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. 2010 Jun;38(6):2167-82.
doi: 10.1007/s10439-010-0028-0. Epub 2010 Apr 9.

Osteochondral interface tissue engineering using macroscopic gradients of bioactive signals

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Osteochondral interface tissue engineering using macroscopic gradients of bioactive signals

Nathan H Dormer et al. Ann Biomed Eng. 2010 Jun.

Abstract

Continuous gradients exist at osteochondral interfaces, which may be engineered by applying spatially patterned gradients of biological cues. In the present study, a protein-loaded microsphere-based scaffold fabrication strategy was applied to achieve spatially and temporally controlled delivery of bioactive signals in three-dimensional (3D) tissue engineering scaffolds. Bone morphogenetic protein-2 and transforming growth factor-beta(1)-loaded poly(D,L-lactic-co-glycolic acid) microspheres were utilized with a gradient scaffold fabrication technology to produce microsphere-based scaffolds containing opposing gradients of these signals. Constructs were then seeded with human bone marrow stromal cells (hBMSCs) or human umbilical cord mesenchymal stromal cells (hUCMSCs), and osteochondral tissue regeneration was assessed in gradient scaffolds and compared to multiple control groups. Following a 6-week cell culture, the gradient scaffolds produced regionalized extracellular matrix, and outperformed the blank control scaffolds in cell number, glycosaminoglycan production, collagen content, alkaline phosphatase activity, and in some instances, gene expression of major osteogenic and chondrogenic markers. These results suggest that engineered signal gradients may be beneficial for osteochondral tissue engineering.

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Figures

FIGURE 1
FIGURE 1
Microparticle and scaffold fabrication process. (a) Microspheres were made from a polymer stream (20% w/v PLGA in DCM) and annular carrier stream (0.5% w/v PVA in ddH2O) with an ultrasonic transducer; (b) Programmable pumps created a gradient in microsphere types based on a time-dependent process; (c) Experimental groups and particle sizes for hBMSCs and hUCMSCs.
FIGURE 2
FIGURE 2
Sectioning and orienting construct slices. Constructs were placed in culture with the “chondrogenic” side upwards and the “osteogenic” side towards the well plate surface. This orientation was maintained throughout the harvesting and histological procedure and for photo orientation.
FIGURE 3
FIGURE 3
Size characterizations for microparticles. Using a multisizer, fabricated particle batches were checked to confirm size distribution. The average diameter of all larger microspheres was approximately 220 µm (BMP-2-loaded and Blank 220 Groups ~215 µm, TGF-β1-loaded ~225 µm), while the smaller microspheres measured near 70 µm.
FIGURE 4
FIGURE 4
Protein release from PLGA constructs. Release samples were taken at 12 h, and 1, 2, 3, 5, 7, 10, 14, and 21 days for (a) TGF-β1 and (b) BMP-2 microparticles and scaffolds. Percent released was calculated from the total amount of detected protein entrapped. Scaffolds showed an accelerated release profile compared to microspheres, and the onset of the third phase of release is evident at day 21. Values are reported as mean ± standard deviation, n = 3.
FIGURE 5
FIGURE 5
Gene expression of collagen type I, Runx2, and Sox9 in hBMSC constructs. Gradient constructs enhanced gene expression of (a) collagen type I, and (c) Sox9 at 6 weeks. Runx 2 expression (b) was not evident in hBMSC Blank 220 constructs at 6 weeks, whereas it was in Gradient groups. Values are reported as mean ± standard deviation, n = 4. Statistically significant change of expression @ = over the calibrator (hUCMSC Blank 220) at that time point (p< 0.05), # = over its value at the previous time point (p < 0.05), and * = over the control (Blank 220) at that time point (p < 0.05).
FIGURE 6
FIGURE 6
DNA content for all hUCMSC and hBMSC groups at 0, 3, and 6 weeks. All groups had statistically significant increases over the week 0 cell number by 6 weeks, hUCMSC growth factor groups and all hBMSC groups had statistically significant increases over the week 3 values, and select hUCMSC growth factor constructs had significantly higher values compared to the control at 6 weeks (hUCMSC Blank 220). Values are reported as mean ± standard deviation, n = 4. Statistically significant change @ = over the week 0 value (Blank 220) (p < 0.05), # = over its value at the previous time point (p < 0.05), and * = over the control (Blank 220) at that time point (p < 0.05).
FIGURE 7
FIGURE 7
GAG content for all hUCMSC and hBMSC groups at 0, 3, and 6 weeks. (a) At 6 weeks, all groups had statistically significant increases in GAG content over the week 0 and week 3 values. Only hUCMSC Biphasic and Gradient constructs had significantly higher GAG content compared to the control at 6 weeks. (b) hUCMSC on Blank 70 scaffolds had a diminished GAG/DNA at week 0, and hUCMSC Gradient constructs had increased GAG/DNA production over the control at 3 weeks. Values are reported as mean ± standard deviation, n = 4. Statistically significant change @ = over the week 0 value (Blank 220) (p < 0.05), # = over its value at the previous time point (p < 0.05), and * = over the control (Blank 220) at that time point (p < 0.05).
FIGURE 8
FIGURE 8
Net hydroxyproline production for all hUCMSC and hBMSC groups at 0, 3, and 6 weeks. (a) Most hUCMSC groups had statistically significant increases in hydroxyproline from week 0 to week 6, but only hUCMSC Gradient constructs had significantly higher production relative to the control at 6 weeks. Notably, this value was at least 2 times higher than any other group at the end of culture. At 6 weeks, the hUCMSC Chondrogenic group also showed a statistical decrease in net hydroxyproline compared to the Blank 220 group at 6 weeks. (b) hUCMSC Gradient constructs produced more HYP/DNA than the control at 3 weeks, although by 6 weeks the levels were not different from the control. Values are reported as mean ± standard deviation, n = 4. Statistically significant change @ = over the week 0 value (Blank 220) (p < 0.05), # = over its value at the previous time point (p < 0.05), * = over the control (Blank 220) at that time point (p < 0.05), and ! = over any other group at that timepoint (p < 0.05).
FIGURE 9
FIGURE 9
Alkaline phosphatase activity of all hUCMSC and hBMSC constructs at 0, 1, 2, 3, and 6 weeks. Only the hUCMSC Biphasic groups showed increases after week 2. Values are reported as mean ± standard deviation, n = 4. Statistically significant change @ = over the week 0 value (Blank 220) (p < 0.05), # = over its value at the previous time point (p < 0.05), and * = over the control (Blank 220) at that time point (p < 0.05).
FIGURE 10
FIGURE 10
Histological staining (Safranin-O/Fast Green and Alizarin Red) of constructs at 6 weeks. Orientation is detailed in Fig. 2 (for biphasic and gradient groups, the chondrogenic side is the top, and the osteogenic side is the bottom). (a) hUCMSC growth-factor constructs showed significantly more GAG formation and calcium deposition. hUCMSC Gradient constructs had GAG formation localized to the scaffold top (red arrows). hUCMSC Osteogenic groups appeared to have more homogenous calcium deposition than any other group. (b) hBMSC Blank 220 scaffolds had little GAG formation and even calcium deposition, whereas the Gradient group had light GAG staining localized to the scaffold top, which dissipated towards the center. Scale bar = 200 µm.

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