The Effect of Mesenchymal Stem Cell Migration and Adhesion on Demineralized Bone Scaffolds under Vacuum Infusion Packaging Technology.

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1 The Effect of Mesenchymal Stem Cell Migration and Adhesion on Demineralized Bone Scaffolds under Vacuum Infusion Packaging Technology. Celeste Abjornson, PhD 1, Sudhanshu Somasundar, MS 2, Antonio Brecevich, BS 1, Frank Cammisa Jr, MD 1 1 Integrated Spine Research Program, Hospital for Special Surgery, New York 2 Biologics Research, Alphatec Spine Inc Abstract The objective of this investigation was to compare the efficacy of a novel vacuum infusion packaging cell seeding technique to the standard drip / soak seeding technique. Demineralized bone scaffold samples from three commercially available products were compared. Group 1 consisted of Grafton DBM Strip samples; Group 2 consisted of Bacterin Osteosponge samples; and Group 3 consisted of 3D ProFuse Bioscaffold (ProFuse) samples. All tissue forms are comparable scaffolds except for aseptic processing and vacuum packaging of the ProFuse scaffolds. All scaffold samples were seeded with human mesenchymal stem cells (hmscs). After incubation for ~24 hours post seeding, scaffold samples from all three groups were prepared for SEM imaging. All scaffold samples were cut for cross-sectional representation. Cross sectional sectioning allowed for imaging of both surface level and core micro-architecture. Lamellipodial and filopodial expression on cell membrane post seeding, being a morphological indication of commitment of the hmscs to osteoblastic differentiation, was studied in all cases. Conclusion: The SEM images revealed that hmscs seeded on ProFuse by the Vacuum infusion packaging seeding technique showed morphology indicating commitment to osteoblastic differentiation. The scaffolds in other groups did not express such morphology. Thus it may be inferred from this study that Vacuum infusion packaging seeding technique promotes homogeneous distribution of mesenchymal stem cells (MSCs) and results in better osteogenic cell functionality immediately (24 hours) post seeding. Introduction The clinical benefit of demineralized bone in stimulating bone formation was first introduced by Marshall Urist and has been subsequently studied by prominent researchers for the last forty years [1, 2, 3]. It is known that demineralized bone forms a natural osteoconductive and osteoinductive scaffold which promotes the migration of stem cells into the scaffold and differentiation of stem cells into osteoblasts. [4, 5, 6, 7, 8, 9, 10]. It has been established that homogeneous distribution of cells on a scaffold, or cell spreading, ensures better cell viability, proliferation and performance [11, 12] Thus, in surgical procedures, consideration of the principle should be made to ensure that both the cells and signals are homogeneously distributed within the scaffold. The Vacuum Infusion Packaging (VIP) system patented by Alphatec Spine, packages the tissue scaffold under near vacuum. This system allows a rapid hydration through a septum, with fluids such as Bone Marrow Aspirate (BMA). It provides effective delivery of cells and inherent signals into the packaged scaffold [13]. This study assessed cell functionality immediately post seeding (~24hours) on a three dimensional scaffold of cancellous bone aseptically processed and packaged under the patented system called Vacuum Infusion Packaging (VIP), in comparison to two other non-vip packaged and sterile-processed scaffolds of demineralized cancellous bone commercially available in the market. It has been shown that the sterile processing of tissue should not affect cell migration or adhesion but has been shown to affect cell proliferation rates. [14] The cell functionality was analyzed by SEM imaging of lamellipodial and filopodial expression by the hmscs seeded on the various scaffold groups. Filopodia activity is indicative of the beginning of MSC commitment to the osteoblastic lineage [15]. Filopodia are thin, dynamic cell extensions associated with lamellipodia and are comprised of tight bundles of long actin filaments covered with cell membrane [16, 17]. Lamellipodia are sheet-like webs of actin filaments at the advancing edge of migrating cells [16, 17]. Lamellipodia are essential for cell movement [17, 18, 19]. The structure of the lamellipodia propels the cell across a substrate [17, 18, 19]. Filopodia are extensions from the lamellipodia and form focal adhesions with the substratum, linking it to the cell surface [16, 17, 18, 19, 21]. Filopodia have roles in sensing, migration and cell-cell interactions [16, 17, 18, 19]. SEM imaging of sections from the edges and core of the scaffold was performed. This was done to assess efficacy of VIP system seeding technique in comparison to standard drip soak seeding technique in delivering cells homogeneously to the core of the scaffold. Additionally, this analysis was conducted to assess the comparative functionality of such cells in the core of the scaffold when seeded by the two different techniques. 1

