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Bone morphogenetic proteins (BMPs) can induce bone formation in vivo when combined with appropriate carriers. Several materials, including animal collagens and synthetic polymers, have been evaluated as carriers for BMPs. We examined alginate, an approved biomaterial for human use, as a carrier for BMP-7. In a mouse model of ectopic bone formation, the following four carriers for recombinant human OP-1 (BMP-7) were tested: alginate crosslinked by divalent cations (DC alginate), alginate crosslinked by covalent bonds (CB alginate), Type I atelocollagen, and poly-D,L-lactic acid-polyethyleneglycol block copolymer (PLA-PEG). Discs of carrier materials (5-mm diameter) containing OP-1 (3–30 μg) were implanted beneath the fascia of the back muscles in six mice per group. These discs were recovered 3 weeks after implantation and subjected to radiographic and histologic studies. Ectopic bone formation occurred in a dose-dependent manner after the implantation of DC alginate, atelocollagen, and PLA-PEG, but occurred only at the highest dose implanted with CB alginate. Bone formation with DC alginate/OP-1 composites was equivalent to that with atelocollagen/OP-1 composites. Our data suggest DC alginate, a material free of animal products that is already approved by the FDA and other authorities, is a safe and potent carrier for OP-1. This carrier may also be applicable to various other situations in the orthopaedic field.
The repair capacity of human bone appears to depend on different very complex processes, such as vascularization, biomechanics, and topography. When damage is severe, as occurs with comminuted fractures or large bone defects after tumor resection, it is difficult for bone union to be achieved . In such cases, autologous or allogenic bone grafting has been used. Autologous bone grafting is common and is still the gold standard, but has several disadvantages, including a limited supply of suitable bone and the risk of chronic pain, nerve damage, fracture, and cosmetic problems at the donor site. Allografts have no donor site problems, but there is the potential risk of disease or an immunologic reaction [10, 21]. For these reasons, the use of bone substitutes such as calcium phosphate-based porous ceramics has been increasing [18, 33]. These bioceramics are highly biocompatible and demonstrate osteoconduction, which is the ability to bind to bone matrix directly. However, they have no osteoinduction, which is the ability to induce new bone formation at ectopic sites.
Bone morphogenetic proteins (BMPs) belong to the transforming growth factor superfamily, are known to elicit new bone formation in vivo, and may play a leading role in bone tissue engineering [36, 38]. To date, three types of BMP-based bone tissue engineering have been tried, which are cell therapy, gene therapy, and cytokine therapy . Cell therapy involves the transplantation of autologous bone marrow mesenchymal cells after differentiation has been induced by BMP, but considerable resources and time are needed to culture the necessary cells [22, 34]. Gene therapy involves the transduction of genes encoding BMPs into cells at the site of damage [2, 7]. BMP-transduced cells may work more efficiently, compared with a single dose of recombinant cytokine therapy. However, gene therapy still has unsolved problems such as tumorigenesis and immunogenicity. Cytokine therapy involves the implantation of BMPs together with a carrier material that acts as a drug delivery system. We believe cytokine therapy is the most promising of these three approaches in terms of practical application. Cytokine therapy seems most convenient and safe, but the cost is very high because a large amount of BMP is required to achieve bone growth in humans. To increase the cost effectiveness of BMP, an appropriate carrier material is necessary.
Previous studies have indicated adequate in vivo new bone formation cannot be obtained by simply injecting a solution of BMP into the area where bone is needed . For cytokine therapy, an appropriate carrier material is needed that retains BMP and releases it slowly, while serving as a scaffold for new bone formation [28, 29]. Several materials have already been evaluated as BMP carriers, including collagen obtained from animal sources [3, 11, 13, 31], synthetic polymers [14, 15, 19], tricalcium phosphate , and other inorganic materials . Atelocollagen is a well-established BMP carrier, and has already been used clinically. PLA-PEG , one of the synthetic polymers, has been reported as a potent carrier for BMPs [23, 25, 26]. Although all of these materials can induce bone formation at ectopic and orthotopic sites, none of them has achieved widespread use because of disadvantages, such as the potential risk of disease transmission, fragility, stickiness, and difficulty in obtaining approval for clinical use [1, 4, 5, 14]. We therefore focused on alginate, which is already approved by the FDA for human use as a wound dressing and food additive [8, 37].
