No description available.

Tissue engineering and ex vivo gene therapy can be used synergically as tool to regenerate bone, which overcome the problems of currently available bone replacements. Recently, a new source of mesenchymal stromal cells (MSCs) has been found in the umbilical cord; human umbilical cord perivascular cells (HUCPVCs) provide an alternative to bone marrow derived MSCs and due to their easy harvest, fast expansion, and non-immunogeneic and immunomodulatory phenotype we hypothesized that HUCPVCs are a putative candidate cell source for osteogenic ex vivo gene therapy. This work proposes the generation of cocktails of genetically modified HUCPVCs and their cryopreservation as an “off the shelf” therapeutic. This approach involves the engineering of osteogenic cell populations, by genetically modifying HUCPVCs using recombinant adenoviruses to deliver four fundamental genes for bone formation: bone morphogenetic protein 2 (BMP-2), runt-related transcription factor 2 (Runx2), Osterix (OSX/SP7) transcription factor and vascular endothelial growth factor (VEGF). Our results show that HUCPVCs can be efficiently modified by adenoviruses and can be cryopreserved without affecting the production efficiency and bioactivity of proteins of interest produced by the cells. Moreover, overexpression of BMP2, Runx2 and SP7 enhances ALP activity levels in HUCPVCs and upregulates ALP, OPN, COL1A1 and OCN gene expression; data that provides the first evidence of the effects of combinational expression of BMP2, Runx2 and SP7. Furthermore, we report for the first time the genetic modification of human BMSCs to express SP7 and Runx2, which enhances their ALP activity and matrix mineralization capacity.

Background. Recently rodent and porcine bone marrow stromal cells (MSCs) have been reported to be uniquely immune tolerant. In order to confirm these findings in human cells, we tested the hypothesis that human MSCs are also immune tolerant, such that they can be useful as "universal donor cells" for myocardial regenerative therapy. / Methods. Immunocompetent female rats underwent left coronary ligations (n=90). They were randomized into 3 groups. In Group I, lac-Z labeled male human MSCs were implanted into the peri-infarcted area. In Group II and III isogenic rat MSCs or culture medium were injected respectively. Echocardiography was carried out to assess cardiac function, and the specimens were examined serially for up to 8 weeks with immunohistochemistry, FISH and PCR to examine MSCs survival and differentiation. / Results. Human MSCs were found to survive within the rat myocardium without immunosuppression. This was confirmed by PCR and FISH test. No cellular infiltration characteristic of immune rejection was noted. Some of these cells appeared to express cardiomyocyte-specific markers such as troponin-Ic and connexin-43. Furthermore, the implanted MSCs significantly contributed to the improvement in ventricular function and attenuated LV remodeling. / Conclusions. Human MSC survived within this xenogeneic environment, and contributed to the improvement in cardiac function. Our findings support the feasibility of using these cells as "universal donor cells" for xeno- or allo-geneic cell therapy, as they can be tested, prepared and stored well in advance for urgent use. Allogeneic MSCs from healthy donors may be particularly useful for severely ill or elderly patients whose own MSCs could be dysfunctional. / Plusieurs études ont récemment démontré la tolérance immunologiquedes cellules souches stromales (CSS) issues de rongeurs et de porcinés. Pour confirmer cesrésultats chez les cellules humaines, l'étude actuelle évalue l'effet des CSS humaines sur larégénération du myocarde chez des rats immunocompétents et étudie la possibilité d'utiliserces CSS comme « donatrices universelles» à la suite d'un infarctus.

