- Email: [email protected]
Absence of bone morphogenetic protein-2 in human breast milk after spinal surgery
The Spine Journal 10 (2010) e17–e20
Case Report
Absence of bone morphogenetic protein-2 in human breast milk after spinal surgery Shiau-Tzu Tzeng, MDa, Jen-Chung Liao, MDb,c, Samuel S. Murray, MDd,e, Elsa J. Brochmann, PhDd,e, Gregory D. Carlson, MDf,g,h, Jeffrey C. Wang, MDb,* a
Department of Orthopedics, Buddhist TzuChi General Hospital-Taipei Branch, TzuChi University, Taiwan, ROC b Department of Orthopedics, UCLA School of Medicine, Santa Monica, CA 90404, USA c Department of Orthopedics, Chang Gung Memorial Hospital, Chang Gung University, Taoyuan, Taiwan, ROC d Department of Medicine, UCLA School of Medicine, Santa Monica, CA 90404, USA e Geriatric Research, Education and Clinical Center, VA Greater Los Angeles Health Care System, Los Angeles, CA, USA f Orthopaedic Specialty Institute, 280 South Main Street, suite 200, Orange, CA 92868, USA g Department of Orthopaedic Surgery, University of California, Irvine, CA 92697, USA h Spine Surgeon, St. Joseph Hospital, Orange, CA 92868, USA Received 5 November 2009; revised 8 February 2010; accepted 29 March 2010
Abstract
BACKGROUND CONTEXT: Bone morphogenetic protein-2 (BMP-2) has been used extensively in recent years to enhance the process of spinal fusion. However, numerous side effects have been reported. This raises concerns regarding the safety of using this material in human beings. There are no published reports, that we are aware of, that demonstrate detectable levels of BMP-2 in human milk. PURPOSE: The purpose of this study was to determine the presence/absence of recombinant human bone morphogenetic protein-2 (rhBMP-2) in milk samples taken from a patient who underwent spinal surgery using rhBMP-2 shortly after delivering a child. STUDY DESIGN/SETTING: The study design comprises a case report dealing with analyzing milk from a patient who underwent spinal surgery using rhBMP-2 shortly after delivering a child. METHODS: We report the analysis of milk samples from a patient who underwent spinal surgery using rhBMP-2 shortly after delivering a child to determine the presence/absence of rhBMP-2. The milk samples were taken before and after the spinal surgery. Enzyme-linked immunosorbent assays were performed repeatedly to the samples. RESULTS: The standard curve for the assay had an r2 value of 0.9853 indicating an acceptable degree of statistical reliability. The dose range for the standard curve was 62.5 to 2,000 pg/mL. None of the samples had an optical density greater than that of the lowest standard. CONCLUSIONS: No rhBMP-2 was detected in human milk in this single case report. Although this result seemed promising, it did not eliminate the concern regarding the potential risk of rhBMP-2 on child development. We still need further studies including more cases to verify this conclusion. Ó 2010 Elsevier Inc. All rights reserved.
