Dynamic Culture Using Novel Slow Perfusion Biroeactor and Porous Polycaprolactone (PCL) Scaffolds for Bone Regeneration and Repair

In the United States nearly 20 million people have osteoarthritis, a disease which is characterized by the degeneration of joints, including bone and cartilage. As a result, back pain is the second leading cause of sick leave. Further, osteoporotic fractures have doubled in the last decade, and 40% of all women over the age of 50 will suffer from an osteoporotic-related fracture in their life-time. Traditional biomaterials and current treatments for bone and cartilage regeneration and/or repair are unsatisfactory, and have lead to a great demand for innovative biomaterials and scaffolds. Porous structure and biological properties of the scaffolds are two of the most important factors in promoting tissue regeneration. Using proprietary 3D Precision Microfabrication Technology, we are able to fabricate porous biodegradable polymer scaffolds with well controlled porous structures that are readily compatible with our novel slow perfusion bioreactor system. Therapies today use bone harvesting, demineralizated freeze-dried cadaver bones, metals, or plastic. Current growth factor delivery to an injured/diseased tissue results in serious systemic side effects associated with high doses of these factors necessary for therapeutic effects. When combined with our bioreactor, these porous PCL scaffolds will promote human stem cell proliferation, osteoblastic differentiation, and the generation of a uniform, cell-derived extracellular matrix (ECM) coating richly incorporated with osteoinductive and osteoconductive factors. Using the microenvironment created by these naturally coated PCL scaffolds, it is now possible to localize and enhance growth factor delivery to specific sites for bone regeneration/repair. Combined, these therapeutic technologies will reduce long-term costs associated with current therapies. They will also increase functional tissue availability, eliminate host immune responses and disease transmission, as well as reduce repeat surgeries, long hospital stays, and rehabilitation. Materials and Methods Cells Human mesenchymal stem cells (hMSCs) and human dermal fibroblast cells (hFB) were purchased from Lonza (Walkersville, MD) and LifeLine Cell Technologies (Walkersville, MD), respectively. According to manufacturer’s instructions, hMSCs and hFB were maintained in growth media. Cells were maintained in a humidified tissue culture incubator at 5% CO2 and 37°C. Polycaprolactone (PCL) Scaffold Microfabrication (3D InsertTM-PCL) Porous polycaprolactone (PCL) scaffolds were engineered using 3D Biotek’s Proprietary Precision Microfabrication Technology (Figure 1A). Uniquely, fiber diameter is controlled by nozzle diameter while spacing between fibers is controlled by a motion control system. Scanning electron micrographs demonstrate that the struts of each layer are oriented 90° relative to the struts of the layer immediately below (Figure 1B-C). Before use, scaffolds are tissue culture surface treated and γ-radiation sterilized. This study implemented 24-well compatible 3D InsertTM-PCL scaffolds, 1.9 cm2 in diameter mm in diameter and 1.6 mm in thickness, with a configuration of 300 μm fiber diameter and 300 μm pore size (PCL3030). The total cell growth area of a 24-well 3D InsertTM-PCL3030 is 18.28 cm2 compared with 1.9 cm2 of total growth area in a traditional 24-well 2D TCP (Figure 1D). 3D Cell Seeding Cells were initially statically seeded onto 3D InsertTM-PCL scaffolds for 24 h according to 3D Biotek’s 3D cell seeding protocol (Figure 2). Cells were resuspended in 1 ml growth media using 5x103 cells/scaffold. To seed each scaffold, 100 μl of the cell suspension was slowly pipetted onto the top surface of each 3D InsertTM-PCL. To ensure high seeding efficiency, the cell suspension droplet was not allowed to contact the sides of the wells. After a 3 h incubation in 5% CO2 at 37oC, 400 μl of media was added to the 3D wells. After 24 h, scaffolds containing cells were loaded into the slow perfusion system according to 3D Biotek’s protocol. Stem Cell Differentiation Immediately upon loading scaffolds containing hMSCs into the bioreactor, hMSC osteoblastic differentiation was begun. hMSC osteoblastic differentiation was performed according to manufacturer’s instructions. Alkaline Phosphatase Activity Assay hMSC lysates were prepared using M-PER (Pierce) followed by a centifugation at 14,000 rpm for 5 min. The lysate in supernatant was collected and analyzed using p-Nitrophenyl Phosphate Liquid Substrate System (pNPP) (Sigma) and 4-nitrophenol solution. Alkaline phosphatase activity was normalized to DNA. Alkaline Phosphatase Activity Assay Scaffolds containing hMSC-osteo and hFB cells were fixed with 10% formalin for 0.5 h. Cultures were rinsed with ddH2O, incubated with 2% silver nitrate, and covered. Scaffolds were incubated for 10 min and then rinsed again with ddH2O, exposed to bright light for 15 min, dehydrated in 100% EtOH for 1 min, and then dried. Scaffolds with cells were imaged with a digital camera. Figure 2. Cell Seeding Flow-Chart Figure 3. 3D PCL scaffolds are compatible with slow perfusion bioreactor system For further information, please contact [email protected] www.3dbiotek.com Figure 1. 3D PCL scaffold