Introduction
Natural biopolymers such as collagen and fibrin have been largely used in bone regenerative applications. Collagen, by definition, is characterized by a unique triple-helix formation that extends over a large portion of its structure, and type I collagen is a main organic component of extracellular matrix in bone tissues. Type I collagen influences the development and maintenance of the osteoblast phenotype and induces differentiation of bone marrow stromal cells along the osteoblast pathway [1,2]. In addition to the specific motifs of collagen that can bind cellular integrin receptors [3,4], its fiber structure can contribute to osteoblast differentiation by stabilizing the Runx2 protein [5]. Fibrous structure of collagen has been noticed to enhance the osteoblast differentiation, and mimicking the fibrous morphology of collagen, fibrous engineered matrices have been developed and have shown good biological performance for bone regeneration [6-8].
Fibrin, a fibrous polymer, has been applied in biomedical research for hard tissue and soft tissue repair including bone, cartilage, skins, neurons, heart valves, blood vessels, and cornea [9]. Many commercially available fibrins made from human fibrinogen and thrombin including several growth factors has been introduced for tissue regeneration in various clinical applications [10]. Generally accepted advantages of fibrin are biocompatibility and biodegradability, because it is a first formed natural scaffold in body that assists wound healing after damage through blood clotting cascade. Controls of polymerization time, pore size, and fiber thickness are relatively simple by modifying mixing technique in fibrin gel, thus it has a mold-ability suitable for injectable scaffolds [11,12]. Moreover, it can be prepared by autologous blood products during operation avoiding an immunogenicity of allogenic application [13,14].
Based on the biological properties, collagen and fibrin have been used as a scaffolding material, as a coating agent covering some synthetic polymers, and as a carrier for delivering genes, drugs, or bioactive agents [9]. Despite the frequent use in bone regenerative approaches, their comparative efficacies need to be evaluated. Previously, we compared the effects of fibrin and collagen on protein adsorption, proliferation, and differentiation of osteoblasts [15]. Compared with collagen, fibrin adsorbed greater amounts of serum fibronectin. Fibrin allowed the proliferation of MC3T3-E1 pre-osteoblasts larger than collagen and promoted the osteoblast differentiation at higher levels than collagen. In this study, we addressed the superiority of fibrin to collagen in supporting bone healing through an in vivo study using a model of critical size calvarial bone defects in rats.
Materials and methods
Reagents
Bovine fibrinogen, thrombin, and aminocaproic acid were purchased from Sigma (St. Louis, MO, USA). Rat tail type I collagen was purchased from BD Bioscience (San Jose, CA, USA). Anti-Runx2, anti-actin, and HRP-conjugated IgG antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Preparation of Matrices
Collagen matrix was prepared by pouring 3 mg/ml type I collagen in PBS (pH 7.4) into a culture dish and was solidified in an incubator at 37°C. Fibrin matrix was prepared by mixing fibrinogen and thrombin solutions, which contained 3 mg/ml fibrinogen in PBS (pH7.4), 1 U/ml thrombin, 50 mg/ml aminocaproic acid, and 1 mM of CaCl2.
In vivo rat calvarial defect model: animals and surgical procedures
All procedures were approved by the institutional review board of animal laboratories at Seoul National University (authorization SNU-050608-3). Sprague-Dawley rats (250-300 g, 6 rats per group) were anesthetized via a peritoneal injection of a mixture of Zoletil (Virbac, Carros, France) and Rompun (Bayer Korea, Ansan, Korea). Surgical procedures were performed, as described in a previous report [16]. In brief, an incision was made along the midline, and the full-thickness skin and periosteum were elevated to expose the calvarial bone surface. Careful drilling using an 8 mm external diameter trephine bur was performed around the sagittal suture to prepare a standardized, round, segmental defect. Then, the defect was injected and filled with prepared fibrin or collagen gels. The periosteum and skin were closed in layers with absorbable 4/0 vicryl (Ethicon, Edinburgh, UK) and non-absorbable 3/0 black silk (Ethicon, Edinburgh, UK).