Keywords:
rhBMP-2; Bone morphogenetic protein; Spinal fusion; Breast milk
FDA device/drug status: not applicable. Author disclosures: SSM (royalties, UCLA; consulting, Lanx; research support: staff and/or materials, Lanx; grants, Lanx; other relationships, IsoTis); GDC (consulting, Vertebral Technology Incorporated; stock ownership, including options and warrants, Alphatec (ATEC)); JCW (royalties, Medtronic, Stryker, SeaSpine, Osprey, Aesculap, Biomet, Amedica, Zimmer, Alphatech; stock ownership, including options and warrants, FzioMed; private investments, including venture capital, start-ups, Promethean Spine, Paradigm Spine, Benevenue, NexGen, K2 Medical, Pioneer, Amedica, Vertiflex, Electrocore, Surgitech, Invuity, Axiomed; board of 1529-9430/$ – see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.spinee.2010.03.034
directors, North American Spine Society, Cervical Spine Research Society; scientific advisory board, VG Innovations, Corespine, Expanding Orthopaedics, Syndicom, Osprey, Amedica, Bone Biologics, Curative Biosciences, Facet Solutions, PearlDiver, Pioneer, SeaSpine, Axis). * Corresponding author. Orthopaedic Spine Service, Department of Orthopaedics and Neurosurgery, UCLA Comprehensive Spine Center, 1250 16th St, Suite 745, Santa Monica, CA 90404, USA. Tel.: (310) 319-3334; fax: (310) 319-5055. E-mail address: [email protected] (J.C. Wang)
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S.-T. Tzeng et al. / The Spine Journal 10 (2010) e17–e20
Introduction Bone morphogenetic protein-2 (BMP-2) has been used extensively in recent years to enhance the process of spinal fusion [1–3]. However, as the use of BMP-2 has become more prevalent, numerous side effects, such as ectopic bone formation, neck swelling, hematoma, dysphagia, airway compression, bony resorption, graft subsidence, and fluid accumulation with mass effect over operative area have been reported [4,5]. This raises concerns regarding the safety of using this material in human beings [6]. In addition, concerns regarding the use of the BMPs in women of childbearing age and possible adverse effects on the fetus are theoretically contraindications for use. There is also the possibility of the transmission of the proteins through breast milk in lactating mothers as another potential adverse issue that may be of concern. There are no published reports, that we are aware of, that demonstrate detectable levels of BMP-2 in human milk or for that matter in human serum (milk is modified serum). This report deals with analyzing milk from a patient who underwent spinal surgery using recombinant human bone morphogenetic protein-2 (rhBMP-2) shortly after delivering a child to determine the presence/absence of rhBMP-2.
Case report A 35-year-old female suffered from postdiscectomy aseptic discitis at the L4/L5 level. She received combined anterior lumbar interbody fusion with posterior instrumentation on April 17, 2008. During the anterior operation, rhBMP-2 (medium-sized INFUSE bone graft kit [Medtronic, Inc., Memphis, TN, USA]) was packed into the middle of the femoral ring allograft to enhance the process of spinal fusion. Before surgery, the patient breast-fed her child for 4 months. A control sample of milk was obtained preoperatively. A few hours after surgery, the patient began pumping milk and continued to do so for several weeks. Each time a sample was collected, it was immediately placed in a freezer and subsequently shipped to the laboratory on dry ice. Each sample was analyzed using the method described below.
Table 1 Sample details Sample no.
Date
Time
Preoperative* 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
April 2 April 18 April 18 April 18 April 18 April 19 April 19 April 19 April 20 April 20 April 21 April 23 April 23 April 27 April 29 April 30 April 30 May 1
— 1:30 AM 8:30 AM 3:10 PM 8:45 PM 2:15 AM 6:30 AM 4:00 PM 9:00 AM 11:00 AM 10:00 AM 1:00 AM 5:00 AM 2:00 PM 3:00 PM 7:00 PM 9:00 PM 7:00 PM
* Date of surgery: April 17, 2008.
to 50-mL conical tubes. If only a small amount of material was available from two bags with identical designations, they were pooled. One milk sample was used for assays at each time point. An aliquot of 10 mL from each of the 18 samples was transferred to a 15-mL centrifuge (Sarstedt, AG & Co., Rommelsdorfer Straße, Nu¨mbrecht, Germany) tube. The processing methods were adapted from those of Oslislo et al. [7]. The 10-mL samples were centrifuged at 2,000 rpm at 4 C using a Sorvall SM-24 rotor in a Sorvall RC-5 centrifuge for 10 minutes (estimated force5500g). Approximately 4 mL of clarified ‘‘milk serum’’ was removed using a thin-tipped transfer pipette. From the approximately 4 mL of isolated ‘‘milk serum,’’ four 1-mL samples were placed into 1.4-mL microfuge tubes and frozen for subsequent use. On the day of assay, one of the four samples was thawed and then spun briefly in a microfuge. A sample of 100 mL was removed from the clarified liquid for assay. Assay methods
Samples were received frozen, on dry ice, in labeled Gerbers plastic bags. Each sample was labeled with a date and a time. Some had other designations as noted below. The time between the ‘‘preoperative’’ sample and the first ‘‘postoperative’’ sample was 16 days. A total of 18 unique time-point samples were received (Table 1).