Tissue engineering and ex vivo gene therapy can be used synergically as tool to regenerate bone, which overcome the problems of currently available bone replacements. Recently, a new source of mesenchymal stromal cells (MSCs) has been found in the umbilical cord; human umbilical cord perivascular cells (HUCPVCs) provide an alternative to bone marrow derived MSCs and due to their easy harvest, fast expansion, and non-immunogeneic and immunomodulatory phenotype we hypothesized that HUCPVCs are a putative candidate cell source for osteogenic ex vivo gene therapy. This work proposes the generation of cocktails of genetically modified HUCPVCs and their cryopreservation as an “off the shelf” therapeutic. This approach involves the engineering of osteogenic cell populations, by genetically modifying HUCPVCs using recombinant adenoviruses to deliver four fundamental genes for bone formation: bone morphogenetic protein 2 (BMP-2), runt-related transcription factor 2 (Runx2), Osterix (OSX/SP7) transcription factor and vascular endothelial growth factor (VEGF). Our results show that HUCPVCs can be efficiently modified by adenoviruses and can be cryopreserved without affecting the production efficiency and bioactivity of proteins of interest produced by the cells. Moreover, overexpression of BMP2, Runx2 and SP7 enhances ALP activity levels in HUCPVCs and upregulates ALP, OPN, COL1A1 and OCN gene expression; data that provides the first evidence of the effects of combinational expression of BMP2, Runx2 and SP7. Furthermore, we report for the first time the genetic modification of human BMSCs to express SP7 and Runx2, which enhances their ALP activity and matrix mineralization capacity.

Bone marrow-derived mesenchymal stem cells (MSC) have been shown to provide neuroprotection after transplantation into the injured nervous system. The present thesis investigates whether adult human and rat MSC differentiated along a Schwann cell lineage could increase their expression of neurotrophic factors and promote regeneration after transplantation into the injured peripheral nerve and spinal cord. Human and rat mesenchymal stem cells (hMSC and rMSC) expressed characteristic stem cell surface markers, mRNA transcripts for different neurotrophic factors and demonstrated multi-lineage differentiation potential. Following treatment with a cocktail of growth factors, the hMSC and rMSC expressed typical Schwann cells markers at both the transcriptional and translational level and significantly increased production of brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF). Age and time in culture are of relevance for clinical settings and growth-promoting effects of hMSC from young donors (16-18 years) and old donors (67-75 years) were compared. Undifferentiated hMSC from both young and old donors increased total neurite length of cultured dorsal root ganglion (DRG) neurons. Differentiation of hMSC from the young donors, but not the eldery donors, further enhanced the neurite outgrowth. Undifferentiated hMSC were cultured for eleven weeks in order to examine the effect of in vitro expansion time on neurite outgrowth. hMSC from the young donors maintained their proliferation rate and their ability to enhance neurite outgrowth from DRG neurons. Using a sciatic nerve injury model, a 10mm gap was bridged with either an empty tubular fibrin glue conduit, or conduits containing hMSC, with and without cyclosporine treatment. Cells were labeled with PKH26 prior to transplantation. At 3 weeks after injury the conduits with cells and immunosuppression increased regeneration compared with an empty conduit. PKH26 labeled human cells survived in the rat model and the inflammatory reaction could be suppressed by cyclosporine. After cervical C4 hemisection, BrdU/GFP-labeled rMSC were injected into the lateral funiculus rostral and caudal to the spinal cord lesion site. Spinal cords were analyzed 2-8 weeks after transplantation. Transplanted MSC remained at the injection sites and in the trauma zone for several weeks and were often associated with numerous neurofilament-positive axons. Transplanted rMSC induced up-regulation of vascular endothelial growth factor in spinal cord tissue rostral to the injury site, but did not affect expression of brain-derived neurotrophic factor. Although rMSC provided neuroprotection for rubrospinal neurons and significantly attenuated astroglial and microglial reaction, cell transplantation caused aberrant sprouting of calcitonin gene-related peptide immunostained sensory axons in the dorsal horn. In summary these results demonstrate that both rat and human MSC can be differentiated towards the glial cell lineage, and show functional characteristics similar to Schwann cells. hMSC from the young donors represent a more favorable source for neurotransplantation since they maintain proliferation rate and preserve their growth-promoting effects in long-term cultures. The data also suggest that differentiated MSC increase expression of neurotrophic factors and support regeneration after peripheral nerve and spinal cord injury.

