The Influence of PTH 1-34 on the Osteogenic Characteristics of Adipose and Bone Marrow Derived Stem Cells from Juvenile and Ovarectomized Rats

Background: Mesenchymal Stem Cells (MSCs) are of growing interest in terms of bone regeneration; the majority of preclinical trials utilise bone marrow derived stem cells (bMSCs), though this is not without isolation and expansion difficulties Objective: We compare the characteristics of bone marrow and adipose derived cells from juvenile, adult and ovarectomized rats; also assessing the effect of hPTH 1-34, on their osteogenic potential. Methods: cells were isolated from the adipose and bone marrow of juvenile, adult and previously ovarectomized wistar rats. Cells were characterised with flowcytometery, proliferation assays, osteogenic and adipogenic differentiation, and migration to SDF-1. Experiments were repeated with and without co-culturing with 50nMol of intermittent PTH 1-34. Results: The juvenile and adult MSCs demonstrated significantly increased differentiation into bone and fat and superior migration towards SDF-1 than ovarectomized groups, this was the case for adipose and bone marrow derived cells equally. PTH increased parameters of osteogenic differentiation and migration to SDF-1, this was significant for all cell types, though had the most significant effect on cells derived from OVX animals. Bone marrow derived cells from all groups, showed increased mineralisation and migration to SDF-1 compared to adipose derived cells. Conclusion: Juvenile MSCs showed significantly greater migration to SDF-1 and showed greater osteogenic and adipogenic differentiation compared to cells from osteopenic rats, this was true for bone marrow and adipose derived cells. The addition of PTH, increased the osteogenic characteristics and migration of all cells, and further illustrates the possible clinical utility of both PTH and MSCs from various sources in bone regenerative therapies


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Multiple influences alter the osteogenic capabilities of undifferentiated mesenchymal stem cells (MSCs); studies 62 have compared the functional differences between cells from adolescent and aged animals, with a smaller body 63 of work exploring the role of source osteoporosis on cell activity (1)(2)(3)(4). Resultantly, the use of cells, be they 64 minimally manipulated or enhanced with pharmacological adjuncts, may be key to orthopaedic therapies 65 particularly in aged and osteoporotic populations; a patient cohort growing in size and worsening the 66 musculoskeletal burden.

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Post-menopausal oestrogen deficiency leads to an uncoupling of the bone remodelling cycle, where upregulated 69 osteoclast activity is matched only with aberrant osteoblast activity-resulting in bone resorption (5,6). As such, 70 type I osteoporosis is synonymous not only with reduced bone strength and thus increased fracture risk, but also 71 is characterised by a reduction in bone mass and altered trabecular microarchitecture hence bone fragility (7).

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In clinical applications utilizing a tissue engineering approach or cell therapy treatments, the majority of pre-73 clinical studies have used mesenchymal stem cells derived from bone marrow (bMSCs). Yet, clinically this is 74 not without problems, including morbidity associated with obtaining cells from iliac crest puncture, low cell 75 yield, and reduced potency following extensive passage (8). Zuk et al (9) initially described the use of cells 76 obtained from subcutaneous adipose liposuction aspirate as a source rich in MSCs (AdMSCs). The initial 77 population isolated contains haemopoietic cells, pericytes, and adipose cells-all of which are plastic adherent; 78 though after in vitro culturing, cells exhibit a homogeneous phenotype that fulfill the criteria for MSCs.

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Moreover, unlike periosteal cells or cells obtained from myogenic sources, adipose tissue is readily available, 80 harvesting carries very limited morbidity, and cell yield is much greater than found from other sources (10,11).

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Reports on the osteogenic capacity of AdMSCs compared to bone marrow derived counterparts are 82 contradictory; BMSC characteristics are thought to be affected by age, unlike AdMSCs where cells are thought 83 to retain all characteristics regardless of age of source. Cell yield is also a fundamental difference between the 84 sources, BMSCs yield 6 × 10 6 nucleated cells per mL of aspirate, with a maximum of 0.01% being MSCs.

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Whereas, 2 × 10 6 cells can be isolated from 1 gm adipose tissue, and 10% are stem like (12,13). As such, 86 investigations into the combined effect of age and ovarectomy on the osteogenic efficacy of these cells is pivotal 87 to fully determine their possible eventual clinical utility.

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In addition, to cell source and age of animal, pharmacological adjuncts can be used to modulate stem cell 90 behavior. Studies, including those conducted on post-menopausal women, demonstrate a profound anabolic 91 effect of parathyroid hormone 1-34 (PTH) (14,15). Moreover, in vitro data has shown PTH to mediate MSC 92 fate, not only increasing MSC number, but also their preferential osteogenic differentiation over adipogenesis 93 (16). Interestingly, these findings have all been reported in cells derived from bone marrow, with very little data 94 on the effect of PTH on adipose derived MSCs.