Bone morphogenetic protein-2 enzyme-linked immunosorbent assay kits were obtained from R&D Systems (Minneapolis, MN, USA). Assays were performed exactly according to the manufacturer’s instructions with the exception that 100 mL of sample was assayed rather than 50 mL. Samples were assayed undiluted. Two assays were performed. The first assay was done to determine if dilution of the samples was required. A second assay was performed to confirm the results of the first. Samples were tested in duplicate within each assay.
Sample preparation
Results
Samples were initially thawed on ice. Final thawing occurred at room temperature. Samples were then transferred
The breast milk was obtained at the stated time points, and each sample was assayed for the presence of BMP-2
Samples received
S.-T. Tzeng et al. / The Spine Journal 10 (2010) e17–e20 Table 2 Sample results Sample no.
BMP-2 concentration (pg/mL)*
Optical density value
Preoperative 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
78.629y 78.629y 78.629y 78.629y 78.629y 78.629y 78.629y 78.629y 55.444y 78.629y 78.629y 55.444y 78.629y 55.444y 55.444y 32.258y 14.113y 55.444y
0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.003 0.002 0.002 0.003 0.002 0.003 0.003 0.004 0.006 0.003
BMP-2, bone morphogenetic protein-2. * Dose range for the standard curve: 62.5 to 500 pg/mL. y Value is outside of standard curve.
with the enzyme-linked immunosorbent assay kits. The linear regression analysis was performed. This calculation used four standards, and the correlation coefficient (r) for the resulting line was 0.9998, indicating an acceptable degree of statistical reliability. The optical density values and the extrapolated BMP-2 concentrations of the samples were measured and listed in Table 2. The dose ranges for the standard curve were 62.5 to 500 pg/mL for the BMP-2 concentration and 0.008 to 0.026 for the optical density value. The optical density values of the samples ranged from 0.002 to 0.006; all of them are less than that of the lowest standard (‘‘limit of detection’’). Using this precise standard curve, 17 of the 18 samples gave negative BMP-2 concentrations (78.629 pg/mL in 11 samples, 55.444 pg/mL in 5 samples, and 32.258 pg/mL in 1 sample), that is, none was detected. One sample gave a positive result (14.113 pg/mL), which was less than one quarter that of the lowest standard (62.5 pg/mL). However, this is a statistical artifact. This cannot be regarded as an accurate measurement and is regarded as below the level of detection for the assay. Therefore, if we took into consideration of this statistical artifact, the BMP-2 was not detected in all the milk samples with a limit of detection of 62.5 pg/mL.
Discussion There has been growing concern regarding the safety of using rhBMP-2 for spine surgery. A single report was located indicating that animal milk proteins enhance bone
e19
growth [8]. To our knowledge, there is no literature focusing on the presence of BMP-2 within human milk. If BMP-2 exists in breast milk, it is possible that it could be transmitted to the baby through breast-feeding. We do not know yet whether BMP-2 could have positive or negative effects on child development. This is a basic fact we must ascertain to avoid potential risks to child development. In our study, BMP-2 was not detected in human milk with a limit of detection of 62.5 pg/mL. Although some statistical artifact was inevitably generated during data analysis, confidence in this negative result is increased by the fact that two assays using different reagents were performed and gave the same results. Pharmacokinetic studies using rat and nonhuman primates have shown rapid systemic clearance of rhBMP-2 after intravenous administration. The use of rhBMP-2 in combination with absorbable collagen sponges has resulted in retention of rhBMP-2 at the site of application and low levels of detectable rhBMP-2 within the systemic circulation. The slow release of the rhBMP-2 from the implant site coupled with the rapid clearance of rhBMP-2 results in low systemic exposure [5]. Besides, these processes still could be dose related. If we use larger amount of rhBMP-2 during surgery, it would increase the chance of BMP-2 to be detected in the assay. For this study, the time between rhBMP-2 implantation and the collection of the first postoperative sample is unknown. Therefore, we cannot rule out the possibility that rhBMP-2 might have been detected shortly after implantation. It is possible that because of rapid clearance of rhBMP-2, systemic concentrations (such as in the serum or breast milk) are virtually undetectable. Other factors must be taken into consideration when interpreting the results of this study. For example, the protein may have been denatured during the process of sample collection and storage. This may have caused false negative results to be obtained. In addition, animal studies using rhBMP-2 have demonstrated that rhBMP-2 does not have teratogenic properties. Furthermore, rhBMP-2 has not demonstrated systemic toxicity in human studies [4,5]. Finally, this study is only a single case report; a stronger conclusion could be generated through completion of a more comprehensive study involving a larger number of patients.