Malignant glioma is a highly lethal and destructive disease with no proper cure. We have investigated some of the hallmarks of cancer in connection to glioma and found ways to disrupt these and prevent tumor growth. The work is done within the context of a glioma subtype distinguished by activation of PDGF signaling termed the proneural subtype. In two of the studies we have investigated mechanisms regulating the glioma cells themselves, and in the other two we have focused on the tumor stroma. In the first study, glioma-initiating cells were isolated in defined serum free culture medium from PDGF-B driven murine glioma and shown to be independent of EGF and FGF2 for self-renewal and proliferation. When cultured in serum the GICs displayed an aberrant differentiation pattern that was reversible. Specific depletion of the transduced PDGF-B caused a loss of self-renewal and tumorigenicity and induced oligodendrocyte differentiation. The transcription factor S-SOX5 has previously been shown to have a tumor suppressive effect on PDGF-B induced murine glioma, and to induce cellular senescence in PDGF-B stimulated cells in vitro. We found that S-SOX5 had a negative effect on proliferation of newly established human glioma cells cultured under stem cell conditions. We also revealed a connection between alterations causing up-regulation of SOX5 with the proneural subgroup and a tendency towards co-occurrence with PDGFRA alterations. Angiogenesis, the formation of new blood vessels from existing ones, is an important hallmark for glioma malignancy. We found that the anti-angiogenic protein HRG had a negative effect on glioma progression in PDGF-B induced experimental tumors and that HRG was able to completely prevent formation of glioblastomas. Subsequently it was shown that HRG could skew pro-tumorigenic tumor associated macrophages into an anti-tumorigenic phenotype. Stromal cells had not previously been fully investigated in gliomas. We observed a correlation between tumor malignancy and increased numbers of tumor-associated macrophages as well as pericytes in PDGF-B induced gliomas. There was also a correlation between tumor grade and vessel functionality that had not previously been shown. Our results offer further understanding of gliomagenesis and present possible future therapies.

Diss. (sammanfattning) Linköping : Linköpings universitet, 2008. / Härtill 5 uppsatser.

