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Migration of Co-cultured Endothelial Cells and Osteoblasts in Composite Hydroxyapatite/Polylactic Acid Scaffolds

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Annals of Biomedical Engineering, Vol. 39, No. 10, October 2011 (Ó 2011) pp DOI: /s z Migration of Co-cultured Endothelial Cells and Osteoblasts in Composite Hydroxyapatite/Polylactic
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Annals of Biomedical Engineering, Vol. 39, No. 10, October 2011 (Ó 2011) pp DOI: /s z Migration of Co-cultured Endothelial Cells and Osteoblasts in Composite Hydroxyapatite/Polylactic Acid Scaffolds AMITA R. SHAH, 1,2,3 SARITA R. SHAH, 2 SUNHO OH, 1 JOO L. ONG, 1 JOSEPH C. WENKE, 2 and C. MAULI AGRAWAL 1 1 Department of Biomedical Engineering, University of Texas at San Antonio, San Antonio, TX, USA; 2 US Army Institute of Surgical Research, San Antonio, TX, USA; and 3 Department of Surgery, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA (Received 8 March 2011; accepted 25 June 2011; published online 16 July 2011) Associate Editor Scott I Simon oversaw the review of this article. Abstract Regeneration of bone in large segmental bone defects requires regeneration of both cortical bone and trabecular bone. A scaffold design consisting of a hydroxy apatite (HA) ring surrounding a polylactic acid (PLA) core simulates the structure of bone and provides an environment for indirect and direct co culture conditions. In this exper iment, human umbilical vein endothelial cells (EC) and normal human primary osteoblasts (OB) were co cultured to evaluate cell migration and interactions within this biphasic composite scaffold. Both cell types were able to migrate between the different material phases of the scaffold. It was also observed that OB migration increased when they were co cultured with ECs, whereas EC migration decreased in co culture. The results show that co culture of ECs and OBs in this composite biphasic scaffold allows for migration of cells throughout the scaffold and that pre seeding a scaffold with ECs can increase OB infiltration into desired areas of the scaffold. Keywords Endothelial cell migration, Osteoblast migration, Ceramic scaffold, Polymer scaffold, Bone regeneration. INTRODUCTION Extremity injuries are prevalent types of injury in civilian trauma and modern warfare, as exemplified in the current wars in Iraq and Afghanistan. 21 Large segmental bone defects as a result of trauma are especially troublesome. Extensive bone debridement, chronic infection, and non-union due to these injuries result in significant morbidity. These defects are difficult to treat, and amputation is recommended for defects larger than 10 cm. 8 Autologous posterior iliac Address correspondence to C. Mauli Agrawal, Department of Biomedical Engineering, University of Texas at San Antonio, San Antonio, TX, USA. Electronic mail: 2501 bone grafts are currently the gold standard treatment for large segmental defects, but this treatment is limited by donor site morbidity and the small amount of bone that is available for harvesting. Each posterior iliac has enough tissue to graft 4 cm of tibia or 8 total cm per person. 11 Other treatment options include vascularized free tissue transfers and the Ilizarov technique, but these two techniques have variable results, long healing times, and significant donor site morbidity. 8 Many tissue-engineering strategies for bone regeneration utilize combinations of scaffolds, cells, and growth factors, and attempts to regenerate mineralized bone matrix have had moderate results. 9 More recently, the focus has shifted to establishing vasculature in the area of regeneration because good vascularization is necessary for the regeneration of bone. Studies have shown improved osteogenesis in vivo through the use of endothelial cell (EC) and osteoblast (OB) co-cultures in scaffolds. 32,33 When segmental bone defects are healed naturally, cortical and trabecular bone are formed, along with vasculature to support them. 8 In current treatment methods for segmental bone defects, avascular scar tissue is removed to expose the vasculature or small holes are drilled in surrounding bone before the placement of the grafts in order to encourage vascular ingrowth. 8 These methods work for smaller defects, but unfortunately, there is no current treatment modality that can reliably heal large defects. Differentiation of cells occurs in response to local biomechanical conditions. 19 Unfortunately, most patients with large defects are unable to apply the appropriate loads needed to initiate this differentiation to the injured areas. A method to circumvent this /11/ /0 Ó 2011 Biomedical Engineering Society Report Documentation Page Form Approved OMB No Public reporting burden for the collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing the collection of information Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for reducing this burden, to Washington Headquarters Services, Directorate for Information Operations and Reports, 1215 Jefferson Davis Highway, Suite 1204, Arlington VA Respondents should be aware that notwithstanding any other provision of law, no person shall be subject to a penalty for failing to comply with a collection of information if it does not display a currently valid OMB control number 1. REPORT DATE 01 OCT REPORT TYPE N/A 3. DATES COVERED - 4. TITLE AND SUBTITLE Migration of co-cultured endothelial cells and osteoblasts in composite hydroxyapatite/polylactic acid scaffolds. 