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Browsing by Author "Ehrhart, Nicole, advisor"

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    ItemOpen Access
    Do mesenchymal stromal cells abrogate the immune response in massive cortical allograft recipients?
    (Colorado State University. Libraries, 2015) McNamara, Kaitlyn Louise, author; Ehrhart, Nicole, advisor; Dow, Steven, advisor; Donahue, Seth, committee member; Duncan, Colleen, committee member; Palmer, Ross, committee member; Page, Rodney, committee member
    OBJECTIVE: To evaluate the humoral and cellular immune response against bone associated antigens when delivered in a vaccine, and to evaluate the immunomodulation on the aforementioned immune response with the addition of adipose-derived mesenchymal stromal cells (AD-MSCs). ANIMALS: 68 C57BL/6 mice PROCEDURES: Femur fragments harvested from Balb/C or C57BL/6 mice were resuspended in PBS and cationic liposomal DNA complexes (CLDC) to create an allograft or autograft vaccine, respectively. A positive control vaccine was created utilizing bovine alkaline phosphatase (ALP) resuspended in PBS and CLDC. Twenty C57BL/6 mice were divided into four treatment groups: non-vaccinated (n=5), ALP vaccine recipients (n=5), allograft bone vaccine recipients (n=5), or autograft bone vaccine recipients (n=5). Forty-eight C57BL/6 mice were divided into the same 4 vaccine treatment groups (n=16), and received either intravenous AD-MSCs (n=8) or a subcutaneous injection of AD-MSCs (n=8). All mice received an initial vaccine on Day 1 and a booster vaccine on Day 7, followed by euthanasia on Day 21. Blood was collected on Day 1 and Day 7 just prior to vaccination, and on Day 21 just prior to euthanasia. Serum was subjected to an antibody detection ELISA to evaluate the humoral response. Spleens were collected and flow cytometry was used to evaluate T cell proliferation as an indicator of the cellular immune response. RESULTS: The bone vaccines did no elicit a detectable humoral immune response to the total bone antigen vaccine used in this model. The addition of AD-MSCs had no effect on the lack of a detectable humoral immune response. The T cell response towards a bone antigen was dampened in mice previously vaccinated with a bone vaccine. This effect was most pronounced when looking at the T cell response towards an allograft bone antigen in mice previously vaccinated with an allograft bone vaccine, particularly with the addition of AD-MSCs. CONCLUSIONS AND CLINICAL RELEVANCE: The bone vaccine model used in this study allowed evaluation of the humoral and cellular immune response towards bone associated antigens. The model suggests that recipients of an allograft bone vaccine will dampen the T cell proliferation seen upon second exposure to the bone antigens. This model could be used in future vaccine studies looking at the effect of vaccinating mice with a bone vaccine prior to undergoing a limb-salvage procedure. If efficacious, the bone vaccine model may provide a new treatment option for decreasing the risk of transplant rejection following massive limb reconstruction.
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    ItemEmbargo
    Engineered mRNA therapeutic encoding beta-catenin increased bone formation in a murine tibial fracture model
    (Colorado State University. Libraries, 2023) Nelson, Anna Laura, author; Ehrhart, Nicole, advisor; Bahney, Chelsea, advisor; Huard, Johnny, committee member; Popat, Ketul, committee member; Prawel, David, committee member
    Fractures continue to be a global economic burden and impaired fracture healing cases, like delayed and non-union, occurring in about 14% of all tibial shaft fractures. Current treatments to aid in fracture healing involve surgical interventions and osteoanabolic, bone-morphogenetic protein-2 (BMP-2), yet is challenged supraphysiological doses and adverse side effects. Given the limited treatment options available, there remains a clinical need to develop injectable therapeutics to accelerate fracture healing in impaired fracture healing cases. Mechanistic data reveals β-catenin as a molecular driver in endochondral ossification. The central hypothesis for this dissertation is a stabilized, non-destructive β-catenin mRNA delivered locally in the fracture callus can accelerate fracture healing in a murine tibia fracture healing model. Using mRNA therapeutically continues to be challenged with stability and immunogenicity of the mRNA. To circumvent these limitations, delivery carriers have been employed to maximize gene stability, minimize off-target effects, and reduce immunogenicity. Recent advancements in liposomal technologies have led to the development of lipid nanoparticles (LNPs), leading to successful clinical translation of several novel and highly effective therapies, like SARS-CoV-2 vaccine. Alternative delivery carriers have emerged involving use of mineral coated microparticles (MCMs) as a biomimetic and biocompatible system to deliver liposomes at the site of a fracture in a controlled manner. Here, we explore mRNA delivery carriers for fracture healing applications, including manufactured cationic