Peripheral nerve injuries are often associated with significant loss of nervous tissue leading to poor restoration of function following repair of injured nerves. Although the injury gap could be bridged by autologous nerve graft, the limited access to donor material and additional morbidity such as loss of sensation and scarring have prompted a search for biosynthetic nerve transplants. The present thesis investigates the effects of a synthetic matrix BD™ PuraMatrix™ peptide (BD)hydrogel, alginate/fibronectin gel and fibrin glue combined with cultured rat Schwann cells or human bone marrow derived mesenchymal stem cells (MSC) on neuronal regeneration and muscle recovery after peripheral nerve injury in adult rats. In a sciatic nerve injury model, after 3 weeks postoperatively, the regenerating axons grew significantly longer distances within the conduits filled with BD hydrogel if compared with the alginate/fibronectin gel. The addition of rat Schwann cells to the BD hydrogel drastically increased regeneration distance with axons crossing the injury gap and entering into the distal nerve stump. However, at 16 weeks the number of regenerating spinal motoneurons was decreased to 49% and 31% in the BD hydrogel and alginate/fibronectin groups respectively. The recovery of the gastrocnemius muscle was also inferior in both experimental groups if compared with the nerve graft. The addition of the cultured Schwann cells did not further improve the regeneration of motoneurons and muscle recovery. The growth-promoting effects of the tubular conduits prepared from fibrin glue were also studied following repair of short and long peripheral nerve gaps. Retrograde neuronal labeling demonstrated that fibrin glue conduit promoted regeneration of 60% of injured sensory neurons and 52% of motoneurons when compared with the autologous nerve graft. The total number of myelinated axons in the distal nerve stump in the fibrin conduit group reached 86% of the nerve graft control and the weight of gastrocnemius and soleus muscles recovered to 82% and 89%, respectively. When a fibrin conduit was used to bridge a 20 mm sciatic nerve gap, the weight of gastrocnemius muscle reached only 43% of the nerve graft control. The morphology of the muscle showed a more atrophic appearance and the mean area and diameter of fast type fibres were significantly worse than those of the corresponding 10 mm gap group. In contrast, both gap sizes treated with nerve graft showed similar fiber size. The combination of fibrin conduit with human MSC and daily injections of cyclosporine A enhanced the distance of regeneration by four fold and the area occupied by regenerating axons by three fold at 3 weeks after nerve injury and repair. In addition, the treatment also significantly reduced the ED1 macrophage reaction. At 12 weeks after nerve injury the treatment with cyclosporine A alone or cyclosporine A combined with hMSC induced recovery of the muscle weight and the size of fast type fibres to the control levels of the nerve graft group. In summary, these results show that a BD hydrogel supplemented with rat Schwann cells can support the initial phase of neuronal regeneration across the conduit. The data also demonstrate an advantage of tubular fibrin conduits combined with human MSC to promote axonal regeneration and muscle recovery after peripheral nerve injury.

<p>Cancer metastasis is the cause of about 90% of cancer patients' deaths. Despite significant improvements in the past three decades in understanding the molecular bases of oncogenic transformation of cancer cells, little is known about the molecular mechanisms underlying tumour cells' alteration of their microenvironment, entrance into the circulation, and colonization of distant organs. In recent years, accumulating evidence has indicated that tumour microenvironment, which consists of a variety of cell types and extracellular matrix components&#65292;plays an important role in regulating the metastatic abilities of carcinoma cells. Co-opted by cancer cells, those stromal cells promote tumour progression via multiple mechanisms, including enhancement of tumour invasiveness, elevation of angiogenesis, and suppression of immune surveillance activity. </p><p>Using a series of human breast cancer cell lines with different metastatic potentials <italic>in vivo</italic>, we performed an unbiased screen examining expression of miRNAs, and found that miR-126 and miR-126*, whose expression are regulated by methylation of the promoter of their host gene Egfl7 inside tumour cells, were significantly negatively correlated with metastatic potential. Using both mouse xenograft models and <italic>in vitro</italic> assays, we showed that this pair of miRNAs suppressed breast cancer metastasis through shaping the tumour microenvironment without changing tumour cell autonomous properties. Specifically, miR-126 and miR-126* act independently to suppress the sequential recruitment of mesenchymal stem cells (MSCs) and inflammatory monocytes into the primary tumour stroma, consequently inhibiting lung metastasis by breast tumour cells. Mechanistically, these miRNAs directly inhibit the production of stromal cell-derived factor-1 alpha (Sdf-1&alpha;, also known as Cxcl12), and indirectly suppress the expression of chemokine (C-C motif) ligand 2 (Ccl2) by the cancer cells within the tumour mass in an Sdf-1&alpha;-dependent manner. In addition, in contrast with the majority of reports which have shown incorporation of only the guiding strand of the miRNA duplex into the mRNA-targeting RNA induced silencing complex (RISC), both strands of the miR-126 RNA duplex are maintained at a similar level and suppress Sdf-1&alpha; expression independently. </p><p>Collectively, we have determined a dynamic process by which the composition of the primary tumour microenvironment could be altered via a change in the expression of two tumour-suppressive miRNAs derived from a single miRNA precursor to favor metastasis by breast cancer cells. Importantly, this work provides a prominent mechanism to explain the clinical correlation between reduced expression of miR-126/126* and poor metastasis-free survival of breast cancer patients.</p> / Dissertation