6. AUTHOR(S) Shah A. R., Shah S. R., Oh S., Ong J. L., Wenke J. C., Agrawal C. M., 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 5d. PROJECT NUMBER 5e. TASK NUMBER 5f. WORK UNIT NUMBER 7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) United States Army Institute of Surgical Research, JBSA Fort Sam Houston, TX 8. PERFORMING ORGANIZATION REPORT NUMBER 9. SPONSORING/MONITORING AGENCY NAME(S) AND ADDRESS(ES) 10. SPONSOR/MONITOR S ACRONYM(S) 12. DISTRIBUTION/AVAILABILITY STATEMENT Approved for public release, distribution unlimited 13. SUPPLEMENTARY NOTES 14. ABSTRACT 15. SUBJECT TERMS 11. SPONSOR/MONITOR S REPORT NUMBER(S) 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT UU a REPORT unclassified b ABSTRACT unclassified c THIS PAGE unclassified 18. NUMBER OF PAGES 10 19a. NAME OF RESPONSIBLE PERSON Standard Form 298 (Rev. 8-98) Prescribed by ANSI Std Z39-18 2502 SHAH et al. problem is to create an environment utilizing materials with mechanical properties that help cells differentiate to the appropriate structure and function. To accomplish this, a biphasic scaffold modeled after the natural structure and function of bone has been developed. This scaffold is composed of a hydroxyapatite (HA) ring and an oxygen gas plasma (GP)-treated polylactic acid (PLA) core. Polylactic acid is a biodegradable polymer widely used for tissue-engineering scaffolds and other in vivo applications. 2,16 Oxygen gas plasma-treated PLA scaffolds were chosen for this application because they have been shown to enhance angiogenesis in vivo in subcutaneous mouse models compared to non-treated PLA scaffolds. 22 Hydroxyapatite (HA) scaffolds similar to the ring used in this scaffold design have been shown to support bone and vessel formation in vivo in a canine model. 3 This scaffold s porosity is 85% with an average pore size of 500 lm. 12 The pore size of calcium phosphate scaffolds is an important factor for vascularization and osseointegration. Although smaller pore sizes have more surface area, pore sizes greater than 210 lm have been found to have increased amounts of bone and vessel formation than scaffolds with smaller pores. 17 It is believed that the configuration of materials with different mechanical properties and the gas plasmatreated inner core will enhance vascular network formation in the middle of the scaffold while supporting the formation of bone on the periphery. In addition to utilizing a scaffold that more closely replicates natural bone, it is also important to create a cell environment that more closely replicates the cellular processes that occurs during the natural bone healing, where multiple cells types migrate into the wound area and interact with each other through various paracrine and juxtacrine pathways and intercellular contacts. 7 Since the goal of bone tissue engineering is to produce vascularized bone tissue, it is important to study EC and OB co-cultures in a scaffold rather than only studying cell monocultures. In existing co-culture studies which use scaffolds, migration patterns of the cells within the scaffolds have not been investigated. 6,23,24,28 It is unknown if the cells have a preferred direction of migration or if the type of material on which the cells are seeded affects the migration patterns. The goal of this study was to evaluate the migration of co-cultured ECs and OBs in a HA and PLA biphasic tissue-engineering scaffold. It was hypothesized that ECs and OBs co-cultured together will interact with each other and alter the cell migration patterns compared to when they are cultured alone. Enhancing our understanding of EC and OB co-cultures is important to the eventual goal of developing a scaffold which supports cortical bone growth on the outside and vasculature formation on the inside for the repair of large segmental defects in bone. MATERIALS AND METHODS Cell Culture Human primary OBs were purchased from Lonza (Allendale, NJ) and cultured in OB growth media containing ascorbic acid and antibiotics (Lonza, Allendale, NJ). All cells were used on first or second passage. Human umbilical vein ECs (HUVEC) were purchased from Invitrogen (Carlsbad, CA) and cultured in Lifeline EC media (Walkersville, MD) with 2% fetal bovine serum, 15 ng ml 1 insulin growth factor, 5 ng ml 1 endothelial growth factor, 5ngmL 1 fibroblast growth factor, 50 lg ml 1 ascorbic acid, 1 lg ml 1 hydrocortisone, 0.75 U ml 1 heparin, 10 mm glutamine, and antibiotics. There was no vascular endothelial growth factor (VEGF) in this media. Co-cultured cells were