Alginate is a water-soluble linear polysaccharide extracted from brown seaweed that is composed of one to four linked α-L-gluronic and β-D-mannuronic acid monomers . Gelation of alginate occurs as a result of crosslinking by divalent cations or covalent bonds . Therefore, two types of alginate wound dressing products are available on the market and both effectively promote wound healing by maintaining a moist environment. One is an alginate crosslinked by divalent cations (DC alginate) and the other is an alginate crosslinked by covalent bonds (CB alginate).
To determine whether alginate can be a carrier for BMP, we compared four materials as carriers for OP-1(BMP-7) using the bone mineral content (BMC) measurement and alkaline phosphatase (ALP) activity measurement of the bone nodules ectopically induced by carrier materials/OP-1 composites. The four materials were DC alginate, CB alginate, atelocollagen, and PLA-PEG. We hypothesized: (1) BMC of bone nodules ectopically induced by DC alginate/OP-1 composite and/or CB alginate/OP-1 composite are equivalent or superior to those by atelocollagen and PLA-PEG; (2) ALP activity of bone nodules ectopically induced by DC alginate/OP-1 composite and/or CB alginate/OP-1 composite are equivalent or superior to those by atelocollagen and PLA-PEG by radiographic appearance and histology of the ectopic bone nodules; and (3) DC alginate and/or CB alginate have appropriate in vitro release kinetics of OP-1 equivalent to atelocollagen and PLA-PEG.
To verify our first hypothesis, we designed the following experiment (Experiment 1; Table 1). For each dose of OP-1 (3, 10, and 30 μg), 24 4-week-old male ICR mice were assigned to four equally sized independent groups after they were housed and acclimatized in cages with free access to food and water for 1 week. The four independent groups were DC alginate group, CB alginate group, atelocollagen group, and PLA-PEG group. The mice were anesthetized by intraperitoneal injection of pentobarbital. As reported previously [14, 15], carrier material/OP-1 composites were implanted beneath the fascia of the back muscles on the left side (one composite per animal). The experiment was designed under the assumption that the justifiable difference (effect size) between the atelocollagen group as a control and the other groups was 6 mg in BMC and the standard deviation within each group was 3 from the result of the previous study . For the experiment to detect the difference at the 5% significance level with 90% power in the one-way analysis of variance, the necessary number of mice per group was six. Three weeks after implantation, these mice were killed and ectopic bone induced at the implantation site was harvested for further evaluation, including BMC measurement, radiography, and histological examination. The experimental protocol was approved by the Animal Experiment Committee of Osaka University, and the experiments were carried out in accordance with the Osaka University guidelines for care and use of laboratory animals.
To verify the second hypothesis, we repeated the Experiment 1 and obtained radiographs and measured ALP activity (Experiment 2; Table 1).
The BMC of the harvested discs was determined by dual-energy xray absorptiometry (DXA) using an animal bone densitometer (PIXImus; Lunar Corp, Madison, WI) and was expressed as milligrams per ossicle. Radiographs were obtained with a soft xray apparatus (MX-20 Faxitron®; Torrex and Micro Focus Systems, Wheeling, IL).
To measure ALP activity, the harvested discs were crushed, homogenized in 0.2% Nonidet® P-40 containing 1 mmol/L MgCl2, and centrifuged at 10,000 rpm for 1 minute at 4°C. The supernatants thus obtained were assayed for ALP activity with an Alkaline Phospha B-Test Wako kit (Wako Pure Chemical Industries, Ltd, Osaka, Japan) using p-nitrophenyl phosphate (p-NP) as a substrate. The protein content was measured with a Pierce® BCA protein assay kit (Thermo Fisher Scientific Inc, Rockford, IL), and ALP activity was standardized by the protein content and expressed as nmol p-NP/minute/mg protein.
After radiography and BMC measurement, the samples were fixed in 10% neutral formalin, decalcified with ethylenediaminetetraacetic acid (pH 7.4), dehydrated in a graded ethanol series, and embedded in paraffin. One section per group with the largest tissue area (5-μm thick) were cut and stained with hematoxylin and eosin for observation under a light microscope. The formation of new bone, new bone marrow, degradation of the materials, and inflammatory change were evaluated by a pathologist (AM) and an orthopaedic surgeon (KN).
To verify the third hypothesis that DC alginate and/or CB alginate have appropriate in vitro release kinetics of OP-1, we incubated carrier materials/OP-1 composites in centrifuge tubes containing 1000 μL phosphate-buffered saline (PBS; Invitrogen, Carlsbad, CA) and kept for 21 days at 37°C. For each composite group, three samples were examined. The PBS in the tubes was replaced every 2 days, and then 100 μL was collected for assay after 24 hours. The amount of OP-1 was determined by measurement with a commercial BMP-7 ELISA kit (R&D Systems Inc, Minneapolis, MN) on days 1, 3, 7, 13, and 21 according to the manufacturer’s instruction.