liposomes, MCMs, LNPs and a combination of these carriers. Manufactured liposome, Lipofectamine™, was found to be prolong transfection when tested in a murine fracture model in vivo as compared to TransIT Transfection Reagent. Using Lipofectamine™ to deliver mRNA, chemically-doped MCMs enhanced transfection and stimulated bone in vitro when delivered in chondrocytes. When testing these platforms in a murine tibia fracture model, chemically-doped MCM did not promote bone expression through testing RNA in the fracture callus for bone-related genes and through histomorphometry of the fracture callus 2 weeks post-fracture. The chemically doped MCM was found to prolong transfection of reporter gene, firefly luciferase mRNA, in vivo when compared to other treatment groups including the liposome and mRNA complex (lipoplex) alone. Ionizable-based LNPs are positively charged at a low pH and net neutral at physiological pH. Two FDA-approved ionizable phospholipids, MC3 and SM-102, were used to generate ionizable LNPs. First, MC3 LNP was tested for transfection capacity when combined with MCMs. While chemically-doped MCMs when combined with firefly luciferase mRNA encapsulated MC3 LNPs showed improved transfection in vitro, no improvements in transfection efficacy were found in vivo. Next, MC3 and SM-102 LNPs were then complexed with reporter gene, firefly luciferase mRNA to test transfection potential, immunogenicity, fracture interference and biodistribution in vitro and in a murine fracture healing model. SM-102 LNPs showed enhanced transfection efficacy in vitro, prolonged transfection in vivo, minimal fracture interference in vivo and showed no localized inflammatory response in the murine fracture callus. Ex-vivo IVIS images of main organs revealed no biodistributive effects when delivering SM-102 complexed with mRNA locally to the site of the fracture callus. Capitalizing on prior mechanistic data showing β-catenin's critical role in chondrocyte to osteoblast transdifferentiation, a non-destructive β-catenin, β-cateninGOF, mRNA transcript was generated using nucleoside modification, N1-methyl-pseudouridine, and cap analog, CleanCap. When testing the generated β-cateninGOF mRNA encapsulated in SM-102 LNPs in vitro for bioactivity, downstream canonical Wnt genes were significantly upregulated. When testing SM-102-β-cateninGOF mRNA therapeutic in murine tibia fracture model, more bone and less cartilage composition compared to PBS control was determined when analyzing histomorphometry at 25 and 45 μg concentrations at 2 weeks post-fracture. To further confirm SM-102-β-cateninGOF mRNA therapy's capabilities to promote bone in vivo, μCT was performed revealing significantly more bone volume over total volume with 45 μg dose as compared to PBS control. Taken together, we generated a novel mRNA based therapeutic encoding a non-destructive β-catenin mRNA and optimized ionizable LNP, SM-102, to maximize transfection efficacy with a localized delivery. This SM-102-β-cateninGOF mRNA therapeutic may accelerate fracture healing in a murine tibia fracture healing model.
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    ItemOpen Access
    Influence of adipose-derived mesenchymal stromal cells on osteosarcoma minimal residual disease
    (Colorado State University. Libraries, 2015) Aanstoos-Ewen, Megan, author; Ehrhart, Nicole, advisor; Kipper, Matthew, committee member; Dow, Steven, committee member; Custis, James, committee member
    Introduction: Mesenchymal stromal cells (MSCs) have been shown to improve bone integration and healing in several preclinical studies and have therapeutic potential in limb salvage following massive bone loss due to tumor resection. However, MSCs have also been shown to promote primary and pulmonary metastatic tumor growth when injected in the presence of gross tumor or when co-injected with tumor cells in rodent models. While these results raise concerns about the safety of using MSCs in sarcoma patients, MSCs are unlikely to be utilized in a clinical setting when gross tumor is present. The objective of this dissertation project was to develop murine models of minimal residual osteosarcoma following primary tumor removal then to utilize these models to determine whether the administration of adipose-derived MSCs with or without chemotherapy treatment in a minimal residual disease setting would promote either pulmonary metastatic osteosarcoma progression or local disease recurrence. We hypothesized that surgical site or intravenous administration of MSCs will influence either osteosarcoma pulmonary metastatic burden or local disease recurrence in a minimal residual disease setting. Materials & Methods: Two syngeneic, orthotopic models of luciferase-expressing osteosarcoma were developed. In the first model, tumor-bearing mice underwent a coxofemoral amputation and were followed to assess development of pulmonary metastases. In the second model, a femorotibial amputation was performed in order to develop a model of consistent local tumor recurrence. In this model, all gross tumor was removed, however, microscopic tumor remained at the surgical margin. In this dissertation