also cultured in this media. Cell Labeling Cells were labeled with PKH lipophilic cell membrane label (Sigma-Aldrich, St. Louis, MO). HUVEC cell membranes were labeled green with PKH 67 at 10 mm concentration. OBs were labeled with PHK 26 at 10 mm concentration which labeled the cell membranes red. At this dye concentration, the cell membranes were fluorescent up to 7 days with no evidence of dye transfer between cells. Scaffold Fabrication The biphasic composite scaffold consisted of a HA scaffold ring with a PLA scaffold core. These scaffolds were fabricated as follows. Hydroxyapatite Scaffold Fabrication The HA scaffold was a ring 10 mm in diameter and 2 mm in height with a 5-mm diameter hole in the center (Fig. 1). This scaffold was fabricated using a polyurethane sponge template with 60 ppi (pores per inch) as previously described by Appleford et al. 3 Briefly, the sponge template for this outer shell was first cut to resemble a cylindrical pipe with a hollow core in the middle. This polyurethane sponge template was 2 mm in height, with an outer diameter of 10 mm and an inner diameter of 5 mm, thereby having a 2.5-mm wall thickness. The sponges were then ultrasonically treated Migration of Co cultured ECs and OBs in Composite HA/PLA Scaffolds 2503 Birmingham, AL) was dissolved in 3.25 ml acetone. The polymer solution was added to 5.5 g sodium chloride while being vibrated under continuous air flow conditions in a Teflon mold. 5-mm diameter disks of 2 mm thickness were punched out of the polymer mixture after drying under vacuum and low heat. The sodium chloride was leached out in sterile deionized water. After 2 days in water, scaffolds were lyophilized. 1 GP treatment was performed in a pure oxygen environment in a glow discharge system (PDC-32G, Plasma Cleaner/Sterilizer; Harrick Scientific Inc., New York) for 3 min at 100 W as a surface treatment and for sterilization. The gas plasma treatment decreases PLA s contact angle from 75 to 30. FIGURE 1. Biphasic composite scaffold design with hydroxyapatite ring (HA) and polylactic acid (PLA) core. The diameter of the scaffold is 10 mm and the height is 2 mm. in 10% sodium hydroxide solution for 20 min, cleaned in flowing water for 40 min, and then rinsed with distilled water. The foam templates were then dried at 80 C in an oven for 5 h. Nano-sized HA powder (Berkeley Advanced Biomaterials, Berkeley, CA) was used for fabrication of the scaffold. A 3% (by mass) polyvinyl alcohol solution with 3% (by mass) carboxymethylcellulose, 7% (by mass) of ammonium polyacrylate dispersant, and 5% (by mass) of N,N-dimethylformamide drying agent was made. HA powder was slowly dispersed into the solution, followed by stirring on low heat until a powder to liquid ratio of 1.50 was obtained. The treated sponge template was immersed in the coating slurry under manual compression until the HA slurry was fully absorbed in the sponge template scaffold. The sponge template was then removed from the slurry and excess slurry removed. The HA slurry-coated sponge template scaffolds was then dried and sintered in a furnace at 1230 C for 3 h, thus removing the sponge. The scaffolds were sterilized with ethylene oxide. The resulting scaffold had a porosity of 84.4%, pore size of 500 lm, compressive strength of 1.5 MPa, and tensile strength of 60 kpa. 12 Polylactic Acid Scaffold Fabrication The 5-mm diameter core of the composite scaffold is a gas plasma-treated PLA scaffolds fabricated using the vibrating particle salt-leaching method to create an open-cell, interconnected scaffold with porosity 90%, permeability of 24.4 ± 11.3 E 08 m 4 N 1 s 1, and pore sizes of lm. 1 Briefly, 0.38 g of poly(d,l-lactic) acid polymer (Mw of 109,500 Da, Mn of 64,900 Da, polydispersity of 1.69) (DURECT Corp, Scanning Electron Microscopy Scanning electron microscopy (SEM) was used to characterize the surface morphology of the biphasic composite scaffold prior to the seeding of cells. The scaffolds in their entirety were sputter-coated with gold palladium and imaged using an EVO40 SEM (Zeiss, Germany). Scaffold Seeding 50,000 cells in 20 ll of media were seeded onto the PLA core, and 150,000 cells in 500 ll of media were seeded onto the HA ring as described below. These cell numbers were chosen to keep the per unit volume cell seeding density consistent between the two scaffolds. The PLA scaffolds were seeded using the drop-wise method in an ultra-low attachment well plate (Corning, Edison, NJ) after degassing the scaffolds under vacuum. The cells were allowed to attach to the scaffold for 45 min before adding additional media. This method results in 90 95% cell attachment to the PLA scaffold. The HA scaffolds were seeded by incubating the scaffold in a cell suspension of 150,000 cells in 500 ll inan ultralow attachment well plate for 24 h which results in 95 98% cell attachment. After 24 h, there was uniform distribution of the cells on the scaffolds (Fig. 2). After incubating the scaffolds for 24 h separately, the scaffolds were assembled together and incubated