| Reconstructing large bone defects is still a major concern in the orthopedic field. Multiple approaches have been attempted using autografts, allografts, and xenografts. Autografts such as iliac crest bone grafts still remain the gold standard in bone replacement, but the available autologous bone supplies are limited and their harvesting may induce donor site morbidity. Surgeons often use allografts and xenografts to overcome the drawbacks of autografts. However, such grafts had limited success because of slow integration, poor remodeling, immunoreactions, and disease transmission. For these reasons, tissue engineering approaches that incorporate osteoblastic cells and porous biocompatible scaffolds are currently being explored as potential alternatives. Although traditional static seeding and static culture have been the most widely used methods of cell seeding and cell/scaffold construct culture, these techniques suffer from poor cellular distribution, low seeding efficiencies and limited diffusion, confining the majority of the cells to the outer surface of the scaffold. In this work, we explored a novel method of three-dimensional cell seeding and growth in a flow perfusion bioreactor which allowed for improved efficiency and uniformity of seeding, enhanced proliferation and homogenous distribution of osteoblasts, and reinforced osteogenic potential of tissue engineered bone to heal the large segmental bone defect.1. Three-dimensional cell seeding of four kinds of porous scaffolds in a flow perfusion bioreactorThe purpose of the present study was to develop a novel flow perfusion bioreactor for the cell seeding of three-dimensional (3D) scaffolds, designed to induce continuous fluid flow of cell suspension through the scaffold pores. Using quantitative biochemical and image analysis techniques, we have evaluated the effects of flow perfusion on seeding efficiency and spatial distribution of human fetal osteoblasts in four kinds of porous scaffolds includingβ-tricalcium phosphate (β-TCP), human allogenic cancellous bone, gelatin sponge and collagen sponge, using statically seeded scaffolds as controls. The effects of scaffold was also evaluated. Perfusion seeding ofβ-TCP and gelatin sponge yielded higher seeding efficiencies and more homogeneous distribution as compared with those of static seeding. There were no significant differences in cell load and cell distribution in cancellous bone scaffolds between the perfusion group and static group. Cell viability, cell seeding efficiency and cell counting in collagen sponge were significantly higher in static group than in perfusion group, but the uniformity of seeding was similar for both groups. It was found that perfusion seeding ofβ-TCP and gelatin sponge was effective. In addition, cancellous bone and collagen sponge did not facilitate perfusion seeding but collagen sponge did facilitate static seeding.2. Flow perfusion bioreactor improves three-dimensional cell seeding and growth in porousβ-TCP scaffolds The purpose of the present study was to create a new perfusion bioreactor in which the full process of tissue regeneration, from cell seeding to cell growth, can be performed in successive steps. The effect of different conditions such as seeding time, density of cell suspension and flow rate on the efficiency of cell seeding and cell growth were also investigated. Human fetal osteoblasts were dynamically seeded and cultured in this system in relevant volumes (1.5cc) of small sized porousβ-TCP scaffolds (3.5～5mm). We have evaluated the effect of different conditions (seeding time, density of cell suspension, rate of fluid flow) on cell seeding and growth by the cell viability, cell seeding efficiency and histological study, using statically seeded scaffolds as controls. It was found that seeding efficiency and cell viability could be alternated by several conditions including seeding time, density of cell suspension, flow rate of cell suspension and culture medium. It was demonstrated that the seeding time of 12～24 hours and the density of cell suspension of 2×10~5/mL～5×10~5/mL was effective, and that the optimal flow rate for cell seeding was 1mL/min. Moreover, the optimal flow rate for cell growth was 0.5mL/min～2mL/min in initial 24 hours and 2mL/min subsequently. These results demonstrated the feasibility and benefit of combing high efficiency cell seeding and cell/scaffold constructs cultivation culture in a flow perfusion bioreactor for bone tissue engineering applications, and which can be improved by optimization and control of several conditions such as seeding time, cell density and flow rate.3. Integrated three-dimensional seeding and culture of osteoblasts in a perfusion bioreactor system for in vitro tissue engineered bone reconstructionIn the present study, a perfusion bioreactor system with integrated seeding and long-term culture functions was utilized for producing 3D engineered bone constructs. In the perfusion seeding and perfusion culture (PSPC) group, human fetal osteoblasts were dynamically seeded in porousβ-TCP scaffolds and the cell/scaffold constructs were cultured under flow perfusion conditions for 8 days. Cell proliferation and distribution were assessed by daily D-glucose consumption, cell viability (MTT assay), histological evaluation and scanning electron microcopy (SEM) observation as compared to the conventional method of static seeding and static culture (SSSC) and the method of static seeding and perfusion culture (SSPC). The daily glucose consumption was much higher in the SSPC and PSPC group than in the SSSC group (P<0.05), and the results of cell viability via MTT colorimetry after 8 days of incubation coincided with those of glucose consumption. Although the daily glucose consumption was significantly higher in PSPC group than in SSPC group after 2, 4 and 6 days of