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RAPID COMMUNICATION |
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;
* corresponding author, jmao2{at}uic.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: suture • bone • osteoblast • craniofacial • tissue engineering
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Much of our knowledge of suture development has derived from studies of heterogeneous congenital disorders collectively known as craniosynostosis or premature suture ossification prior to the completion of craniofacial growth (Cohen and MacLean, 2000; Opperman, 2000). Although many craniosynostosis phenotypes are linked to an increasing number of gene mutations, such as FGFRs and MSX2, over 50% of current craniosynostosis cases appear to be sporadic (Liu et al., 1995; Cohen and MacLean, 2000; Wilkie and Morriss-Kay, 2001; Ignelzi et al., 2003). Craniosynostosis occurs in one of approximately every 2500 live human births, and clinical phenotypes may manifest as visible craniofacial disfigurations, high intracranial pressure, and severe neurological disorders such as mental retardation, blindness, and seizure (Cohen and MacLean, 2000). Craniofacial surgery is the primary choice for correcting visible craniofacial disfigurations and relieving abnormally high intracranial pressure (Marsh, 2000; Tessier, 2000). Surgeons typically perform craniotomy in early childhood by dissecting fused sutures and leaving gaps of empirical size between involved calvarial bones, anticipating that the surgically created gaps will accommodate both brain growth and calvarial bone growth (Marsh, 2000; Tessier, 2000). Due to the unpredictability of craniofacial growth from the time of the first surgical correction of craniosynostosis, frequently performed in infants, to growth completion in adolescents, surgically created gaps may re-synostose. Secondary surgeries are then needed for re-synostosis, probably attributable to the fact that a synostosed suture with missing mesenchymal and fibrous interface is replaced by a surgically created gap still lacking a sustainable mesenchymal and fibrous interface (Marsh, 2000; Tessier, 2000).
In the present study, we attempted to tissue-engineer a composite construct utilizing autologous dermal fibroblastic cells isolated subcutaneously from the anterior tibial region, culture-expanding them, and seeding them in a gelatin scaffold sandwiched between two microporous collagen sponges loaded with recombinant human BMP2 (rhBMP2). The tissue-engineered composite construct was implanted into a surgically created calvarial defect in the center of the parietal bone devoid of natural sutures in the donor rabbit from which dermal fibroblasts had been isolated. The rationale for the present model was to create a tissue-engineered cranial suture from autologous cells and a potent osteogenic stimulant, BMP2, in an approach that is potentially aligned with eventual therapeutic application in patients with craniosynostosis. It was hypothesized that a cranial suture can be tissue-engineered from autologous fibroblasts and BMP2 loaded in biocompatible polymers.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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). Upon 90% confluence, the fibroblasts were trypsinized and subcultured at a density of 106 cells/100-mm dish. An absorbable gelatin scaffold (Pharmacia, Kalamazoo, MI, USA) was trimmed to 2 x 2 x 6 mm3 and immersed in cell suspension at 107 cells/mL in DMEM supplemented with 10% FBS under a light vacuum created by a 20-mL syringe for 1 hr (Fig. 1B), followed by two-hour incubation at 37°C.
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). We prepared recombinant human bone morphogenetic protein-2 (rhBMP2) by dissolving 2.5 µg rhBMP2 in 15 µL PBS at 4°C overnight (R&D Systems, Minneapolis, MN, USA) (Fig. 2A). The rationale for incorporating BMP2 was to simulate the high osteogenic potential of synostosed cranial sutures (De Pollack et al., 1996). The fibroblast-gelatin construct as prepared above was sandwiched between two rhBMP2-loaded collagen sponges to create a composite construct with an overall dimension of 6 x 2 x 8 mm3 (Fig. 2A).
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). Care was taken to ensure that the surgically created full-thickness calvarial defect was devoid of the adjacent coronal, sagittal, or lamboidal suture, and also to maintain the underlying dura mater intact (Fig. 2C). Three groups were devised: (1) two rhBMP2-loaded collagen constructs without either gelatin scaffold or autologous fibroblasts (no Y component in Fig. 2A) (N = 3); (2) two rhBMP2-loaded collagen constructs without autologous fibroblasts, but with an intervening gelatin scaffold (Y component without cells in Fig. 2A) (N = 3); and (3) two rhBMP2-loaded collagen constructs with autologous fibroblasts seeded in an intervening gelatin scaffold (Y component present in Fig. 2A) (N = 3). In the experimental group (Group 3), the tissue-engineered composite construct (Fig. 2D) was implanted into the surgically created calvarial defect of the corresponding donor rabbit from which the autologous fibroblasts had been harvested. In all 3 groups following placement of constructs, the previously deflected periosteum, subcutaneous tissue, and scalp were replaced. The incision was closed with 4-0 absorbable gut surgical suture. The rabbits were housed in a temperature- and light-controlled room (23–25°C, 14 hrs light/day), and given standard daily amounts of food and water.