It has been shown that stem cells exist within adult adipose tissue. These stem cells are named adipose-derived mesenchymal stem cells (ASCs), are derived from the mesoderm, and can differentiate into a number of cells including osteoblasts, chondrocytes, and adipocytes. However, before these cells can be used clinically it is important that we understand how factors like age, sex, and ethnicity affect ASC number and potential. Additionally, since men and women vary in their distribution of adipose tissue, it will be important to see if the ideal source of ASCs is different for each sex. The goal of this study was to assess how age and sex affects ASCs. We used flow cytometry to investigate how age and sex affected the number of ASCs in adipose tissue. Additionally, we plated these cells in culture and treated them with an osteogenic media (OM) with the intention of pushing them towards osteoblast differentiation. The purpose of this was to see if age or sex affected the potential of the ASCs to undergo osteogenesis in culture. For this study we used real-time PCR and biochemical assays to look at markers of early and late osteogenic differentiation. Finally, we used immunohistochemistry to demonstrate where in adipose tissue the CD73 and CD271 positive cell population exists. It is our hope that this work will shed light on how age and sex affect ASCs so that clinicians can optimize their ASC harvest depending on the patient's physiology.

As the prevalence of osteoporosis is expected to increase over the next few decades, the development of novel therapeutic strategies to combat this disorder becomes clinically imperative. These efforts draw extensively from an expanding body of knowledge pertaining to the physiologic mechanisms of skeletal homeostasis. To this body of knowledge, we contribute that cells of hematopoietic lineage may play a crucial role in balancing osteoblastic bone formation against osteoclastic resorption. Specifically, our laboratory has previously demonstrated that megakaryocytes can induce osteoblast proliferation in vitro, but do so only when direct cell-to-cell contact is permitted. To further investigate the nature of this interaction, we have effectively neutralized several adhesion molecules known to function in the analogous interaction of megakaryocytes with another cell-type of mesenchymal origin - the fibroblast. Our findings implicate the involvement of fibronectin/RGD-binding integrins including á3â1 (VLA-3) and á5â1 (VLA-5) as well as glycoprotein IIb (CD41), all of which are known to be expressed on megakaryocyte membranes. Furthermore, we demonstrate that IL-3 can enhance megakaryocyte-induced osteoblast activation in vitro, as demonstrated in the megakaryocyte-fibroblast model system. Taken together, these results suggest that although their physiologic and clinical implications are very different, these two models of hematopoietic-mesenchymal cell activation are mechanistically analogous.

