Osteogenic protein-1 (OP-1) is a member of the bone morphogenetic protein (BMP) family9 and otherwise is known as BMP-7. Bone morphogenetic protein family members in turn are members of the TGF-β superfamily of growth and differentiation factors.20,22 Several BMPs form bone at an ectopic site but not at all doses.18,19,21 In addition, some studies have examined the effect of BMPs given as a single bolus dose (one-time dose), and the effect of a continuous versus a one-time dose of OP-1 is unclear.7,10,11,13–16 Therefore, the effect of OP-1 dose and dosing regimen on ectopic bone formation has not been examined completely in the rat ectopic model. The purpose of the current study was to determine the effect of varying doses and dosing regimens of OP-1 on ectopic bone formation.
MATERIALS AND METHODS
All chemicals were obtained from Sigma (St Louis, MO). Osmotic pumps were obtained from Alzet (Cupertino, CA). Demineralized bone matrix was prepared as described previously.7,11 Briefly, tibial and femoral bones from adult male Sprague Dawley rats (200–300 g) were removed and cleaned of adherent tissue. The diaphyseal shafts were cleared of bone marrow, dehydrated with ethanol, and defatted with diethyl ether. The diaphyseal shafts were ground and sieved to a particle size < 420 μm, demineralized with 0.5 normal HC1, washed with water and dried, but not sterilized additionally.7,11
Adult male Sprague Dawley rats (200–300 g) were acclimated for approximately 5 days before surgery. Immediately before surgery, the rats were anesthetized with intraperitoneal pentobarbital at the approximate dosage of 75 mg/kg. The abdomen was shaved, and a 3-cm parasagittal skin incision was made. A 1-cm2 subcutaneous pouch was dissected at the distal end of the incision, and 10 to 15 mg demineralized bone matrix were deposited in each subcutaneous pouch. The demineralized bone matrix therefore was used as a control in the demineralized bone matrix only group. Demineralized bone matrix is known to possess growth factors although the amounts of these factors do not induce significant bone formation as shown in this study. Demineralized bone matrix however is an effective carrier and the effect of OP-1 on ectopic bone formation without a Type I collagen carrier is markedly diminished.1
The rats were divided into four groups: demineralized bone only, single bolus of OP-1 and demineralized bone, continuous infusion of OP-1 and demineralized bone, and a combination of one bolus and continuous infusion of OP-1 with demineralized bone. For the single bolus group there were three subgroups with the OP-1 delivered onto the demineralized bone matrix by micropipette at doses of 0.1, 1.0, and 10 μg (Table 1). For the continuous infusion group there also were three subgroups (Table 1). An Alzet® osmotic pump, model 1002, was loaded with a total dose of either 0.1, 1.0, or 10 μg of OP-1, and a Silastic catheter was attached and primed with OP-1. The distal end of the catheter was implanted into the demineralized bone matrix pouch, and the catheter and pump were secured to the abdominal wall with 3–0 polypropylene sutures. The osmotic pumps delivered the cumulative doses during a 14-day period. The bolus plus infusion group received 1.0 μg OP-1 immediately by micropipette plus 0, 0.1, 1.0, or 10 μg OP-1 for 14 days by the osmotic pump (Table 1). Every group received 10–15 mg demineralized bone matrix. Each dosing subgroup contained six rats and the OP-1 was delivered in 5% lactic acid at a constant volume (100 μL). However, animals were excluded if at the time of sacrifice the surgical wound was not healed or they died before the day of sacrifice. There also were additional groups in which the 1-μg OP dose was left at 37°C for 0, 1, or 3 days before single bolus. On postoperative Day 15, the animals were euthanized by CO2 inhalation and the subcutaneous implants were dissected away from the abdominal wall. The specimens were cut in half with part being fixed in 10% phosphate buffered formalin and prepared histologically using toluidine blue staining. The histologic specimens were assessed qualitatively for bone formation. The remaining part of each specimen was homogenized in hypotonic buffer (0.15 mol/L NaCl, 3 mmol/L NaHCO3, pH 7.4) as described previously.7 These homogenates were centrifuged at 20,000 g for 30 minutes and the alkaline phosphatase activity of the supernatant was determined with 0.1 mol/L p-nitrophenylphosphate as substrate at pH 9.3 at 37° for 30 minutes. One unit of enzyme liberates 1 μmol of p-nitrophenol under the assay conditions and the colorimetric change (optical density) was measured on a spectrophotometer (Beckmann) at 405 nm.7 Each sample was normalized for total protein content.6 All implants in the vicinity of infected wounds or delayed wound healing were excluded from the analysis. An additional study was done to examine if the OP-1 incubated in the osmotic pumps at 37°C for 1 or 3 days before implantation with a single bolus still was effective in ectopic bone formation. This study was done to see if there was an effect from the osmotic pump on the OP-1 that rendered it less effective with time.
