increased perfusion rate and cell-seeding ...increased perfusion rate and cell-seeding density...

INCREASED PERFUSION RATE AND CELL-SEEDING DENSITY ENHANCE TISSUE-ENGINEERING OFHUMAN BONE

1 Grayson WL, 1Bhumiratana S, 1Chao PHG, 2CanniZzaro C, 1Liu X, 1Guo E, 3Caplan A, and 1* Vunjak-Novakovic G

1 Department of Biomedical Engineering, Columbia University New York NY, USA , 2Harvard-MIT Division of Health Sciences & Technology,Cambridge MA, USA, 3Skeletal Research Center, Case Western University, Cleveland OH USA: *gv2131@columbia.edu

Introduction:The ability of human mesenchymal stem cells (hMSC) to differentiateinto osteoblastic phenotypes has been rigorously demonstrated (1).However, the specific set of molecular factors and physical signalsoptimized to orchestrate hMSC differentiation into engineered boneconstructs remain unresolved. For practical applications, it is critical todevelop engineered constructs that would be immediately capable ofload bearing upon implantation into host tissue, and have the capacityfor structural integration and remodeling. We chose scaffolds fabricatedfrom fully decellularized, trabecular bone because of their biomimeticstructure, composition and mechanical properties. In our previousstudies, we have established that hydrodynamic shear stresses andimproved nutrient transfer associated with medium perfusion enhancethe spatial uniformity and structural fidelity of hMSC-based engineeredbone constructs. We also established that these constructs can repaircritical size calvarial (2) and femoral (3) defects in a nude rat model.The present study was designed to determine the role of (a)hydrodynamic shear (by varying the rate of medium perfusion throughthe developing tissue) and (b) cell-cell contacts (by varying the initialcell density within scaffolds), by using an advanced modular bioreactor.

Methods:hMSCs were isolated from fresh human bone marrow aspiratesfollowing an established protocol (4), and expanded to the 4th passage(P4) in basal medium supplemented with FBS from lots selected fortheir capacity to support proliferation and osteogenesic differentiation.Trabecular bovine carpal bones were cored into discs (4 mm ∅ x 4 mmthick) and decellularized using high-velocity streams of water. Theresulting scaffolds were completely free of cellular material and hadvoid volumes of ~ 70%. Scaffolds were seeded with P4 hMSCs andcultured for a period of 5 weeks in bioreactors with medium perfusionthrough the constructs (Fig. 1A,B). Osteogenic medium (DMEM withFBS, β-glycerophosphate, dexamethasone, and ascorbic acid 2-phosphate) was used and replaced at a rate of 50% twice weekly. Fourexperimental groups were established by varying the initial cell densityand perfusion rate through the constructs: (A) – unseeded scaffoldscultured at high flow rate (400 µm/s); (B) – low cell density (30 x 106

cells/ml) - high flow rate (400 µm/sec); (C) – low cell density (30 x 106

cells/m) – low flow rate (100 µm/s); (D) – high cell density (60 x 106

cells/ml) – low flow rate (100 µm/s). Constructs were assessedbiochemically (DNA; alkaline phosphatase, AP), histologically (H&E)and structurally (microcomputer tomography (µCT)).

Results:Up to six scaffolds were placed in each bioreactor module and perfuseduniformly with culture medium (Fig. 1B). Cells within constructsmaintained their viability through the seeding process and subsequentculture (Fig. 1C,D). As expected, the weight, DNA content and APactivity after 5 weeks of culture were significantly higher for cell-basedconstructs than unseeded scaffolds (Table 1). Among the seededsamples, the DNA content was highest for group B, whereas the wetweights and AP contents were all comparable. µCT imaging indicatedthat group D had significantly more mineral than other groups and thatgroups B, C and D contained greater mineralized deposits than group Ademonstrating that hMSCs are creating a bone-like tissue matrix (Fig. 2A-D). Histological analyses confirmed the ability of cells to occupy thescaffold pores and secrete matrix components. In some regions the celland matrix densities approached those of native tissues (Fig. 2E-H).

Discussion:Decellularized bovine trabecular bone provided excellent scaffolds forthe growth and differentiation of hMSCs into bone tissue. On averagethe construct wet weight increased 29% over the 5-week culture period,consistent with the measured increases in construct cellularity andmatrix deposits. The AP activity in all seeded groups was considerably

elevated at 5 weeks. Interestingly, the group with the highest seedingdensity (D) did not exhibit the most elevated DNA content at the end ofthe culture period, but did appear in µCT to have much highermineralization than the other groups. This may be related either to thefact that hMSCs proliferate more quickly at low seeding densities, orthat differentiation and proliferation are mutually exclusive and at higherseeding densities hMSCs tend toward differentiation. Consistent with theprevious reports that increased shear stresses enhanced mineralization ofosteo-induced hMSCs (5), we observed that higher flow rates resulted inhigher construct cellularity, due to either higher shear alone or also toimproved nutrient delivery. Histological analyses corroborated thesefindings. Although medium perfusion enabled the maintenance of viablecells throughout the volumes of these rather thick constructs (4 mm), thecell distributions were not completely uniform. In all cell-seededconstructs, the top and bottom regions contained areas with high celldensities, while some of the pores at the construct centers appearedacellular. At higher perfusion rate, the central acellular regions weresmaller, and the high cell density regions were thicker. Taken together,these data support the utility of decellularized bone and hMSCs fortissue engineering, and suggest that the properties of engineered boneimprove when cell density and perfusion rates are increased.

Figure 1. Validation of the bioreactor system (A) Single modularbioreactor unit with decellularized bone scaffolds seeded with hMSCs.(B) Dye-transport study showing uniform perfusion of all scaffolds. (C)Live-stain of cells seeded into bone scaffolds after 1 week. (D) Deadstain of same region indicating very few dead cells

Table 1: Construct weights and compositionsConstructproperties Group A Group B Group C Group D

Wet weight ( mg) 63.3 ± 1.1 79.8 ± 2.7a

85.8 ± 2.4 a

78.1 ± 7.7 a

DNA (ng/mg ww) 0.96 ± 0.22 41.66 ± 2.16 a

29.59 ± 5.48 a, b

34.58 ± 8.31 a

AP (umol pNP / mg ww)

0.00 ± 0.00 0.30 ± 0.01 a

0.29 ± 0.02 a 0.29 ± 0.04

a

Data are the Avg ± SD (n=3).a -Statistically different from Group A: b -Statistically different from Group B

Fig. 2. Construct structures. Planar µCT scans. (A) Scaffold (B) Highflow-low cell density (C) Low flow-low cell density (D) Low flow-highcell density H&E staining (E) Unseeded bone (F) High flow-low celldensity (G) Low flow-low cell density (H) Native bone

Acknowledgements:The work was funded by the NIH (5R01DE016525 and P41-EB002520).

References:(1) Caplan AI., Tiss Eng 11: 1198-1211, 2005. (2) Meinel et al, Bone 37:688-698, 2005 (3) Meinel et al, June 3 2006 [Epub ahead of print] 2006(4) Lennon et al, In Vitro Cell Dev Biol 32: 602-611, 1996 (5) Sikavitsaset al, PNAS 100: 14683 – 14688, 2003.

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53rd Annual Meeting of the Orthopaedic Research Society

Paper No: 0124