This study investigated the effect of bone marrow derived mesenchymal stem cells (MSCs) in a PEGylated fibrin gel (PEG) seeded into a decellularized extracellular matrix (ECM) on recovery of skeletal muscle following a volumetric muscle loss (VML) injury. Six to nine month old male Sprague-Dawley rats were used in this study. Approximately one-third of the skeletal muscle mass of the lateral gastrocnemius (LGAS) was removed from the LGAS, which was immediately replaced with an acellular ECM of the same dimensions. Seven days after injury, animals were injected with one of four solutions: saline (SAL), MSCs (MSC), PEGylated fibrin hydrogel (PEG), or MSCs in PEG (PEG+MSC). Maximal isometric tetanic tension (Po) of the LGAS was assessed fifty-six days after VML injury, followed by histological evaluation. VML injury resulted in a functional impairment of the LGAS capable of producing 76.1± 4.9% of the force generated in the non-injured contralateral LGAS. Tetanic tension of the PEG+MSC treated group was significantly higher compared to all other treatment groups (p < 0.05), although specific tension (N/cm2) in the PEG+MSC group (79.7±4.0%) was only significantly higher compared to SAL (58.2±3.0) and PEG (64.0±2.1%) treated groups (p < 0.05). However, LGAS mass was significantly higher in the PEG+MSC group compared to all other groups (p < 0.05). These findings suggest the combination of the PEG+MSC did not lead to a significant increase in muscle function compared to MSC treatment alone, and demonstrates the importance of MSCs in skeletal muscle regeneration in VML injury models. However, as evident by the significant increase in LGAS mass, PEG+MSC treatment may lead to histological differences not evaluated in this study. Gross morphology of the repaired gastrocnemius was indistinguishable from the contralateral control. / text

I developed a system to release multiple growth factors from PEGylated fibrin gels with varying profiles to induce vasculogenesis from embedded human MSCs. Zero-order release can be obtained by conjugating a growth factor with a homobifunctional, amine-reactive, PEG derivative. Growth factors can be entrapped during thrombin-mediated crosslinking and released rapidly. Growth factors with physical affinity for fibrinogen or fibrin can be sequestered within the matrix and released via degradation and/or disassociation. PDGF-BB was loaded via entrapment while TGF-β1 was sequestered through a combination of physical affinity and conjugation. The affinity of TGF-β1 and fibrinogen had never been previously examined or quantified. I aimed to determine the Ka and Kd between TGF-β1 and fibrinogen through a variety of assays. Binding ELISAs were developed for TGF-β1 and fibronectin, a protein associated with fibrin gels, and TGF-β1 and fibrinogen. However, background was high due to insufficient blocking agents. Other assays explored included western blots, surface plasmon resonance, and radiolabeled TGF-β1 with limited success. The affect of TGF-β1 on human MSC differentiation towards vascular cell phenotypes was examined both in 2D and fibrin gels embedded with MSCs. With exposure to TGF-β1, MSC proliferation was significantly inhibited in both 2D and within fibrin gels indicating that loaded TGF-β1 maintained bioactivity for at least 7 days. Gene expression of MSCs exposed to TGF-β1 demonstrated inhibited endothelial cell differentiation and stimulated smooth muscle cell differentiation. However, confocal and light microscopy indicated that endothelial cell differentiation is maintained with TGF-β1 loaded PEGylated fibrin gels. The system developed is highly modular and can be applied to other tissue engineering systems. Furthermore, other growth factors could be incorporated to promote vascular cell differentiation. / text

No description available.

No description available.