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The ability for intermittent PTH (iPTH) to mobilise/increase migration of cells from the haemopoietic niche, is 96 particularly significant in the context of sites of increased bone turnover (fractures and peri-implant). Cells are 97 initially "mobilised" from their niche into the circulation, "home" across the tissue endothelium and mature into 98 active cell types-eventually "modulating" the local environment. The SDF-1/CXCR4 axis has been found to be 99 an important regulator of stem cell migration. Stromal derived factor-1/CXC1L2 (SDF-1) is produced by a 100 multitude of tissue types including fracture endosteum and in its active form is bound to the CXCR4 receptor

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As such, although comparisons have been made between the osteogenic potential of different stem cell sources; 110 a very limited body of work compares these differences across juvenile, adult and ovarectomized animals, nor 111 does this work elucidate their capacity to migrate to SDF-1. Moreover, no study has explored this in the context 112 of PTH administration. We hypothesise that mesenchymal stem cells isolated from bone marrow aspirates and 113 adipose tissue of juvenile female Wistar rats will have greater tridiffrentiation, migration and proliferation than 114 cells isolated from adult and ovarectomized rats, and that co-culture with intermittent PTH will upregulate the 115 osteogenic characteristics of both adipose and bone marrow derived cells.

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Female Wistar rats were used throughout this study, animals were classed as "juvenile" at 2-4 weeks or "adult" 123 at 6-9 months, one group of animals ("OVX") were supplied immediately post ovarectomy (all animals supplied 124 by Charles River Laboratories, Harlow, Essex). These animals were housed for 16 weeks in pairs and osteopenia 125 confirmed by assessing their femoral, lumbar 3rd and 4 th vertebrae and humeri mineral density with pQ-CT 126 compared to age matched non-ovarectomized controls; a reduction of 22% in bone mineral density was 127 confirmed, as such our model was one of osteopenia rather than osteoporosis.

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Husbandry conditions for all animals used throughout the thesis were standardised, whereby animals were    with sterile scissors, In a 20ml universal tube, 8mls of warmed 0.1% collagenase-type II (Sigma-Aldrich) was 146 added to the adipose tissue and agitated in a 37°C water bath for 60minutes. Samples were then centrifuged at 147 5000rpm for 5minutes, forming a gelatinous pellet. The supernatant was aspirated and discarded, leaving the 6 148 remaining pellet in the tube, to which a further 5mls of standard media was added, followed by repeat 149 centrifuge.

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Following this, the supernatant was discarded, and the pellet resuspended in 5mls of fresh standard media, the 151 suspension was then cultured in standard media, and again used for experimental procedures at passage 3-4 (40).

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To quantify staining, 10% cetylpyridinium chloride (CPC) was added to 10mM of sodium phosphate to obtain a 170 working solution of pH 7. Following imaging, PBS was discarded from wells, and 200µl of the CPC solution 171 added to each well for 15minutes, agitated at room temperature and covered in foil. Absorbance was then read 172 on a plate reader at wavelength 570nm (Tecan infinite Pro, Tecan trading, Switzerland).

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A standard curve was made by serial dilution of alizarin red working solution in CPC and read again at 570nm.

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The CD expression was compared with the isotype control. Cells were fixed in 4% formalin for 15 minutes at

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Expression of CD markers: There was no significant difference in CD marker expression by cells 271 obtained from any group, regardless of tissue source. Mean CD marker expression is outlined in Table 1 272 273 274 Table 1 275 Cluster differentiation marker expression showed no significant difference between groups.

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There was large variability in the ALP expression from day three to day 14 across all groups. no difference 306 between ALP expression from cells derived from adipose or bone marrow was seen, but as with calcium 307 phosphate deposition, juvenile and adult cells expressed significantly more ALP than OVX cells at day 14 when 308 production peaked for cells from both tissue sources (p<0.04). iPTH led to increased ALP production peaking

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The rate of lipid accumulated from day 7 onwards was greater in cells isolated from juvenile rats.

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Additionally, lipid droplet accumulation significantly accelerated from day 14 to day 21 for juvenile bMSCs, 335 while bMSCs from adult and ovX rats showed no increase in lipid accumulation after day 7. Cells isolated 336 from adipose tissue regardless of donor age or whether they were derived from OVX rats continued to show 337 increased lipid formation over the 21 day period, though juvenile cells were always more productive than the 338 other groups.

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When comparing bone marrow to adipose derived cells, there was no difference in adipogenic differentiation,  animals rather than their juvenile counterparts. We also found differences between cells isolated from juvenile 374 and ovarectomized animals for each cell source, with a reduction in osteogenic and migrative characteristics in 375 OVX derived cells although these cells showed similar proliferative capacity and CD marker expression.

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Finally, this work demonstrated increased osteogenic capabilities of young and OVX cells from both tissue 377 sources, when dosed with pulsatile PTH 1-also resulting in increased migration of all cells, though had no effect 378 on cellular proliferation. Interestingly this effect was often greater with OVX cells, and on cells obtained from 379 bone marrow rather than adipose tissue.

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My data showed no difference in CD marker expression from the cells independent of source or age or OVX

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Very little work has explored the effect of PTH on adipose derived cells; we found these cells also reacted to 465 PTH in a similar way to bone marrow derived cells with the percentage increase in migration was greatest in 466 OVX cells-compared to untreated cells.

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The findings of this chapter confirm the ability to obtain stem cells from the adipose and bone marrow of Wistar