OP-1 (BMP-7 in a lyophilized 5% lactose formulation) was provided by Stryker Biotech (Hopkinton, MA). OP-1 was dissolved in distilled water at a concentration of 2 μg/μL. DC alginate (ARGODERM®; crosslinked by Ca2+), CB alginate (KURABIO®), and atelocollagen (INSTAT®) were purchased from Smith & Nephew (London, UK), Koyo Sangyo Co, Ltd (Tokyo, Japan), and Johnson & Johnson (New Brunswick, NJ), respectively. PLA-PEG with a total molecular weight of 11,400 Da and a PLA:PEG molar ratio of 51:49 was synthesized and provided by Taki Chemicals Co, Ltd (Hyogo, Japan).
To prepare carrier material/OP-1 composites, sheets of DC alginate, CB alginate, and atelocollagen were cut into discs (5-mm diameter). Then 25 μL of a solution containing 3, 10, or 30 μg OP-1 was added dropwise to each disc, after which the discs were freeze-dried and stored at −20°C until implantation into mice. All procedures were carried out under sterile conditions.
PLA-PEG/OP-1 composites were prepared as described previously . Briefly, 10 mg of the polymer was liquefied in 50 μL acetone and mixed with 3, 10, or 30 μg OP-1. Each mixture was evaporated to dryness to remove acetone in a safety cabinet, fabricated into a disc-shaped implant, and stored at −20°C until implantation into mice.
To verify the first and second hypotheses, we used one-way analysis of variance (ANOVA), followed by a post hoc Scheffe’s test. For each of these statistical analyses, the data sets met the assumptions of normality (p > 0.15 by the Jarque-Bera test ) justifying the use of parametric models. All analyses were performed using the R software program (Version 2.8.1;R Foundation for Statistical Computing).
With 3 μg OP-1, BMC of the new bone in the DC alginate group was greater than that in atelocollagen group (p = 0.0234) and PLA-PEG group (p = 0.0009). With 30 μg OP-1, however, we observed no differences among the DC alginate, atelocollagen, and PLA-PEG. On the other hand, BMC of CB alginate group was very low compared with the other groups (Fig. 1). The results suggest that the BMC of DC alginate group was superior to those of atelocollagen and PLA-PEG groups, especially with a low dose of OP-1.
In the DC alginate group, ALP activity was high independent of the OP-1 dose. With 3 μg OP-1, DC alginate/OP-1 composites exhibited higher ALP activity than atelocollagen group (p = 0.0071) and PLA-PEG group (p = 0.0001) (by Scheffe’s test). ALP activity of the CB alginate group was very low compared with the other groups (Fig. 2). The results suggest that ALP activity of the DC alginate group was superior to those of atelocollagen and PLA-PEG groups, especially with a low dose of OP-1.
In the release study of OP-1, the maximum concentration of OP-1 in the supernatant was detected on Day 1, followed by a steady decline. The decline of OP-1 levels in the atelocollagen group was faster than that in the other groups. In the DC alginate group, the decrease of OP-1 levels was the slowest and the concentration of OP-1 was still higher than 200 ng/mL on Day 21 (Fig. 3). These data suggested that DC alginate retains OP-1 and releases it most slowly compared to atelocollagen and PLA-PEG.
In the additionally performed radiographic examination of the bone nodules, obvious bone formation was only detected in the DC alginate and atelocollagen groups with 3 μg OP-1 (Fig. 4A–D). In the CB alginate group, new bone formation was observed only with 30 μg OP-1. The results of the additionally performed histological examination were consistent with the radiographic findings. In the DC alginate and atelocollagen groups, abundant new bone formation that contained normal hematopoietic bone marrow was observed even at low dose of OP-1. In the CB alginate group, however, new bone formation was very poor at low dose of OP-1. With 30 μg OP-1, irrespective of the carrier materials, newly formed bone had a thin cortex surrounding cancellous bone that contained hematopoietic bone marrow, and no inflammatory change was observed (Fig. 5A–D). These additional results were compatible with the results of BMC and ALP activity, suggesting that DC alginate can be an equivalent or superior carrier for a low dose of OP-1 compared with atelocollagen and PLA-PEG.