Assessment of de novo Formation of Tissue-engineered Constructs
Four weeks after in vivo implantation, all rabbits were killed by pentobarbital overdose (300 mg/kg, i.v.). The entire calvarium was harvested with an orthopedic saw, fixed in 10% paraformaldehyde, demineralized in equal volumes of 20% sodium citrate and 50% formic acid, and embedded in paraffin. Serial 8-µm-thick sections were cut in the parasagittal plane by means of a microtome.
Mineral deposition in surgically implanted grafts was grossly examined by radiophotographic imaging at 90 kV and 15 mA with 10-second exposure. Three 8-µm-thick microscopic sections in the parasagittal plane were stained with hematoxylin and eosin: one from the center of the implanted graft and two from both sides of the graft, each 2 mm from the parasagittal midline. Quantitative histomorphometric analysis was performed on H&E-stained microscopic sections under a research microscope equipped with a digital camera. The widths of the fibrous interface between two active bone formation fronts were measured from no fewer than 6–8 lines per specimen, by computerized image analysis (cf. Fig. 3A'). By ANOVA with Bonferroni tests at an alpha level of P < 0.05, the average widths of the fibrous interface (f in Fig. 3C') in the tissue grafts were compared among those with seeded autologous fibroblasts in an intervening gelatin scaffold and those either without autologous fibroblasts or fibroblast-free gelatin scaffold.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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), indicating ossification of the surgically created calvarial defect without delivery of autologous fibroblasts, as corroborated by the corresponding microscopic image (Fig. 3A'). This was further confirmed by ossification of defects upon delivery of two rhBMP2-loaded microporous collagen sponges with an intervening fibroblast-free gelatin scaffold (Fig. 3B) and corresponding microscopic image (Fig. 3B'). In contrast, a band of radiolucency (between opposing white arrows in Fig. 3C) was present in the center of each of the tissue-engineered composite constructs, consisting of a fibroblast-seeded gelatin scaffold sandwiched between two rhBMP2-loaded microporous collagen sponges. The corresponding microscopic section (Fig. 3C') corroborated the radiographic image (Fig. 3C) by demonstrating a fibrous tissue interface (f in Fig. 3C') between the two osteogenic bone formation fronts (b in Fig. 3C'), indicating that autologous fibroblasts are essential for de novo formation of a tissue-engineered cranial-suture-like structure.
High-power microscopic examination of the tissue-engineered cranial suture indicated de novo formation of a non-mineralized, fibrous interface between two apparently new bone formation fronts in the surgically created calvarial defects devoid of natural cranial sutures (Fig. 4A). Fibroblast-like cells resided among apparent collagen fibers, with areas of angiogenesis in the tissue-engineered cranial suture (Fig. 4A). Columnar osteoblast-like cells lined up along the surface of the bone formation front with osteocyte-like cells embedded in mineralized bone that possessed distinct lacunae-like structures (Fig. 4A). These microscopic characteristics of the tissue-engineered cranial suture were similar to those of the adjacent natural sagittal suture, in which osteogenic cells lined the surface of the sutural bone formation front with embedded osteocytes also possessing lacunae (Fig. 4B). In comparison with the tissue-engineered cranial suture in Fig. 4A, tissue grafts consisting of two rhBMP2-loaded collagen sponges lacking an intervening fibroblast-free gelatin scaffold led to complete ossification of the surgically created defect (Fig. 4C), similar to complete ossification by tissue grafts consisting of two rhBMP2-loaded collagen sponges with an intervening fibroblast-free, gelatin scaffold (data not shown but cf. Fig. 3B'). Histomorphometric data measured from multiple microscopic sections demonstrated that the average width of tissue-engineered cranial sutures consisting of an 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 tissue grafts either without an intervening autologous fibroblast-gelatin scaffold (0.006 ± 0.004 mm) or with an intervening fibroblast-free gelatin scaffold (0 ± 0 mm; N = 3) (Fig. 4D).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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The make-up of cells in natural suture mesenchyme is not entirely clear, although there are certainly multiple cell types, such as mesenchymal cells, fibroblast-like cells, osteoblast-like cells, and any blood-vessel-borne cells (Rice et al., 2000; Wilkie and Morriss-Kay, 2001). Type I collagen is the most abundant collagen phenotype in cranial sutures (Meikle et al., 1982; Yen et al., 1989; Zimmerman et al., 1998; Rafferty and Herring, 1999). Thus, cranial sutures likely contain cells that produce type I collagen fibrils. The lack of ossification of suture mesenchyme during normal development indicates the presence of cells in suture mesenchyme capable of producing type I collagen fibrils and a mechanism(s) preventing their mineralization. Accordingly, autologous fibroblasts have been delivered in the present tissue-engineered composite constructs to initiate the fibrous interface between mineralized bones. The presently delivered fibroblasts are autologous in that tissue-engineered cranial sutures are formed de novo in rabbits from which dermal fibroblasts have been isolated. This autologous tissue-engineering approach may eliminate the potential problem of immune rejection by allografts or xenografts. The osteogenic cells lining the osteogenic fronts in the tissue-engineered cranial suture can derive from the overlying periosteum or underlying dura mater, or from mesenchymal cells resident in the dermal fibroblast population that differentiated into osteogenic cells in the presence of rhBMP2. Cell labeling is necessary to differentiate among these cell sources.