Conclusion In this single case report, rhBMP-2 was not detected in human milk before or after the use of the growth protein in a spine surgery. Although this result seemed promising, it did not eliminate the concerns regarding the potential side effects of transmission of rhBMP-2 on child development. We still need further studies including larger case numbers to verify this conclusion.
e20
S.-T. Tzeng et al. / The Spine Journal 10 (2010) e17–e20
Acknowledgments Key points
Bone morphogenetic protein-2 has been used extensively in recent years to enhance the process of spinal fusion. However, numerous side effects have been reported. This raises concerns regarding the safety of using this material in human beings. It is possible that because of rapid clearance of recombinant human bone morphogenetic protein-2 (rhBMP-2), systemic concentrations (such as in the serum or breast milk) are virtually undetectable. The protein may have been denatured during the process of sample collection and storage. This may have caused false negative results to be obtained. No rhBMP-2 was detected in human milk in this single case report. This promising result did not mean that we do not need to concern about the potential risk of rhBMP-2 on child development. Further studies including more cases are needed to verify this conclusion.
The authors thank Dr Shireen Guide for her contributions in this report. References [1] Carlisle E, Fischgrund JS. Bone morphogenetic proteins for spinal fusion. Spine J 2005;5:240S–9S. [2] Sandhu HS. Bone morphogenetic proteins and spinal surgery. Spine 2003;28:S64–73. [3] Gautschi OP, Frey SP, Zellweger R. Bone morphogenetic proteins in clinical applications. ANZ J Surg 2007;77:626–31. [4] Poynton AR, Lane JM. Safety profile for the clinical use of bone morphogenetic proteins in the spine. Spine 2002;27:S40–8. [5] Benglis D, Wang MY, Levi AD. A comprehensive review of the safety profile of bone morphogenetic protein in spine surgery. Neurosurgery 2008;62(5 Suppl 2):ONS423–31. [6] FDA public health notification: life-threatening complications associated with recombinant human bone morphogenetic protein in cervical spine fusion. 2008 July. [7] Oslislo A, Czuba Z, Slawska H, et al. Decreased human milk concentration of epidermal growth factor after preterm delivery of intrauterine growth-restricted newborns. J Pediatr Gastroenterol Nutr 2007;44: 464–7. [8] Aoe S, Toba Y, Yamamura J, et al. Controlled trial of the effects of milk basic protein (MBP) supplementation on bone metabolism in healthy adult women. Biosci Biotechnol Biochem 2001;65:913–8.