Micro-computed tomography (micro-CT)
The rats were sacrificed at 4 or 8 weeks after surgery. The calvarial bone was excised, trimmed, and fixed in 4% paraformaldehyde for 24 hours at 4°C. The specimens were scanned using a micro-CT scanner (Skyscan, Aartselaar, Belgium) and then reconstructed with TomoNT software (Skyscan), in a previous report [16]. The volume of radiopaque matter in the regions of interest (ROI) was measured for four animals in each group. The data are presented as the mean ± standard error of the mean (SEM).
Histology
After the micro-CT measurements, the specimens were decalcified in 10% EDTA at 4°C for 4 weeks with constant gentle rocking, after which they were dehydrated with ascending graded alcohol, embedded in paraffin, sectioned at 5 μm intervals and stained with hematoxylin and eosin (H&E).
Immunostaining
Immunostaining was performed for Runx2. The sections were deparaffinized, washed in PBS, and heated in a microwave for 2 min. The sections were incubated in a 3% H2O2 solution to quench any endogenous peroxidases in the section. The sections were blocked with 1% anti-goat serum in PBS for 30 min at room temperature, and incubated in primary anti-Runx2 antibody at 4°C overnight. The sections were also incubated in isotype-control antibodies (anti-rabbit IgG for Runx2), which served as a negative control for immunostaining. After washing with PBS, the sections were incubated in horseradish peroxidase-conjugated secondary antibodies for 30 min at room temperature; this was followed by treatment with diaminobenzidene solution (Dako Cytomation, Carpinteria, CA, USA).
Statistical analysis
All of the results are expressed as the means ± S.E.M. (n=4). All experiments were repeated three times. Significant differences were analyzed using ANOVA. P values < 0.05 were considered to indicate a statistically significant difference.
Results
To compare the effects of fibrin and collagen on bone defect healing, critical-sized calvarial defects were made and filled with fibrin and collagen gels. A micro-CT analysis was performed 8 weeks after surgery. Three-dimensional images were reconstructed and bone volume fractions were measured in the ROIs. In the non-filled group, a bony defect remained with a slight ingrowth of marginal bone (data not shown). There was an approximately 30% increase of the bone volume fraction in the ROI of the fibrin-filled group, compared with collagen-filled group (Fig. 1). Histological observation revealed active bone formation in the fibrin-filled bone sections (Fig. 2). Discrete regions of immature bone containing numerous bone cells were observed in the fibrin-filled defects. These results were consistent with the in vitro culture experiments [15]. Previously, we observed that both collagen and fibrin increase the levels of Runx2 protein in cultures [5,15,17]. Immunostaining for Runx2 was also performed to compare the Runx2 levels during the healing of bony defects in vivo. The cells in the regenerative tissues of the fibrin-filled defects were immunostained strongly for Runx2, while collagen-placed defects were stained weakly, indicating that there was active osteogenic differentiation in the fibrin– filled defects (Fig. 3).
Discussion
Our results in this study together combined with previous in vitro ones consistently indicate that fibrin is a better biopolymer than collagen in terms of the features of protein adsorption, enhancing osteoblast proliferation and differentiation, and supporting bone healing. These findings suggest that fibrin can be considered preferentially to collagen in the use as a component of composite scaffolds for enhancing the biological performance of synthetic polymer scaffolds or as a sole material in various bone regenerative applications. Furthermore, fibrin can be prepared by autologous blood products, which highlights its importance in clinical applications [13,14]. The biological properties of fibrin can be modulated by ingredients [17-19], and adequate mechanical strength and timely degrading properties are also needed for the bone regenerative approaches. Further studies are required for optimization of fibrin-based biomaterials.
Runx2 is widely accepted as an early osteogenic transcription factor that can switch on osteoblast differentiation [20-23]. Runx2-knockout mice display complete bone loss due to arrested osteoblast maturation, and Runx2 plays a critical role in osteoblast marker gene expression, including ALP, osteocalcin, and bone sialoprotein [20,21]. Most osteogenic factors, such as BMPs, can induce the expression of Runx2, and various extracellular signals can affect the transcriptional activity of Runx2 [22,23]. In this study, we showed that the level of Runx2 was higher in the regenerative tissues of fibrin-filled defects than in those of collagen-filled ones, consistent with in vitro results [15]. These results suggest that the signals from the fibrin matrix may increase the levels of Runx2 which can contribute to the biological performance of fibrin-based biomaterials.
