Identification of the Optimal Biologic to Enhance Endogenous Stem Cell Recruitment

Authors:
Hannah L. Holmes Brooke Wilson Jesse L. Silverberg Julian Goerger Lisa A. Fortier.
Cornell University Ithaca NY USA.

Disclosure:
 H.L. Holmes: None. B. Wilson: None. J.L. Silverberg: None. J. Goerger: None. L.A. Fortier: 3B; Arthrex Consultant.

Introduction: In situ tissue engineering approaches can be used to recruit the body’s endogenous reservoir of stem cells to a site of injury. Biologics such as bone marrow aspirate concentrate (BMAC) and platelet rich plasma (PRP) are commonly used to enhance recovery from musculoskeletal injuries. These biologics contain growth factors such as platelet-derived growth factor (PDGF) and transforming growth factor- β (TGF-β) which could act as chemoattractants for bone marrow derived mesenchymal stem cells (MSCs). The purpose of this study was to identify the optimal biologic for recruitment of MSCs. Our hypothesis was that PRP would recruit more MSCs and to a greater distance of migration compared to BMAC.
Methods: MSCs were obtained by sternal bone marrow aspirate (BMA) collection and isolation of plastic adherent cells from 4 mature horses. Biologics were generated from different horses. Leukocyte poor PRP (PRP-1) was generated from blood using Double Syringe Autologous Conditioned Plasma Systems (Arthrex Naples FL). Leukocyte rich PRP (PRP-2) was generated from blood using GPS III Kits (Biomet Warsaw IN). BMA was obtained by sternal bone marrow aspiration. Bone marrow aspirate concentrate (BMAC) was produced by centrifugation of BMA using SmartPReP2 BMAC Packs (Harvest Technology Corp Plymouth MA). Human recombinant PDGF was the positive control. Media containing 10% FBS was the neutral control. Negative controls such as 0% FBS media alone resulted in MSC death during the experiments. Complete blood counts were performed on all biologics and they were stored at -80°C until sufficient stem cells were available for the experiments. Animal use was approved by IACUC.
MSCs were seeded in the center well of the u-slide chemotaxis device (ibidi LLC Verona WI) at an average concentration of 166 cells/cm2 and approximately 10-15% confluence (Figure 1). Biologics were thawed and centrifuged at 12 000xg for 20 minutes to remove cell debris. In each chemotaxis device PDGF or biologic was placed on one side and 10% FBS media was placed on the other side so that a direct competition of chemoattraction could be measured between a biologic and the neutral control (Figure 1). The device was then imaged using live cell imaging on an inverted bright field microscope at 10X magnification every ten minutes for a period of twenty four hours. Time-lapse images were analyzed by tracking the migratory patterns of the individual cells by use of a custom-written code in ImageJ (NIH Bethesda MD). The program tracked the location of the computer cursor as the time-lapse videos were played and recorded (x y) coordinates for every frame. The (x y) coordinates were then used to determine displacement of each cell from its location at time 0 to the final location at time 24 hours. The number of cells migrating in each direction was also recorded. The chemokinetic properties of the biologics were assessed by calculating cell flux (%migrated * µm/24hrs) to represent the rate of movement of MSCs within the device. Cells were tracked up to the point of division death or 24 hours of migration.
The number of cells that migrated in either direction was first normalized by calculating the percentage of cells that migrated in order to control for differences in seeding density of the MSCs between devices. The number of migrated cells was then compared using a Wilcoxon rank sum test. Cell flux was also compared using a Wilcoxon rank sum test. A p value of <0.05 was considered significant.
Results: A significantly greater number of MSCs migrated toward the biologics than the neutral control (Figure 2; p = 0.03). On average biologics attracted 78% of migrating cells. There were no significant differences in number of migrating cells between the biologics. The cell flux was significantly different between all the biologics and the neutral controls (Figure 3). However within the biologics BMAC resulted in significantly greater cell flux than PRP-1 (p = 0.028) as did PDGF (p = 0.028) but there were no other differences between biologics.
Discussion: The data indicates that all tested biologics have the ability to recruit MSCs. However they did not significantly increase cell flux. Certain growth factors such as IGF-1 and PDGF-BB have been shown to have both chemotactic and/or chemokinetic effects on malignant mesothelioma cells (1). Although not measured in this experiment all biologics would contain comparable levels of IGF-1 because of the basal level of IGF-I in serum of both blood and bone marrow (2). In the groups tested PRP-2 contained the highest concentration of platelets and therefore PDGF-BB so it was hypothesized that PRP would result in the greatest chemotaxis of MSCs. There are many potential chemotactic and chemokinetic molecules in biologics so it is overly simplified to attribute our results to only IGF-1 or PDGF-BB when all biologics resulted in increased recruitment of MSCs.
Significance: Biologics such as PRP or BMAC can be used as chemotactic agents to recruit MSCs to a site of injury. This information will reduce the need and associated risks and costs associated with direct stem cell delivery.
Acknowledgments: Harry M. Zweig Fund (LAF)
Supported by the Empire State Stem Cell Fund through New York State Department of Health Contract # C028097. (HLH)
References: 1)Liu Z Klominek J. Chemotaxis and chemokinesis of malignant mesothelioma cells to multiple growth factors. Anticancer Res 2004 24(3a):1625-1630.
2)Schnabel L.V Mohammed H.O Miller B.J McDermott W.G Jacobson M.S Santagelo K.S Fortier L.A. Platelet rich plasma (PRP) enhances anabolic gene expression patterns in flexor digitorum superficialis tendons. J Orthop Res .2007; 25(2):1-11.
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