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Tissue-engineered Rabbit Cranial Suture from Autologous Fibroblasts and BMP2
L. Hong and
J.J. Mao*
Tissue Engineering Laboratory, Rm 237, Departments of Orthodontics, Bioengineering, and Anatomy and Cell Biology, Univ. of Illinois at Chicago, MC 841, 801 South Paulina Street, Chicago, IL 60612-7211, USA;
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Figure 1. Isolation, culture-expansion, and seeding of autologous dermal fibroblasts. A 5-mm incision was made in the anterior tibia. A small piece of subcutaneous fibrous tissue (approx. 3 x 3 mm2) was removed. The isolated dermal fibroblasts were plate-cultured and expanded (A). (B) An absorbable gelatin scaffold was trimmed to 2 x 2 x 6 mm3 and immersed in the suspension of culture-expanded dermal fibroblasts at a density of 107 cells/mL.
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Figure 2. Experimental procedures. (A) Dermal fibroblast-seeded gelatin scaffold (Y component) is sandwiched between two microporous collagen sponges loaded with rhBMP2 (two X components). (B) Location of surgically created calvarial defect in the parietal bone in relation to the natural sagittal suture (s) and coronal suture (c). (C) Surgical creation of full-thickness calvarial defect with a dimension of 6 x 2 x 8 mm3 in the center of the rabbit parietal bone devoid of natural cranial sutures. The adjacent sagittal suture (s) is shown. The dura mater was kept intact. Following creation of surgical calvarial defects in the corresponding rabbits from which dermal fibroblasts had been obtained, composite tissue grafts were implanted into the center of the parietal bone devoid of natural cranial sutures (N = 3). In one control group, tissue grafts consisting of two rhBMP2-loaded collagen sponges without autologous fibroblasts were implanted in age- and sex-matched rabbits (N = 3). In another control group, tissue grafts consisting of two rhBMP2-loaded collagen sponges with an intervening fibroblast-free gelatin scaffold were implanted in age- and sex-matched rabbits (N = 3). (D) The surgically created calvarial defect was filled with a composite tissue construct consisting of autologous autologous fibroblast-seeded gelatin scaffold (Y component) that was sandwiched between two rhBMP2-loaded microporous collagen sponges (two X components). The adjacent sagittal suture (s) was not part of the surgically created calvarial defect.
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Figure 3. Radiographic and photomicrographic images of tissue-engineered cranial suture and controls. (A) Representative radiographic image showing a lack of radiolucency in the tissue graft with two rhBMP2-loaded microporous collagen sponges without intervening autologous fibroblasts loaded in a gelatin scaffold. The adjacent coronal suture is labeled as ‘c’. (A') Corresponding microscopic image showing ossification of the surgically created calvarial defect without delivery of autologous fibroblasts or an intervening gelatin scaffold. (B) Representative radiographic image showing a lack of radiolucency in the tissue grafts with an intervening fibroblast-free, gelatin scaffold between two rhBMP2-loaded microporous collagen sponges. The adjacent coronal and sagittal sutures are labeled as ‘c’ and ‘s’, respectively. (B') Corresponding microscopic image showing ossification of the surgically created calvarial defect with fibroblast-free gelatin scaffold. (C) Representative radiographic image showing a band of radiolucency (between opposing white arrows) corresponding to the area of autologous fibroblasts loaded in a gelatin scaffold intervening between two rhBMP2-loaded microporous collagen sponges, indicating a fibrous tissue interface between mineralized bones. The adjacent coronal suture is labeled as ‘c’. (C') Corresponding microscopic image showing de novo formation of a fibrous tissue interface (f) between two mineralized bone segments (b) in the calvarial defect filled with fibroblast-seeded gelatin scaffold sandwiched between two rhBMP-loaded collagen sponges.
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Figure 4. High-power examination of tissue-engineered cranial suture and controls. (A) The tissue-engineered cranial suture consisted of collagen-fiber-like structures (c), fibroblast-like cells (Fb), osteoblast-like cells (Ob), and osteocyte-like cells (Oy) in apparently mineralized bone. Osteoblast-like cells formed an approximate layer on the surface of an apparent bone formation front. (B) The adjacent natural sagittal suture showed fibroblast-like cells in suture mesenchyme, osteoblasts lining the sutural bone formation front, and osteocytes (Oy) in the mineralized bone. (C) Complete bony fusion occurred in the surgically created calvarial defect filled with tissue grafts consisting of two rhBMP-loaded collagen sponges, but without the intervening fibroblast-seeded gelatin scaffold. (D) Histomorphometric analysis and statistical comparison of the widths of the fibrous tissue interface between two mineralized bone formation fronts. The average width of tissue-engineered sutures consisting of autologous fibroblast-populated gelatin scaffold sandwiched between two rhBMP2-loaded microporous collagen sponges was 1.13 ± 0.39 mm (SD), significantly greater than the average widths of either tissue grafts without intervening autologous fibroblast-gelatin scaffolds (0.006 ± 0.004 mm) or an intervening fibroblast-free gelatin scaffold (0 ± 0 mm; N = 3) **P < 0.01.
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Journal of Dental Research, Vol. 83, No. 10,
751-756 (2004)
DOI: 10.1177/154405910408301003
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