project, three principle projects were completed to test our hypothesis. The first project explored the use of MSCs delivered either to the surgical site or intravenously to ascertain their influence on pulmonary disease burden. A follow-on pilot explored concurrent MSC and chemotherapy treatment on development of pulmonary disease. The second project evaluated the use of MSCs delivered either to the surgical site or intravenously on local recurrence of osteosarcoma at the surgical site. Gross recurrent tumor size was measured for comparison between treatment groups. The third project examined the use of cisplatin and MSCs on survival of mice following removal of primary osteosarcoma. Data were expressed in mean +/- SD or median with 95% CI. ANOVA test, Kruskal-Wallis test, Fisher’s Exact test, Welch’s test, t-test, and Mann Whitney test were used for statistical analysis. Significance was set at p<0.05. Results: Mice treated with intravenous MSCs had a faster time to first pulmonary metastatic disease detection than mice treated with MSCs injected into the surgical site or control mice (no MSCs) (p=0.022). No treatment effect was seen between groups with respect to time to tumor recurrence or size of recurrent tumor in the second study. Survival curves were significantly different when comparing cisplatin, cisplatin and MSC treatment, MSC alone treatment and untreated mice (p<0.001) as well as in pairwise comparisons. Mice treated with MSCs had a 73% chance of earlier death than untreated controls. Discussion/Conclusion: Intravenous administration of MSCs in a minimal residual osteosarcoma environment resulted in a faster time to first detection of pulmonary disease and in a higher chance of earlier death compared to untreated mice. However, administration of MSCs locally in a surgical site following sarcoma excision appears to be safe, even in the setting of known residual microscopic disease. Further, the use of cisplatin treatment appeared to ameliorate the effects of intravenous MSCs on survival. Based on these results, further study is warranted to evaluate the influence of intravenously administered MSCs on minimal residual pulmonary metastatic disease.
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    Mesenchymal stem cell rescue for bone formation following stereotactic radiotherapy of osteosarcoma
    (Colorado State University. Libraries, 2013) Schwartz, Anthony L., author; Ehrhart, Nicole, advisor; Ryan, Stewart, committee member; James, Susan, committee member; Goodrich, Laurie, committee member; Custis, Jamie, committee member
    Background: Osteosarcoma (OSA) is the most common form of primary bone cancer in dogs and humans. Curative-intent treatment options include amputation, radiation therapy or surgical limb salvage for local tumor control combined with adjuvant chemotherapy for prevention or delay of metastatic disease. Stereotactic radiotherapy (SRT) delivers high dose per fraction radiation to a defined tumor volume with relative sparing of surrounding normal tissues. It has been successfully used as a non-surgical limb salvage procedure to achieve local tumor control of spontaneous OSA in dogs. The most common complication observed with this treatment is pathologic fracture of the irradiated bone. Mesenchymal stem cells (MSCs) are multipotent stem cells that have the capability to differentiate into many cell types including bone. The ability of MSCs to differentiate into bone suggests that they should be investigated as a potential therapy to regenerate bone in SRT treated bone. Methods: In experiments described herein, we developed an orthotopic model of canine osteosarcoma in athymic rats and evaluated the ability of SRT to achieve local tumor control. We then evaluated the ability of MSCs to regenerate bone after SRT treatment of OSA. Results: We demonstrated that the canine OSA cell line reliably engrafted in the rat femur. We characterized progression in order to create a reproducible model in which to replicate a clinical scenario to test MSC behavior following SRT of OSA. Two weeks after OSA cell inoculation was identified as the time period when the same clinical characteristics were observed as in canine OSA cases and was chosen to be an appropriate time for SRT treatment. The optimal SRT protocol to achieve local tumor control while minimizing acute radiation effects was determined to be 3 fractions of 12 Gy delivered on consecutive days. MSCs administered either intravenously or intraosseously 2 weeks after SRT revealed no new bone formation; however, decreased tumor necrosis was observed after MSC treatment. Conclusion: The results herein describe the characterization of an orthotopic rat model of canine OSA. This model was useful for the evaluation of different dose and fractionation SRT protocols along with combination adjuvant therapies that may be clinically relevant for canine or human OSA. The administration of MSCs following SRT did not induce new bone growth. The lack of efficacy is most likely due to the radiation-induced alterations to the bone microenvironment that resulted in conditions poorly suited to MSC survival and/or differentiation.