2 Methods Utilizing commercially prepared human MSCs (hmscs), sourced from AllCells (Alameda, CA) three vials stored at -80 C, containing 750,000 hmscs each, were thawed to 37 C in a water bath. All vials underwent standard processing to prepare 2.0mL of hmsc concentrated (approx. 250,000 hmscs) solution per scaffold. Under sterile conditions, three groups of comparative products as shown in Table 1 were seeded and placed in a 37 C incubator with 100% humidity for a minimum of twenty-four (24) hours. Scaffold samples in groups 1 and 2 were drip seeded per standard seeding technique. Scaffold samples in group 3 (ProFuse) were seeded through the vacuum packaging, as per package IFU. Results SEM imaging illustrated considerable differences in MSC migration, adhesion and functional cell cluster formation characteristics between vacuum and non-vacuum packaging conditions. Group 1 Grafton Lamellipodia and Filopodia Inferior with membrane ruffling Cell Penetration Section N/A* Surface 1a Inferior Yes Core 1b Figure Group Product Scaffold Sample Size 1 Grafton DBM Strips 3 2 Bacterin Osteosponge 3 3 3D ProFuse Bioscaffold in VIP 3 Table 1. Test Groups and Sample Size 2 Bacterin Inferior N/A* Surface 2a Inferior with membrane ruffling Negligible Core 2b Healthy N/A* Surface 3a The seeded samples were prepared for scanning electron microscope (SEM) imaging. The demineralized bone scaffold samples in the three groups, comparable in all aspects except for aseptic processing and vacuum packaging (Group 3), are comprised of 100% cancellous human bone. All scaffold samples were cut for cross-sectional representation and stored for least twenty-four (24) hours prior to imaging. Cross sectioning allowed for imaging of both surface level and core micro-architecture. Lamellipodial and filopodial expression on cell membrane of seeded cells were studied. The study was performed at Hospital for Special Surgery, New York. 3 VIP Healthy Yes Core 3b * Not applicable because cells are seeded onto surface. Penetration is measurement of cell migration to core. 2

3 Scaffold Surface/Edge Sections Membrane Ruffling Figure 1a. Group 1 (Grafton ) Inferior signs of lamellipodial and filopodial expression and membrane ruffling observed. Membrane Ruffling Figure 2a. Group 2 (Bacterin ) Inferior signs of lamellipodial and filopodial expression. Figure 2b. Group 2 (Bacterin ) Inferior signs of lamellipodial and filopodial expression and membrane ruffling observed. Figure 2b. Group 2 (Bacterin ) Few MSCs seen adhered with 2 out of the 3 MSCs in this image showing Inferior signs of lamellipodial and filopodial expression and membrane ruffling. Figure 3a. Group 3 (Profuse VIP) Figure 3b. Group 3 (Profuse VIP) Figure 3b. Group 3 (Profuse VIP) Images captured of MSCs seeded on ProFuse in the least density areas on the scaffold surface. Even these cells confirm expression of healthy morphology indicating commitment of the MSCs to Osteoblastic differentiation, when seeded onto 3D ProFuse via VIP. High MSC density observed on ProFuse in VIP, homogeneously distributed over the scaffold area post seeding through VIP, and showing healthy morphology indicating commitment of the MSCs to Osteoblastic differentiation. 3