The present approach appears to complement previous meritorious efforts on suture transplantation or delaying the rate of suture synostosis. Surgical replacement of synostosed rabbit suture with an allogeneic suture graft, including dura mater from the wild-type rabbit, allows post-operative sutural growth to occur (Mooney et al., 2001), representing perhaps the most direct approach to the replacement of synostosed sutures. However, this allogeneic suture transplantation approach necessitates the creation of secondary bony defects, requires donor availability, and has the potential of immune rejection for human applications. Placement of e-PTFE membrane in a surgically created calvarial defect involving the rat sagittal suture led to a suture-like tissue similar to the original sagittal suture, and it was suggested that this suture-like tissue likely developed by migration of cells from the remaining portion of the sagittal suture (Mardas et al., 2002). The exogenously delivered rhBMP2 in the present work, known as a potent osteo-inductive factor, may have multiple effects. The rationale for incorporating BMP2, in the present work, was to simulate the high osteogenic potential of synostosed cranial sutures (De Pollack et al., 1996). The bone formation rate of synostosed sutures can be 50% higher than that of normal sutures (De Pollack et al., 1996). Even in the presence of rhBMP2, dermal fibroblasts seeded in gelatin scaffold intervening between two BMP2-loaded collagen sponges are capable of maintaining the presence of the tissue-engineered cranial suture. Application of rhBMP2 may also have implications in providing osteogenic stimulation in the adult craniosynostosis patient, due to a potential shortage of bone during surgical skull reshaping. The application of certain doses of TGFß-3 loaded in collagen gel placed over the suture delays the timed fusion of the rat interfrontal suture (Opperman et al., 1999, 2002), further substantiating the potential role the TGFß superfamily plays in regulating sutural fusion (Bradley et al., 2000; Greenwald et al., 2000; Moursi et al., 2003). It is probable that many of the currently pursued approaches toward regeneration of cranial sutures may find their applications among heterogeneous clinical phenotypes of craniosynostosis.
Much additional work is necessary before a tissue-engineered cranial suture from autologous cells is available for clinical applications. The long-term outcome must be evaluated. The effects of mechanical stresses on its development should be explored, because sutures also function as articulations for the transmission of mechanical stresses (Rafferty and Herring, 1999; Mao, 2002; Kopher et al., 2003; Kopher and Mao, 2003; Mao et al., 2003; Collins et al., 2004). The mechanical properties of a tissue-engineered cranial suture should be compared with those of natural cranial sutures (Radhakrishnan and Mao, 2004). The selection of biomaterials in the present study—namely, microporous collagen and gelatin scaffolds—was based on the rationale that they are biocompatible, biodegradable, and have been widely used in biomedical applications. Analysis of the present data, taken together, may serve as a ‘proof of concept’ for additional studies leading to tissue-engineered cranial sutures that surgeons can use to replace synostosed cranial sutures in craniosynostosis patients.
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ACKNOWLEDGMENTS |
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Received June 11, 2003; Last revision July 15, 2004; Accepted July 16, 2004
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Cohen MM Jr, MacLean RE, editors (2000). The biology of sutures. In: Craniosynostosis: diagnosis, evaluation, and management. New York: Raven Press, pp. 81–103.
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