Baigiamajame darbe įvertintas skirtingų titano implantų paviršiaus modifikavimo strategijų poveikis pirminių dantenų fibroblastų savybėms – adhezijai, proliferacijai ir diferenciacijai. Tyrimuose naudoti titano mėginiai, kurių paviršiaus šiurkštumas pakeistas fizikiniais metodais – smėliasrove ir lazeriu. Iš gingivektomijos metu paimto ţmogaus dantenų subepitelinio audinio buvo išskirtos ir charakterizuotos dantenų fibroblastų ląstelės. Tiriant šių ląstelių adheziją ant modifikuotų titano mėginių paviršių nustatyta, kad ankstyvuoju laikotarpiu dantenų fibloblastų adhezija buvo panaši ant visų tirtų įvairiai modifikuotų titano mėginių paviršių, tačiau vėliau efektyviausias ląstelių prikibimui paviršius buvo lazeriu suformuotos grotelės. Siekiant pagerinti modifikuotų titano paviršių adhezines savybes, jie papildomai buvo padengti tarpląstelinio uţpildo baltymais – kolagenu ir lamininu, įvertintas tokio padengimo poveikis ląstelių prikibimui ir augimui. Analizuojant mechanizmus, reguliuojančius ląstelių adhezijos ant modifikuotų paviršių procesus, buvo tirta FAK ir Akt kinazių raiška ir aktyvinimas. Vertinant karkasų paviršiaus topografijos poveikį ląstelių diferenciacijai, buvo palygintas osteogeninės diferenciacijos laipsnis ląstelėse augintose ant įvairiai modifikuotų titano mėginių paviršių. Darbą sudaro 6 dalys: įvadas, literatūros apţvalga, medţiagos ir metodai, rezultatai ir jų aptarimas, išvados, literatūros sąrašas. Darbo apimtis – p. teksto be priedų, 24 pav., 2 lent... [toliau žr. visą tekstą] / The influence of different modification strategies of titanium implant surfaces on gingival fibroblasts properties - adhesion, proliferation, and differentiation was studies in this final master thesis. Different titanium surface roughness modifications using physical methods such as sand-blasting as well as laser irradiation were developed. Gingival fibroblasts derived from human gingival subepithelial tissues obtained during gingivectomy were isolated and characterized. The data suggested that the initial adhesion between tested cells and various modified titanium surfaces was similar, but the most efficient surface for subsequent cell attachment was laser-ablated holey arranged in grid-like structures. Additionally, in order to improve the modified titanium surface adhesion properties, these surfaces were coated by extracellular matrix proteins - collagen and laminin. The coating influence on the cell growth and adhesion was evaluated. To analyze the mechanisms regulating cell adhesion processes on the modified surfaces, FAK and Akt kinase expression as well as activation were studied. In order to evaluate the effect of surface topography on cell differentiation, the level of osteogenic differentiation was compared. Structure: introduction, literature review, materials and methods, results and discussion, conclusions, references.

The objective of the present study was to synthesize porous, biodegradable poly (D, l- lactide-co-glycolide) PLGA-B-HA (Bovine hydroxyapatite) composite and evaluate the effect of ceramic content on bone marrow cell differentiation in vitro. A macroporous biodegradable PLGA-B-HA composite with the pore size varying from 0.1 to 1000?? and a highly interconnected structure was fabricated using the freeze-drying/lyophilization technique. A pilot study was done to determine the effects of B-HA on to the osteoblast function. The main study was done to determine the effect of the increase in B-HA concentration on to the mesenchymal stem cell differentiation. Morphological characteristics of the composites were analyzed using FTIR and SEM/EDX analysis. The composites were seeded with neonatal rat calvarial osteoblasts (NRCO). The polymer: ceramic ratio in this study was 35%:65%. For comparison parallel experiments involving pure HA-200 discs were performed. SEM results indicated a higher proliferation and mineralization on PLGA-B-HA composites than pure HA discs. In addition, we evaluated the in vitro characteristics of PLGA-B-HA composites with varying ratios, i.e., 1:1, 1:2 and 1:3, seeded with rat marrow cells. FTIR indicated an increase in the area under the ceramic peak as ceramic concentration was increased. In addition, the average roughness values increased in the order of 1:3 andgt; 1:2 andgt; 1:1. Both compressive strength and modulus of 1:1 were significantly higher than 1:2 and 1:3 PLGA-B-HA composites. No significant difference in compressive modulli and strengths could be observed for 1:2 and 1:3 PLGA-B-HA composites. Cellular activity was determined by measuring AP activity, total protein analysis and osteocalcin concentration. Evaluation of alkaline phosphatase activity showed bone cells attached to 1:3 (PLGA-B-HA) expressed significantly higher alkaline phosphatase as compared to 1:1 and 1:2 PLGA-B-HA composites. In addition, cells seeded on to 1:3 composites secreted significantly higher osteocalcin and at a relatively short time period as compared to the other samples. Corrosion studies (ICP) and pH values indicate minimal difference in the concentration of Ca and P and pH in tissue culture media for all the samples at the end of all time periods. Hence we conclude that an increase in the ceramic concentration stimulated mesenchymal stem cell differentiation thereby promoting osteogenesis.