Submitted by Maria Anália Conceição (marianaliaconceicao@gmail.com) on 2016-06-22T18:00:41Z No. of bitstreams: 1 Publico-novo-01.pdf: 1012668 bytes, checksum: e3115efd76da55d6f5c2b7ab64ba97cc (MD5) / Approved for entry into archive by Maria Anália Conceição (marianaliaconceicao@gmail.com) on 2016-06-22T18:02:06Z (GMT) No. of bitstreams: 1 Publico-novo-01.pdf: 1012668 bytes, checksum: e3115efd76da55d6f5c2b7ab64ba97cc (MD5) / Made available in DSpace on 2016-06-22T18:02:06Z (GMT). No. of bitstreams: 1 Publico-novo-01.pdf: 1012668 bytes, checksum: e3115efd76da55d6f5c2b7ab64ba97cc (MD5) Previous issue date: 2014 / Introdução: A reconstrução óssea de áreas consideradas críticas pode ser realizada com o uso de biomateriais substitutos do tecido ósseo como, por exemplo, os materiais xenógenos, porém sabe-se que uma das limitações destes materiais é a sua incapacidade de ser osteogênico. De outra forma, frequentemente nestes procedimentos são utilizadas membranas ou barreiras teciduais, caracterizando uma regeneração óssea guiada, com o intuito de se obter resultados com maior previsibilidade. Objetivo: Avaliar a associação do aspirado de medula óssea a um enxerto ósseo xenógeno na técnica de regeneração óssea guiada em calvária de coelhos. Métodos: Dez coelhos foram distribuídos aleatoriamente em dois grupos com cinco animais cada. Vinte defeitos ósseos críticos bilaterais, com diâmetro de 12 milímetros, foram criados nos ossos parietais. Foram preenchidos com xenoenxerto ósseo, associado ou não à medula óssea, e sempre um dos defeitos em cada animal realizado de forma aleatória, recoberto por uma membrana de colágeno, formando assim os seguintes grupos: grupo controle com membrana (GC CM), grupo controle sem membrana (GC SM), grupo experimental com membrana (GE CM) e grupo experimental sem membrana (GE SM). Os coelhos foram eutanasiados oito semanas após a cirurgia e, em seguida, as suas calvárias foram removidas e analisadas por tomografia computadorizada. Na sequência, os ossos parietais foram removidos e as áreas de enxertia foram selecionadas e analisadas histologicamente por histomorfometria. Resultados: A tomografia mostrou uma menor área de defeito ósseo remanescente (p<0,05) no grupo experimental coberto com membrana de colágeno (56,46 ± 9,05 mm2 ), quando comparado com o grupo controle, com e sem membrana (68,01 ± 2,86 mm2 e 72,64 ± 5,59 mm2 , respectivamente). A histomorfometria não mostrou diferença entre os grupos (p> 0,05) em relação à área de tecido mineralizado não vital. Para a área de tecido mineralizado vital, o grupo experimental coberto com membrana obteve uma área maior (21,16 ± 3,76% ), quando comparado com o grupo controle, com e sem membrana (12,78 ± 5,9% e 6,31 ± 1,29%, respectivamente) (p <0,05 ). Para a área de tecido não mineralizado, o grupo experimental coberto com membrana obteve uma área menor (20,29 ± 4,17%), quando comparado com o grupo controle, com e sem membrana ( 67,95 ± 7,03% e 81,41 ± 3,25% , respectivamente) (p <0,05). Conclusão: A associação do aspirado de medula óssea a um enxerto xenógeno na regeneração óssea guiada em calvária de coelhos, aumentou significativamente a quantidade de tecido mineralizado vital, bem como diminuiu significativamente a quantidade de tecido não mineralizado. / Objective: The aim of this study was to evaluate the bone healing after the usage of a scaffold enriched with bone marrow. Methods: Ten rabbits were randomly divided into 2 groups of 5 animals. Bilateral 12 mm diameter defects were created in the parietal bones. In control group Bio-Oss® were inserted in both defects and, in experimental group, xenograft enriched with autologous bone marrow were inserted in both defects. In these two groups, one of the calvarial defects was randomly covered with collagen membrane. The rabbits were sacrified 8 weeks after surgery, their heads were removed and analysed by CT. Then, their parietal bones were harvested and analysed by histomorphometrics. Results: The CT showed a lower remaining defect area (p<0.05) in the experimental group covered with collagen membrane (56.46 + 9.05 mm2) when compared with control group, with and without xenograft (68.01 + 2.86 mm2 and 72.64 + 5.59 mm2, respectively). The histomorphometrics showed no difference between groups (p>0.05) regarding the non-vital mineralized tissue area. For vital mineralized tissue area, the experimental group covered with membrane obtained a higher percentage area (21.16 + 3.76%) when compared with control group, with and without membrane (12.78 + 5.9 % and 6.31 + 1.29 %, respectively) (p<0.05). For non-mineralized tissue area, the experimental group covered with membrane obtained a lower percentage area (20.29 + 4.17%) when compared with control group, with and without membrane (67.95 + 7.03 % and 81.41 + 3.25 %, respectively) (p<0.05). Conclusion: The combination of aspirate a xenogeneic bone graft for guided bone regeneration in rabbit calvaria, bone marrow significantly increased the amount of living mineralized tissue, as well as significantly reduced the amount of non-mineralized tissue.