4 Scaffold Core Sections Figure 1b. Group 1 (Grafton ) Inferior signs of lamellipodial and filopodial expression. Figure 1b. Group 1 (Grafton ) Inferior signs of lamellipodial and filopodial expression. Figure 2b. Group 2 (Bacterin ) Even at a low magnification (800x) looking at a comparatively large area, low cell density. Typical amounts of cells found in pockets on this scaffold. Inferior signs of lamellipodial and filopodial expression. Figure 3b. Group 3 (Profuse VIP) Figure 3b. Group 3 (Profuse VIP) Cells confirm expression of healthy morphology and interaction showing commitment to Osteoblastic differentiation when seeded onto 3D ProFuse via VIP. 4

5 The generation of filopodia and lamellipodia to the scaffold surface using vacuum packaged samples demonstrated the most mature and significant cell attachment to the scaffold, indicting better cell functionality. Under non-vacuum packaging, cell migration and adhesion were not so robust and resulted in inferior levels of cellscaffold integration. Discussion Group 3 (tissue packaged under vacuum) exhibited more cell migration and attachment morphology than the other groups. It has been shown, under SEM imaging, that MSCs on the path to osteogenic differentiation show specific morphology of expression of lamellipodial and filopodial extensions from cell membrane [15, 20]. Filopodia expression of participating cells is required in most cases for efficient tissue repair, regeneration and wound healing [16, 17, 18, 19]. Membrane ruffles are compartments of inhibited actin filament turnover and look like dark waves arising at the leading edge of lamellipodia [20 ]. Presence of membrane ruffling denotes the absence, or failure, of lamellipodia adhesions from cell to scaffold [20]. This ruffling may indicate sub-optimal substrate microenvironment, which retards or delays sustained cell migration. (Figure 4.) Cell migration is a vital step in the tissue repair and regeneration process [11, 12, 15, 16, 17, 18, 19, 20]. Without filopodial expression, the cells cannot migrate, or spread, throughout the substrate. [11, 12, 15, 16, 17, 18, 19, 21]. Impaired cell spreading affects cell performance and viability by triggering apoptosis [12]. SEM analysis of surface/edge sections (Table 2) showed that hmscs seeded on vacuum packaged demineralized bone, ProFuse (Group 3), showed morphology indicating commitment to osteoblastic differentiation, while in the case of demineralized bone that was not vacuum packed (Group 1 and 2) the hmscs showed no morphological indication of commitment to osteoblastic differentiation. Cell ruffling features were also observed in the case of hmscs seeded on scaffold samples in groups 1 and 2, indicating impaired lamellipodial adhesion to scaffold. SEM analysis of scaffold core sections (Table 3) showed cell penetration to the core of scaffold samples in Group 1 and Group 3. Commitment to osteoblastic differentiation could be inferred morphologically in the case of hmscs delivered to the core of ProFuse (Group 3) but no such morphological indicators were observed in case of Group 1. There was negligible cell penetration seen in the case of scaffold in group 2. Membrane ruffle like features were observed in the cells seeded on scaffold samples in group 2, indicating impaired lamellipodial adhesion to scaffold. The observations above may indicate that scaffolds in group 1 and 2 do not form an ideal environment for hmsc function towards osteogenesis. It is apparent from this SEM study that the vacuum infusion packaging system promotes a homogenous distribution of mesenchymal stem cells (MSCs) and results in better cell functionality immediately (24hours) post seeding. Conclusion Healthy cell motility and viability within the scaffold are key factors for initiation of healthy bone remodeling. These results suggest that cell delivery throughout the scaffold when hydrated with VIP ensures better cell viability and advanced cell commitment to osteogenesis versus standard cell seeding techniques. Further clinical evaluation will need to be conducted to confirm these results. Better cell migration, integration and functionality to the demineralized bone interface were observed in the 3D Profuse Bioscaffold packaged in VIP. Figure 4. Schematic illustration [Borm et al] summarizing the correlation between adhesion dependent lamellipodia persistence, migration efficiency, and ruffling rates. A suboptimal substrate will lead to higher rates of membrane ruffling, which is an indicator of impending cellular apoptosis; a better substrate will provide higher levels of cell migration and adhesion. 5

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