Statistics and Units of Measurement
Alkaline phosphatase is reported in international units per milligram of protein, and sample means from each group were analyzed by analysis of variance (ANOVA) (Statview, Cary, NC).
In preliminary experiments, it was determined that 15 days after implantation represented adequate time for initiation of bone formation (data not shown) and there was relatively little bone formation in the group treated with demineralized bone matrix alone (control group). We therefore thought that the demineralized bone matrix alone group would serve as an effective control despite the fact that there are growth factors present in this material. These findings were in keeping with previous studies.7,9–11,13–17
Osteogenic protein-1 dosing groups were: bolus alone, bolus with continuous infusion, and continuous infusion alone. For the single bolus group there were three subgroups with the OP-1 delivered onto the demineralized bone matrix by micropipette at doses of 0.1, 1.0, and 10 μg. For the continuous infusion group there also were three subgroups. An Alzet® osmotic pump, model 1002, was loaded with a total dose of either 0.1, 1.0, or 10 μg of OP-1, The bolus plus infusion group also had three subgroups but received 1.0 μg OP-1 immediately by micropipette and 0.1, 1.0, or 10 μg OP-1 for 14 days by the osmotic pump. After animals were excluded for failure of wound healing or death before the day of sacrifice, there were five animals in each group except the single high-dose group (six animals) and the continuous low-dose group (four animals). Every group received 10 to 15 mg demineralized bone matrix. Demineralized bone matrix alone was used as a control. The biochemical results showed that the most bone formation was in the 1.0- and 10-μg single bolus subgroups and the least bone formation was in the demineralized bone matrix alone and 10-μg continuous infusion groups (p < 0.05) (Fig 1). The 0.1-μg single bolus subgroup and the continuous infusion 0.1- and 1.0-μg subgroups also had more bone formation than the demineralized bone matrix alone group (p > 0.05) (Fig 1). In the additional study done to examine if the OP-1 left at 37° for 1 or 3 days before the single bolus still was effective in ectopic bone formation, leaving the OP-1 at 37°C for 1 or 3 days before insertion did not decrease bone formation significantly (Fig 2). This finding indicated that the OP-1 was not likely degraded or otherwise rendered less effective while sitting in the pump before its infusion. The single infusion plus continuous infusion subgroups all caused more bone formation than the demineralized bone matrix alone group (p < 0.05) (Fig 3) but not more than the single bolus of OP-1, indicating that again most of the effect of OP-1 on bone formation is related to the initial bolus dose.
Histologically the most bone formation was in the single bolus group, followed by the continuous infusion group and the demineralized bone matrix or control group had the least bone formation. In the single bolus group, there was a cortical shell of bone surrounding what seemed to be marrow elements. In the continuous infusion group, there was a mixed pattern of bone formation and fibrous tissue. In the demineralized bone matrix group, there was minimal to no bone formation present.
The delineation of optimal dose and dosing frequency is prerequisite to the safe and successful use of BMPs to heal large bone defects. Data from the current study show optimization of dosing regimen of OP-1 and that there is no added benefit to a continuous dosing regimen instead of or in conjunction with a bolus dose. Therefore carriers that release OP-1 rapidly or at least rapidly in the presence of demineralized bone matrix would seem to be optimal. It is somewhat counterintuitive that a higher cumulative dose in the bolus with continuous infusion group did not lead to more bone formation than the single bolus group and that the same cumulative dose given in the continuous infusion group led to less bone formation than in the single bolus group. This study also provides a framework for the rational design of carriers of OP-1 by elucidation of optimal temporal bioavailability of the OP-1 ligand in the rat ectopic assay of bone formation. Although there are a plethora of preclinical studies and some clinical studies using OP-1 the optimal dosing pattern and carrier still are unclear.4,5,12,18
Osteogenic protein-1 is present in the osteoblast and periosteum of the developing limb and early in the process of endochondral ossification but whether supraphysiologic or sustained OP-1 dosing or both improves bone formation is unclear.3,8 The current study does not show an added benefit beyond a 1-μg single bolus dose either by adding more OP-1 (the 10-μg single bolus subgroup) or by more sustained release of the OP-1 (the continuous infusion subgroups).
