Osteoarthritis (OA) is a degenerative disease of diarthrodial joints causing pain and loss of joint function. Etiology is heterogeneous, but commonly involves inflammation arising from impairment of normal tissue homeostasis and/or function. A cycle of low-grade inflammation and global tissue degradation causes alteration of tissue morphology and function via primary mechanisms or inability to withstand physiological forces. Current therapies variably ameliorate symptoms but do not modify progression. Mesenchymal stem cells (MSCs) have multi-modal properties but are ineffective in ameliorating equine OA. However, anti-inflammatory activities of bone marrow derived MSCs (BMSCs) are enhanced by three-dimensional spheroid culture so equine BMSC (eBMSC) spheroids could inhibit intra-articular inflammation.
The overarching hypothesis is that eBMSCs can be enhanced to produce an allogeneic eBMSC therapy that inhibits intra-articular inflammation. In vitro experiments compared differences in anti-inflammatory phenotype between spheroid and traditionally cultured monolayer eBMSCs, the viability and health of eBMSC spheroids administered through needles, and the effects of allogeneic donor on the anti-inflammatory potential of eBMSC spheroids. A model of equine LPS induced synovitis was used to investigate anti-inflammatory efficacy of spheroid eBMSCs compared to placebo or monolayer eBMSCs in vivo.
eBMSCs aggregate into spheroids that have stable stem cell marker expression with increased secretion and gene expression of IL-6 and PGE2, and gene expression of SDF-1 and TSG-6. IFN𝛾 and TNFα were not produced by eBMSC spheroids and IL-10 production varied between individuals. Spheroids maintain higher viability and lower senescence than monolayer eBMSCs after injection through a needle and form in high-throughput culture without detrimental effects on expression of TSG-6, IL-6 and PGE synthases that denote an anti-inflammatory phenotype. Additionally, there is significant variation in this phenotype depending on the eBMSC donor. eBMSC spheroids reduced total nucleated cell counts and objective lameness measurements at peak levels of intra-articular inflammation compared to monolayer cultured eBMSCs in vivo.
In summary, spheroids increase anti-inflammatory potential of eBMSCs and are practical for clinical use. Increased anti-inflammatory efficacy was demonstrated in a model of in vivo inflammation. This dissertation provides an understanding of the anti-inflammatory activities of eBMSC spheroids that can be used to develop an OA therapy. / Ph. D. / Osteoarthritis (OA) is a progressive disease of joints causing pain and loss of function. Multiple factors cause OA including inflammation, tissue destruction from enzymes, and breakdown due to reduced strength with continued use. This cycle of inflammation and joint tissue degradation causes joint tissue damage despite treatment with symptom relieving therapies. Mesenchymal stem cells (MSCs) are a multi-modal therapy, but have been ineffective to relieve equine OA. However, MSCs derived from bone marrow (BMSCs) have enhanced anti-inflammatory activity when produced by three-dimensional culture so BMSCs from horses could reduce joint inflammation better as three-dimensional spheroids.
The overarching goal of these studies was to produce an “off the shelf” horse BMSC therapy that reduces joint inflammation both for horse treatment, and as a model for human OA. These studies compared differences between spheroid and traditionally grown (monolayer) BMSCs to reduce inflammation, survival of spheroids administered through needles, and the variability between different horse donors on the ability of spheroids to reduce inflammation. The ability of spheroids to reduce joint inflammation was determined in live horses compared to control or monolayer BMSCs.
Horse BMSCs form spheroids that retain the properties that define stem cells, plus spheroid BMSCs produce factors that stem cells use to reduce the inflammatory response. Spheroids have enhanced survival compared with monolayer BMSCs after injection through a needle and spheroids can be produced in large quantities without affecting their potential to reduce inflammation. Additionally, BMSCs from different horse donors have varied potential to reduce inflammation. In live horses, donor horse BMSC spheroids reduced signs of joint inflammation and pain when inflammatory levels were highest compared to monolayer BMSCs. This dissertation demonstrates enhanced ability of spheroid BMSCs to reduce inflammation and provides key information that will be used to develop OA therapies.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/81746 |
| Date | 19 April 2017 |
| Creators | Bogers, Sophie Helen |
| Contributors | Veterinary Medicine, Barrett, Jennifer G., Furr, Martin O., Eyestone, Willard H., White, Nathaniel A., Byron, Christopher R. |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Detected Language | English |
| Type | Dissertation |
| Format | ETD, application/pdf, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
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