Various materials have already been evaluated as carriers for BMPs, but they all have some disadvantages as mentioned previously. This study was designed to examine whether alginate, a material with no animal product content, is an equivalent or superior carrier for OP-1(BMP-7) compared with atelocollagen and PLA-PEG. Specifically we hypothesized: (1) BMC of bone nodules ectopically induced by DC alginate/OP-1 composite and/or CB alginate/OP-1 composite are equivalent or superior to those by atelocollagen and PLA-PEG; (2) ALP activity of bone nodules ectopically induced by DC alginate/OP-1 composite and/or CB alginate/OP-1 composite are equivalent or superior to those by atelocollagen and PLA-PEG; and (3) DC alginate and/or CB alginate have appropriate in vitro release kinetics of OP-1 equivalent to atelocollagen and PLA-PEG.
This study has several limitations. First, DC alginate was originally approved for clinical use as a cutaneous wound dressing [8, 37]. Therefore, its biodegradability and immunogenicity are unclear during use at a deeper site. Second, in the histological examination, DC alginate remained at the center of the new ectopic bone, indicating it had not degraded within 3 weeks. Although no inflammatory reaction was found, longer observation will be necessary before this material can be used with confidence at deeper sites. Third, ALP activity is a marker for osteoblastic differentiation, and is high in the early stage of osteoblast lineage. ALP activity is not necessarily parallel to the activity of bone formation. Fourth, in the release study, a commercial BMP-7 ELISA kit can only detect the amount of BMP-7(OP-1) protein, but cannot evaluate the activity of OP-1. The result of a release test may not reflect the actual activity of OP-1 released from carriers in vivo.
To determine whether DC alginate and/or CB alginate are equivalent or superior carriers for OP-1 compared with atelocollagen and PLA-PEG, we measured BMC of ectopic bone nodules as a primary research question. A previous study  reported that BMC of the ectopic bone induced by PLA-PEG/BMP-2 composite is about 6 mg higher than that by atelocollagen/BMP-2 composite. In our study, the BMC of DC alginate/OP-1 (3 μg) composite was about 6 mg higher than that of the atelocollagen/OP-1 (3 μg) composite and even much higher than that of CB alginate and PLA-PEG. The result of BMC measurement suggested that DC alginate is a highly effective carrier that enhances the bone-inducing effect of OP-1 even when OP-1 content is low.
The result of ALP activity measurement was compatible with the result of BMC, which reinforced the hypothesis that DC alginate is an equivalent or superior carrier compared with the other materials. Upon histological examination, not only trabecular bone but also normal hematopoietic bone marrow was observed, and we found no accumulation of inflammatory cells, such as monocyte/macrophages. The histological appearance of the ectopic bone induced by DC alginate/OP-1 composite seemed similar to that by atelocollagen/OP-1 composite, which is considered a safe biomaterial in terms of immunological response. These data suggested that DC alginate appears likely a safe material with no inflammatory response even when used in a deep site.
In contrast, CB alginate achieved relatively poor bone formation, especially with a low dose of OP-1. DC alginate and CB alginate only differ in the mode of crosslinking, but the release of OP-1 from these two alginates was quite different. It is known crosslinking by divalent cations forms a characteristic egg box structure that is suitable for trapping proteins in alginate . Thus, the difference of bone formation between these two types of alginate may be partly due to a difference in their ability to retain OP-1 and release it slowly. It is also known the number of carboxyl residues in DC alginate is larger than that in CB alginate. The carboxyl residues induce apatite nucleation followed by the deposition of hydroxyapatite crystals on the alginate . Furthermore, the Ca2+ contained in DC alginate can be utilized for new calcified bone, which is an advantage compared with CB alginate.
In conclusion, our data suggest DC alginate, a material with no animal product content that is approved by the FDA and other authorities, is a safe and potent carrier for OP-1. It is of note that DC alginate strongly potentiates osteoinduction of OP-1 even at a low dose. Thus, its use may reduce the cost of OP-1-based bone regeneration therapy.
We thank Stryker Biotech, Smith & Nephew, Koyo Sangyo Co, Ltd, Johnson & Johnson, and Taki Chemicals Co, Ltd, for kindly providing the chemicals and materials.
One or more of the authors have received funding from Stryker Biotech K. K. (Tokyo, Japan) (AM) and from the Japan Science and Technology Agency (AM).
Each author certifies that his or her institution has approved the animal protocol for this investigation and that all investigations were conducted in conformity with ethical principles of research.
This work was performed at Department of Orthopaedic Surgery, Osaka University Graduate School of Medicine, Osaka, Japan.