Submitted by Maria Anália Conceição (marianaliaconceicao@gmail.com) on 2016-06-23T14:07:32Z No. of bitstreams: 1 Publico-NOVO-06.pdf: 3014625 bytes, checksum: 7a5c61cc40f9cd02772425bc1b416a0d (MD5) / Approved for entry into archive by Maria Anália Conceição (marianaliaconceicao@gmail.com) on 2016-06-23T14:14:50Z (GMT) No. of bitstreams: 1 Publico-NOVO-06.pdf: 3014625 bytes, checksum: 7a5c61cc40f9cd02772425bc1b416a0d (MD5) / Made available in DSpace on 2016-06-23T14:14:50Z (GMT). No. of bitstreams: 1 Publico-NOVO-06.pdf: 3014625 bytes, checksum: 7a5c61cc40f9cd02772425bc1b416a0d (MD5) Previous issue date: 2014 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Introdução: A manutenção das características estruturais do tecido ósseo é almejada quando substitutos ósseos são utilizados em procedimentos de enxertia aposicional. Dentro deste escopo, a utilização da fração de células mononucleares da medula óssea visa incrementar os resultados da enxertia óssea com o aumento do contato entre osso e titânio. Objetivo: Avaliar a associação da fração de células mononucleares da medula óssea com enxerto ósseo xenógeno em reconstrução aposicional na calvária de coelhos. Métodos: Doze coelhos da raça Nova Zelândia foram distribuídos, randomicamente, em dois grupos, Grupo Controle (n=6 - substituto ósseo xenógeno) e Grupo Experimental (n=6 - substituto ósseo xenógeno enriquecido com a fração de células mononucleares da medula óssea). Foi criado um modelo de regeneração óssea guiada por meio de dois cilindros instalados na calvária de cada um dos animais. A eutanásia ocorreu após oito semanas sendo analisado: (1) mensuração do volume tecidual formado no interior dos cilindros e (2) Histomorfometria dos parâmetros: Tecido Mineralizado Não Vital (TMNV); Tecido Mineralizado Vital (TMV); Tecido Não Mineralizado (TNM) e Contato Tecido Mineralizado Vital - Titânio (CTMVT). Resultados: As mensurações do volume tecidual formado no interior dos cilindros dos grupos Controle e Experimental demostraram manutenção do volume não havendo diferença entre os grupos. A histomorfometria não demonstrou diferença para os parâmetros TMNV, TMV havendo diferença para o parâmetro CTMVT. Conclusão: A associação do enxerto xenógeno com a fração de células mononucleares da medula óssea, em modelo de regeneração óssea guiada, aumenta a área de contato do tecido mineralizado vital com o titânio. / Purpose: This study investigated the combination of a bone marrow mononuclear fraction with a bone xenograft material in an appositional bone regeneration technique. Methods: Twelve New Zealand rabbits were randomly divided into two groups of six animals. Bone reconstruction situations were created using titanium cylinders filled with xenograft in Control group and xenograft enriched with the bone marrow mononuclear fraction in Experimental group. Two cylinders were adapted onto the calvaria of each animal. The bone marrow aspirate was obtained from the tibia of every animal. After 8 weeks, the animals were sacrificed and their parietal bone fixed in 10% formalin for analysis: (1) clinical measurement of the bone volume formed inside the cylinders and (2) histomorphometric evaluation of parameters such as vital mineralized tissue (VMT), non-vital mineralized tissue (NVMT), non-mineralized tissue (NMT) and vital mineralized tissue in contact with titanium (VMTCT). Results: The clinical measurements of groups 1 and 2 demonstrated a bone volume gain of 88.36+25.97% and 98.96+0.00%, respectively. Histomorphometry demonstrated a VMT of 18.96+9.00% and 28.02+8.76%; NVMT of 28.43+2.44% and 25.57+2.33%; NMT of 52.61+10.80% and 46.42+10.06%; VMTCT of 4.98+4.30% and 27.29+9.58%, respectively. Conclusion: The results from this study suggest that the use of the bone marrow mononuclear fraction can improve bone healing and the level of osseointegration.

Master of Science / Department of Clinical Sciences / Walter Renberg / Objective: To evaluate the safety and effect of a single simultaneous intra-articular and intravenous injection of autologous adipose-derived stromal vascular fraction (SVF) and platelet rich plasma (PRP) on coxofemoral osteoarthritis (OA) in dogs. Methods: This was a randomized, double-blind, placebo-controlled prospective pilot trial of simultaneous intra-articular and intravenous SVF and PRP for coxofemoral OA. Dogs with coxofemoral OA causing signs of lameness or discomfort were evaluated by orthopedic exam, visual lameness score, Canine Brief Pain Inventory (CBPI), goniometry, visual analogue scale (VAS), and pressure-sensitive walkway (PSW) at week 0 (baseline), and at 4, 8, 12 and 24 weeks after injection. Joint radiographs were scored at 0 and 24 weeks. Results: Twenty two client-owned dogs with naturally occurring OA of the coxofemoral joints were enrolled (12 placebo-control, 10 SVF-treated). CBPI pain severity scores were lower in the treatment group at 24 weeks compared to the placebo group (p=0.042). The VAS score for the treatment group was significantly greater at 0 weeks than at 4, 8, or 24 weeks (p<0.05). When dogs with low quartile baseline PVF (25th percentile) were compared, the treatment group had statistically higher PVF at all post-injection time points when compared to the placebo group. After SVF injection, fewer dogs in the treated group were lame compared to the control group. Clinical Significance: This study is the first to utilize objective data from PSW as an outcome measure for dogs treated with SVF and PRP for coxofemoral OA. No adverse events were noted. Improvements in some measured parameters in the treated dogs compared to those in the placebo group.