Case Report
Absence of bone morphogenetic protein-2 in human breast milk after spinal surgery Shiau-Tzu Tzeng, MDa, Jen-Chung Liao, MDb,c, Samuel S. Murray, MDd,e, Elsa J. Brochmann, PhDd,e, Gregory D. Carlson, MDf,g,h, Jeffrey C. Wang, MDb,* a
Department of Orthopedics, Buddhist TzuChi General Hospital-Taipei Branch, TzuChi University, Taiwan, ROC b Department of Orthopedics, UCLA School of Medicine, Santa Monica, CA 90404, USA c Department of Orthopedics, Chang Gung Memorial Hospital, Chang Gung University, Taoyuan, Taiwan, ROC d Department of Medicine, UCLA School of Medicine, Santa Monica, CA 90404, USA e Geriatric Research, Education and Clinical Center, VA Greater Los Angeles Health Care System, Los Angeles, CA, USA f Orthopaedic Specialty Institute, 280 South Main Street, suite 200, Orange, CA 92868, USA g Department of Orthopaedic Surgery, University of California, Irvine, CA 92697, USA h Spine Surgeon, St. Joseph Hospital, Orange, CA 92868, USA Received 5 November 2009; revised 8 February 2010; accepted 29 March 2010
Abstract
BACKGROUND CONTEXT: Bone morphogenetic protein-2 (BMP-2) has been used extensively in recent years to enhance the process of spinal fusion. However, numerous side effects have been reported. This raises concerns regarding the safety of using this material in human beings. There are no published reports, that we are aware of, that demonstrate detectable levels of BMP-2 in human milk. PURPOSE: The purpose of this study was to determine the presence/absence of recombinant human bone morphogenetic protein-2 (rhBMP-2) in milk samples taken from a patient who underwent spinal surgery using rhBMP-2 shortly after delivering a child. STUDY DESIGN/SETTING: The study design comprises a case report dealing with analyzing milk from a patient who underwent spinal surgery using rhBMP-2 shortly after delivering a child. METHODS: We report the analysis of milk samples from a patient who underwent spinal surgery using rhBMP-2 shortly after delivering a child to determine the presence/absence of rhBMP-2. The milk samples were taken before and after the spinal surgery. Enzyme-linked immunosorbent assays were performed repeatedly to the samples. RESULTS: The standard curve for the assay had an r2 value of 0.9853 indicating an acceptable degree of statistical reliability. The dose range for the standard curve was 62.5 to 2,000 pg/mL. None of the samples had an optical density greater than that of the lowest standard. CONCLUSIONS: No rhBMP-2 was detected in human milk in this single case report. Although this result seemed promising, it did not eliminate the concern regarding the potential risk of rhBMP-2 on child development. We still need further studies including more cases to verify this conclusion. Ó 2010 Elsevier Inc. All rights reserved.
Keywords:
rhBMP-2; Bone morphogenetic protein; Spinal fusion; Breast milk
FDA device/drug status: not applicable. Author disclosures: SSM (royalties, UCLA; consulting, Lanx; research support: staff and/or materials, Lanx; grants, Lanx; other relationships, IsoTis); GDC (consulting, Vertebral Technology Incorporated; stock ownership, including options and warrants, Alphatec (ATEC)); JCW (royalties, Medtronic, Stryker, SeaSpine, Osprey, Aesculap, Biomet, Amedica, Zimmer, Alphatech; stock ownership, including options and warrants, FzioMed; private investments, including venture capital, start-ups, Promethean Spine, Paradigm Spine, Benevenue, NexGen, K2 Medical, Pioneer, Amedica, Vertiflex, Electrocore, Surgitech, Invuity, Axiomed; board of 1529-9430/$ – see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.spinee.2010.03.034
directors, North American Spine Society, Cervical Spine Research Society; scientific advisory board, VG Innovations, Corespine, Expanding Orthopaedics, Syndicom, Osprey, Amedica, Bone Biologics, Curative Biosciences, Facet Solutions, PearlDiver, Pioneer, SeaSpine, Axis). * Corresponding author. Orthopaedic Spine Service, Department of Orthopaedics and Neurosurgery, UCLA Comprehensive Spine Center, 1250 16th St, Suite 745, Santa Monica, CA 90404, USA. Tel.: (310) 319-3334; fax: (310) 319-5055. E-mail address: [email protected] (J.C. Wang)
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S.-T. Tzeng et al. / The Spine Journal 10 (2010) e17–e20
Introduction Bone morphogenetic protein-2 (BMP-2) has been used extensively in recent years to enhance the process of spinal fusion [1–3]. However, as the use of BMP-2 has become more prevalent, numerous side effects, such as ectopic bone formation, neck swelling, hematoma, dysphagia, airway compression, bony resorption, graft subsidence, and fluid accumulation with mass effect over operative area have been reported [4,5]. This raises concerns regarding the safety of using this material in human beings [6]. In addition, concerns regarding the use of the BMPs in women of childbearing age and possible adverse effects on the fetus are theoretically contraindications for use. There is also the possibility of the transmission of the proteins through breast milk in lactating mothers as another potential adverse issue that may be of concern. There are no published reports, that we are aware of, that demonstrate detectable levels of BMP-2 in human milk or for that matter in human serum (milk is modified serum). This report deals with analyzing milk from a patient who underwent spinal surgery using recombinant human bone morphogenetic protein-2 (rhBMP-2) shortly after delivering a child to determine the presence/absence of rhBMP-2.