incubation, the results of the 8th day were similar for both groups. SEM and histological analysis showed that cells were distributed throughout the entire scaffold by 8 days of perfusion culture in both the SSPC and PSPC groups whereas they were located only along the scaffold perimeter in the SSSC group. The results of histomorphometry showed a significantly higher cell quantity in SSPC and PSPC group as compared to SSSC group. In summary, we demonstrated that the PSPC method was superior to the SSPC method in tissue engineered bone reconstruction. both SSPC and PSPC were effective for 8 days of construct cultivation, but the perfusion seeding resulted in a high efficiency and a uniform distribution of cells throughout the scaffold, which should accelate engineered tissue reconstruction. Moreover, this superiority would become more obvious as the size and porosity volume of the scaffold increased.4. The development of constructing large tissue engineered bone by flow perfusion culture of osteoblasts inβ-TCP scaffolds with controlled architectureLarge-scaleβ-TCP scaffolds with tightly controlled architectures were fabricated using rapid prototyping (RP)technique and a custom designed perfusion bioreactor was developed in the present study. Human fetal osteoblasts were seeded onto the scaffolds, cultured for up to 16 days in static or flow perfusion conditions. After 4, 8, 16 days of incubation, proliferation and distribution of osteoblasts were determined by daily D-glucose consumption, cell viability (MTT assay), histological evaluation and SEM observation. Sphere like structures observed in the SEM images were assessed by energy dispersive X-ray (EDX) analysis. The daily D-glucose consumption and cell viability were significantly higher in the perfusion culture than in static culture (P<0.05). Flow perfused constructs demonstrated improved cell proliferation and a homogeneous layer composed of cells and extracellular matrix in channels throughout the whole scaffold. However, cells were biased to periphery in scaffolds culture statically. Sphere like structures present in the matrix were identified as calcium phosphate nodules via EDX analysis. It was found that flow perfusion culture combined with well-defined 3D interconnected channel environments mitigated nutrient transport limitation, enhanced the proliferation and improved the distribution of osteoblasts in large scaffolds, and provided mechanical stimulation to seeded cells in the form of fluid shear stress, resulting in improved cell osteodifferentiation. The interconnected channel geometry of the scaffold may facilitate uniform media flow and cell feeding and the internal architecture appears to induce de novo tissue modeling and affect the morphology and distribution of the newly formed tissue.5. Repair of radius segmental defect in rabbits using tissue engineered bone graft obtained by a combined perfusion seeding/culture system The aim of this study was to investigate if in vitro pre-culture period in a combined perfusion seeding/culture system of rabbit osteoblasts influence the tissue engineered bone ability to regenerate bone when implanted in a long segmental radius defect in rabbits.β-TCP scaffolds with controlled architectures were fabricated using RP technique. Three groups were assessed over 8 days in vitro:β-TCP scaffolds with cells that were dynamically seeded and proliferated in a perfusion seeding/culture bioreactor system (PSPC group);β-TCP scaffolds with cells that were statically seedied and then proliferated in a perfusion culture system (SSPC group); andβ-TCP scaffolds with cells that were statically seeded and proliferated under static conditions (SSSC group). Cell load and cell distribution were shown using daily D-glucose consumption, cell viability (MTT assay) and histological analysis. These constructs were implanted in 1.5 cm segmental bone defects in radius of New-Zealand white rabbits for 12 weeks, using scaffolds alone, autografts, and empty defects as controls. The daily D-glucose consumption and cell viability were significantly higher in PSPC group than in SSPC group and SSSC group (P<0.05). Histological study showed that PSPC and SSPC group produced a more uniform distribution of cells throughout the scaffold than SSSC group did, but the cell quantity analysis indicated that PSPC group yielded more cells than SSPC group did. The healing rates of autografts, PSPC group, SSPC group, SSSC group and scaffolds without cells were 6/6, 4/6, 2/6, 0/6 and 0/6 respectively at the 12th week. Significantly more new bone formation was found in PSPC group than in SSPC group. The residual material percentage in PSPC group was similar to SSPC group but significantly lower than SSSC group and simple scaffolds. In conclusion, tissue engineered bone graft obtained by the perfusion seeding/culture system is feasible in treating long segmental bone defects. However, although the rate of bone healing and new bone formation were strikingly improved when PSPC was used as compared with SSPC and SSSC group, it was still less satisfactory than that obtained with autografts.In summary, the results of above studies indicated that the perfusion bioreactor system developed in our lab is useful for the dynamical seeding of porous biomaterials and subsequent cell/scaffold constructs cultivation. The perfusion seeding/culture system generated constructs with remarkably uniform cell distribution at high efficiencies, and permitted the persistent nutrition supply and gas exchange into the centre of 3D scaffold, resulting in cells proliferation throughout the whole scaffold. In addition, this system significantly enhanced the osteogenic ability of the engineered bone grafts which could repair the long segmental radius defect efficiently in the rabbit.
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