The data presented here show a benefit to a single bolus dose when delivered in the presence of demineralized bone matrix and do not support the continuous infusion of OP-1 for bone formation. The apparent maximal effect of OP-1 with a single bolus dose indicates the importance of the early presence of this growth factor for bone formation and possible desensitization at the receptor level or upregulation of intracellular or extracellular inhibitors with the continuous infusion of OP-1.2
1. Cullinane DM, Lietman SA, Inoue N, Deitz LW, Chao EY: The effect of recombinant human osteogenic protein-1 (bone morphogenetic protein-7) impregnation on allografts in a canine intercalary bone defect. J Orthop Res 20:1240–1245, 2002.
2. Ebara S, Nakayama K: Mechanism for the action of bone morphogenetic proteins and regulation of their activity. Spine 27(Suppl):S10–S15, 2002.
3. Helder MN, Ozkaynak E, Sampath KT, et al: Expression pattern of osteogenic protein-1 (bone morphogenetic protein-7) in human and mouse development. J Histochem Cytochem 43:1035–1044, 1995.
4. Hollinger JO, Leong K: Poly(alpha-hydroxy acids): Carriers for bone morphogenetic proteins. Biomaterials 17:187–194, 1996.
5. Howell TH, Fiorellini J, Jones A, et al: A feasibility study evaluating rhBMP-2/absorbable collagen sponge device for local alveolar ridge preservation or augmentation. Int J Periodontics Restorative Dent 17:124–139, 1997.
6. Lowry O, Rosebrough N, Farr A, Randall R: Protein measurement with the folin phenol reagent. J Biol Chem 193:265–275, 1951.
7. Muthukumaran N, Ma S, Reddi AH: Dose-dependence of and threshold for optimal bone induction by collagenous bone matrix and osteogenin-enriched fraction. Coll Relat Res 8:433–441, 1988.
8. Onishi T, Ishidou Y, Nagamine T, et al: Distinct and overlapping patterns of localization of bone morphogenetic protein (BMP) family members and a BMP type II receptor during fracture healing in rats. Bone 22:605–612, 1998.
9. Ozkaynak E, Rueger DC, Drier EA, et al: OP-1 cDNA encodes an osteogenic protein in the TGF-beta family. EMBO J 9:2085–2093, 1990.
10. Reddi AH, Huggins C: Biochemical sequences in the transformation of normal fibroblasts in adolescent rats. Proc Natl Acad Sci USA 69:1601–1605, 1972.
11. Reddi AH, Huggins CB: Citrate and alkaline phosphatase during transformation of fibroblasts by the matrix and minerals of bone. Proc Soc Exp Biol Med 140:807–810, 1972.
12. Saito N, Okada T, Horiuchi H, et al: A biodegradable polymer as a cytokine delivery system for inducing bone formation. Nat Biotechnol 19:332–335, 2001.
13. Sampath TK, DeSimone DP, Reddi AH: Extracellular bone matrix-derived growth factor. Exp Cell Res 142:460–464, 1982.
14. Sampath TK, Muthukumaran N, Reddi AH: Isolation of osteogenin, an extracellular matrix-associated, bone-inductive protein, by heparin affinity chromatography. Proc Natl Acad Sci USA 84:7109–7113, 1987.
15. Sampath TK, Nathanson MA, Reddi AH: In vitro transformation of mesenchymal cells derived from embryonic muscle into cartilage in response to extracellular matrix components of bone. Proc Natl Acad Sci USA 81:3419–3423, 1984.
16. Sampath TK, Reddi AH: Distribution of bone inductive proteins in mineralized and demineralized extracellular matrix. Biochem Biophys Res Commun 119:949–954, 1984.
17. Sampath TK, Wientroub S, Reddi AH: Extracellular matrix proteins involved in bone induction are vitamin D dependent. Biochem Biophys Res Commun 124:829–835, 1984.
18. Schmitt JM, Hwang K, Winn SR, Hollinger JO: Bone morphogenetic proteins: An update on basic biology and clinical relevance. J Orthop Res 17:269–278, 1999.
19. Wang EA, Israel DI, Kelly S, Luxenberg DP: Bone morphogenetic protein-2 causes commitment and differentiation in C3H10T1/2 and 3T3 cells. Growth Factors 9:57–71, 1993.
20. Wang EA, Rosen V, Cordes P, et al: Purification and characterization of other distinct bone-inducing factors. Proc Natl Acad Sci USA 85:9484–9488, 1988.
21. Winn SR, Uludag H, Hollinger JO: Carrier systems for bone morphogenetic proteins. Clin Orthop 367(Suppl):S95–106, 1999.
22. Wozney JM, Rosen V, Celeste AJ, et al: Novel regulators of bone formation: Molecular clones and activities. Science 242:1528–1534, 1988.