric values between groups. Results: At 4 weeks after surgery, a significant difference in osteotomy gap density was noted between TTA-GS (8.4 millimeters of Aluminum equivalent [mmAleq]) and TTA-S (6.1mmAleq), and between TTA-GS (8.4 mmAleq) and TTA-O (6.4 mmAleq). There were no significant differences noted between groups at the 8 week recheck. Clinical Significance: There were no significant differences in the osteotomy gap density at 8 weeks after surgery regardless of the treatment modality used. The combination of ACBG and ESWT may lead to increased density of the osteotomy gap in the first 4 weeks after surgery. Densitometry using an aluminum step wedge is a feasible method for comparison of bone healing after TTA in dogs. / Master of Science

Le Laboratoire de génie tissulaire est reconnu pour ses réalisations en ce domaine. Le principal défi soulevé par cette approche est le choix de la matrice des tissus reconstruits. Mes travaux ont consisté à établir une technologie de synthèse de collagène humain recombinant à des fins expérimentales et cliniques. Ce collagène sera utilisé éventuellement pour produire des substituts du ligament croisé antérieur (LCA) du genou, par génie tissulaire. Ces substituts ligamentaires pourraient être une alternative de remplacement des LCA rupturés. Le Dr. Nazrul Islam a conceptualisé une stratégie moléculaire pour construire un plasmide incluant les gènes codant pour les deux chaînes du collagène humain de type 1 et les deux sous-unités de l'enzyme prolyl-4-hydroxylase. Des cellules d'insecte ont été transfectées avec ce vecteur, en exploitant le système de bacul ovi rus, pour synthétiser le collagène recombinant. J'ai participé à chaque étape et à la mise au point des protocoles optimisé à grande échelle de cette nouvelle technologie, pour ensuite purifier le collagène et le caractériser biochimiquement. Mes superviseurs et moi-même considérons que ces travaux ont réussi et que bientôt, des substituts ligamentaires humains seront greffés pour évaluer leur intégration dans une articulation du genou in vivo.

<p>Uvod: magnetno rezonantni imidžing (MR) je u &scaron;irokoj uporebi u dijagnostici patolo&scaron;kih poremećaja ekstremiteta. Postoji interes da se redukuje vreme trajanja snimanja tokom pregleda kako bi se povećao komfor pacijenata i redukovali problemi koji su u vezi sa klaustrofobijom i artefaktima koji nastaju pri pomeranju pacijenta tokom dugih snimanja. Trodimenzionalne (3D) izovoksel sekvence pokazale su značajne mogućnosti u redukciji vremena snimanja MR pregledom zgloba kolena bez smanjenja kvaliteta dijagnostičkih mogućnosti. Cilj: uporediti dijagnostičke mogućnosti rutinskog, 2D MR protokola, na 3.0 Tesla MR aparatu, sa 3D True fast imaging, TrueFISP, izovoksel sekvencom u detekciji hondralnih o&scaron;tećenja, ruptura prednjeg ukr&scaron;tenog ligamenta (LCA), ruptura meniskusa i abnormalnosti subhondralne kosti, u korelaciji sa artroskopijom, kao referentnim standardom. Materijal i metode: Studija je odobrena od institucionalnog etičkog odbora. Pacijenti su informisani o studiji i potpisali pristanak za uče&scaron;će u istraživanju. 76 zgloba kolena kod 76 pacijenata (34 ženskog pola; prosečne starosti 36 godina) uključeno je u prospektivnu studiju, snimljeno je standardnim 2D MR protokolom snimanja i 3D TrueFISP sekvencom u sagitalnoj ravni. Svim pacijentima je urađena artroskopija u periodu od maksimum 30 dana nakon snimanja. Dva radiologa, nezavisno jedan od drugog, su evaluirala dobijene snimke. Preciznost u detekciji hondralnih o&scaron;tećenja, ruptura LCA i meniskusa, i abnormalnosti subhondralne kostne srži je određena podudarno&scaron;ću radiolo&scaron;kih i artroskopskih nalaza kao i proverom podudaranja ove dve metode snimanja. Rezultati: prosečna senzitivnost i specifičnost 3DTrueFISP sekvence je bila 75%, 94% respektivno, u dijagnostici o&scaron;tećenja hrskavice, 97%, 97% u detekciji ruptura LCA, 65%, 89% u postavljanju dijagnoze ruptura meniskusa, a dobra podudarnost radiolo&scaron;kih nalaza je dobijena u interpretaciji o&scaron;tećenja subhondralne kosti. Standardnim 2D MR protokolom prosečna senzitivnost i specifičnost je bila 70%, 93%, respektivno, u postavljanju dijagnoze hondralnih o&scaron;tećenja, 94% i 100% u postavljanju dijagnoze rupture LCA, 65%, 88% u detekciji ruptura meniskusa. Zaključak: dijagnostičke mogućnosti upotrebom 3DTrueFISP sekvence su uporedive sa standardnim, 2D, MR protokolom u snimanju zgloba kolena.</p> / <p>Introduction: Magnetic resonance imaging (MR) is widely used to assess internal derangements of the extremities. There is an interest in decreasing examination times to improve patient comfort and reduce the problems related to claustrophobia and motion artifacts, which occur more commonly with long examinations. The three-dimensional (3D) isovoxel true FISP sequence facilitates a noticeable reduction in acquisition time for MR imaging of the knee without reducing diagnostic performance. Objective: To compare the diagnostic performance of conventional, 2D, MR protocol, at 3.0 Tesla MR, with 3D water-excitation true fast imaging with steady-state precession, TrueFISP, an isotropic resolution sequence for detecting articular cartilage defects, anterior crucial ligament tears, meniscal tears and subhondral bone changes of the knee joint, with arthroscopy as reference standard. Materials and methods: The study was institutional review board approved. Written informed consent was obtained from all patients. 76 knees of 76 patients (34 females; mean age 36 years) were prospectively examined by using a conventional 2D MR protocol and sagittal 3D TrueFISP sequence. All patients underwent arthroscopy within a maximum period of 30 days after imaging. Two blinded readers evaluated the MR images. Accuracy for detection of cartilage defects, anterior cruciate ligament and meniscal tears, and subchondral bone abnormalities interobserver agreement, and intermethod agreement were calculated. Results: Overall sensitivity and specificity of 3DTrueFISP sequence were, respectively, 75%, 94% for diagnosis cartilage defects, 97%, 97%, for a diagnosis ACL tears, 65%, 89% for diagnosis of meniscal tears and with good interobserver agreement in interpretation of subhondral bone abnormalities. The standard MR protocol had overall sensitivities and specificities 70%, 93%, respectively, for diagnosis of cartilage defects, 94% and 100% for diagnosis ACL tears and 65%, 88% for diagnosis meniscal tears. Conclusion: The diagnostic perfomance of knee MR imaging performed by using a 3DTrueFISP sequence is comparable to the diagnostic performance of the conventional, 2D, MR protocol.</p>

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