Novel Cell Seeding System into a Porous Scaffold Using a Modified Low-Pressure Method to Enhance Cell Seeding Efficiency and Bone Formation

Abstract

The efficient seeding of cells into porous scaffolds is important in bone tissue engineering techniques. To enhance efficiency, we modified the previously reported cell seeding techniques using low-pressure conditions. In this study, the effects of low pressure on bone marrow-derived stromal cells (BMSCs) of rats and the usefulness of the modified technique were assessed. There was no significant difference found in the proliferative and osteogenic capabilities among various low-pressure (50-760 mmHg, 1-10 min) conditions. To analyze the efficacies of the cell seeding techniques, BMSCs suspended in the plasma of rats were seeded into porous β-tricalcium phosphate (β-TCP) blocks by the following three procedures: 1) spontaneous penetration of cell suspension under atmospheric pressure (SP); 2) spontaneous penetration and subsequent low pressure treatment (SPSL), the conventional technique; and 3) spontaneous penetration under low pressure conditions (SPUL), the modified technique. Subsequently, these BMSCs/β-TCP composites were used for the analysis of cell seeding efficiency or in vivo bone formation capability. Both the number of BMSCs seeded into β-TCP blocks and the amount of bone formation of the SPUL group were significantly higher than those of the other groups. The SPUL method with a simple technique permits high cell seeding efficiency and is useful for bone tissue engineering using BMSCs and porous scaffolds.

Fresh bone marrow introduction into porous scaffolds using a simple low‐pressure loading method for effective osteogenesis in a rabbit model

“…We devised a simple lowpressure cell-loading method to ameliorate these problems. 19 This method differs from other procedures in that cell soaking is performed under low pressure, whereas the low pressure is applied after cell soaking in the other conventional procedures. This ULP method enhances the cell-loading efficiency and in vivo bone formation capability of expanded BMSCs with porous scaffolds nearly twofold compared with the conventional lowpressure methods.…”

“…The harvested marrow was immediately introduced into highly porous b-TCP (Osferion 1 : Olympus Biomaterial, Japan), which had a 3D structure with 70% porosity with interconnecting macropores (200-400 mm) and micropores (<5 mm); this introduction was done under atmospheric pressure (AP group) or under low pressure (ULP group). The ULP method is a quite simple procedure modified from the described method, 19 using disposable syringes and stopcocks, which are available in any operating room. Briefly, the b-TCPs are set in the 20-mL syringe at the 1-mL level (Fig.…”

“…Quantification of bone marrow penetration into the block implants was calculated according to a previously described method. 18,19 Briefly, the block b-TCPs were divided into five sections at equal intervals. The area of bone marrow penetration within the macropores was manually selected and measured using image-editing software (Photoshop, Adobe Systems, San Jose, CA) and image analysis software (Scion Image, Scion Corp., Frederick, MD).…”

“…15,16 Researchers previously reported that a low-pressure environment enhances cell-loading efficiency and bone formation. 17,18 We have further developed an ameliorated low-pressure cell-loading procedure 19 in which cell soaking is conducted under a low-pressure environment, whereas previous methods used low pressure after cell soaking to remove remaining air. This ''under low pressure'' (ULP) loading method is more effective for loading efficiency and bone formation of in vitro expanded BMSCs when compared with conventional low-pressure methods.…”

“…This ''under low pressure'' (ULP) loading method is more effective for loading efficiency and bone formation of in vitro expanded BMSCs when compared with conventional low-pressure methods. 19 We hypothesized that this method would also be beneficial for enhancing osteogenesis using fresh bone marrow combined with porous scaffolds.…”