for two more days at 37 C and 5% CO 2 on a shaker plate at a low setting to ensure flow of media throughout the scaffold. On either day 3 or day 5, the scaffolds were removed from the media, rinsed gently with phosphate buffered solution, and fixed with 4% paraformaldehyde. The scaffolds were seeded in six different seeding arrangements as described in Fig. 3. This allows for six different groups to assess OB and EC migration: four monoculture groups and two co-culture groups. The groups were named X-OB (n = 6), X-EC (n = 6), OB-X (n = 9), EC-X (n = 6), OB EC (n = 9), and 2504 SHAH et al. FIGURE 2. Confocal microscopy image of HA ring and PLA core at day 1 showing coverage of scaffold struts with cells (blue nuclei of EC). migration from the HA ring into the PLA core was evaluated by comparing monocultured ECs (EC-X) with co-cultured ECs (EC OB). EC migration from the core into the ring was evaluated by comparing monocultured ECs in the core (X-EC) with co-cultured core ECs (OB EC) (Fig. 3). FIGURE 3. Cell seeding diagrams. Shaded areas are seeded with endothelial cells, black areas are seeded with osteoblasts, and blank areas are not seeded with any cells. EC OB (n = 6). The first part of the designation refers to the type of cell seeded on the HA scaffold phase, and the second part refers to the cell type seeded on the PLA scaffold phase. Areas not seeded with cells were designated X. To evaluate OB migration into the PLA core, OBs were seeded on the HA scaffold ring. The core was left unseeded for the monoculture group (OB-X) and seeded with ECs for the co-culture group (OB EC). Migration of OBs from the PLA scaffold core to the HA ring was assessed by seeding the PLA core with OBs and leaving the ring blank (X-OB) or seeding it with ECs (EC OB). The co-cultured biphasic scaffolds from the OB migration experiments could also be used for the endothelial migration experiments. EC Imaging and Image Analysis Prior to imaging, all cell nuclei were counterstained with DAPI. Confocal 3D imaging was performed on the scaffolds with multiple images taken of each phase of the scaffold on days 1 and 3 on a Zeiss 510 LSCM (Zeiss, Germany) at 109. There were six images per sample with three randomly chosen areas on each phase of the scaffold. The images were reconstructed and analyzed using IMARIS (Bitplane, St. Paul, MN) and the number of cells migrating to the other scaffold phase counted per low power field (LPF) using the spots function. Images of the surface of the scaffolds were also taken using fluorescence microscopy on an inverted fluorescence microscope (Nikon, Melville, NY) on days 1, 3, and 5. Statistical Analysis Statistical analysis was performed using Student s t test with significance determined at p Results are reported as mean ± standard error. RESULTS Scaffold Imaging SEM images show that the PLA and HA phases of the scaffold were well approximated. The HA and PLA Migration of Co cultured ECs and OBs in Composite HA/PLA Scaffolds 2505 FIGURE 4. Scanning electron microscopy images of (a) HA scaffold, (b) the interface of hydroxyapatite (HA) ring and polylactic acid (PLA) core, and (c) PLA scaffold. FIGURE 6. Osteoblast migration on day 3, cells/lpf 6 SE (n 5 6 9, p 0.05). There is a statistically significant increase in migration when osteoblasts are co-cultured with endothelial cells (OB EC, EC OB). FIGURE 5. Confocal microscopy three-dimensional reconstruction image of the interface between the HA ring and PLA core at day 1 showing no cell migration or contamination during the assembly of the biphasic scaffold. Blue line approximates the actual interface (red and white dots osteoblasts; green endothelial cells). scaffolds both have interconnected open pores. The HA scaffolds have struts of lm thickness and round pores whereas the PLA scaffolds have square pores with thin walls between each pore (Fig. 4). Osteoblast Migration At day 1, which was directly after the two scaffolds were assembled, there was uniform coverage of the scaffold on which the OBs were seeded. No OBs were present in the other section, suggesting that there was no cell migration or contamination during the assembly of the biphasic scaffold (Fig. 5). At day 3, there was a statistically significant increase in OB migration from one scaffold phase to another (p 0.05) in the OB EC co-culture group. In monoculture (X-OB), 2.7 ± 0.7 OBs per low power field (LPF) migrated from the PLA core into the HA ring. In contrast, when OBs were co-cultured with ECs (EC OB), there was an increase of cell migration to 10.0 ± 2.2 OBs per LPF into the HA ring. Similarly, there was a significant increase in OB migration from the HA ring into the PLA core when the cells were co-cultured (OB EC). In monoculture (OB-X), 15.2 ± 2.7 OBs migrated into the core compared to 24.2 ± 1.7 OBs in co-culture (Fig. 6). Fluorescence and confocal images of the center of the EC-seeded PLA core on day 3 show the presence of OBs, confirming the ability of the OB to migrate int
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