Case report A 35-year-old female suffered from postdiscectomy aseptic discitis at the L4/L5 level. She received combined anterior lumbar interbody fusion with posterior instrumentation on April 17, 2008. During the anterior operation, rhBMP-2 (medium-sized INFUSE bone graft kit [Medtronic, Inc., Memphis, TN, USA]) was packed into the middle of the femoral ring allograft to enhance the process of spinal fusion. Before surgery, the patient breast-fed her child for 4 months. A control sample of milk was obtained preoperatively. A few hours after surgery, the patient began pumping milk and continued to do so for several weeks. Each time a sample was collected, it was immediately placed in a freezer and subsequently shipped to the laboratory on dry ice. Each sample was analyzed using the method described below.
Table 1 Sample details Sample no.
Date
Time
Preoperative* 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
April 2 April 18 April 18 April 18 April 18 April 19 April 19 April 19 April 20 April 20 April 21 April 23 April 23 April 27 April 29 April 30 April 30 May 1
— 1:30 AM 8:30 AM 3:10 PM 8:45 PM 2:15 AM 6:30 AM 4:00 PM 9:00 AM 11:00 AM 10:00 AM 1:00 AM 5:00 AM 2:00 PM 3:00 PM 7:00 PM 9:00 PM 7:00 PM
* Date of surgery: April 17, 2008.
to 50-mL conical tubes. If only a small amount of material was available from two bags with identical designations, they were pooled. One milk sample was used for assays at each time point. An aliquot of 10 mL from each of the 18 samples was transferred to a 15-mL centrifuge (Sarstedt, AG & Co., Rommelsdorfer Straße, Nu¨mbrecht, Germany) tube. The processing methods were adapted from those of Oslislo et al. [7]. The 10-mL samples were centrifuged at 2,000 rpm at 4 C using a Sorvall SM-24 rotor in a Sorvall RC-5 centrifuge for 10 minutes (estimated force5500g). Approximately 4 mL of clarified ‘‘milk serum’’ was removed using a thin-tipped transfer pipette. From the approximately 4 mL of isolated ‘‘milk serum,’’ four 1-mL samples were placed into 1.4-mL microfuge tubes and frozen for subsequent use. On the day of assay, one of the four samples was thawed and then spun briefly in a microfuge. A sample of 100 mL was removed from the clarified liquid for assay. Assay methods
Samples were received frozen, on dry ice, in labeled Gerbers plastic bags. Each sample was labeled with a date and a time. Some had other designations as noted below. The time between the ‘‘preoperative’’ sample and the first ‘‘postoperative’’ sample was 16 days. A total of 18 unique time-point samples were received (Table 1).
Bone morphogenetic protein-2 enzyme-linked immunosorbent assay kits were obtained from R&D Systems (Minneapolis, MN, USA). Assays were performed exactly according to the manufacturer’s instructions with the exception that 100 mL of sample was assayed rather than 50 mL. Samples were assayed undiluted. Two assays were performed. The first assay was done to determine if dilution of the samples was required. A second assay was performed to confirm the results of the first. Samples were tested in duplicate within each assay.
Sample preparation
Results
Samples were initially thawed on ice. Final thawing occurred at room temperature. Samples were then transferred
The breast milk was obtained at the stated time points, and each sample was assayed for the presence of BMP-2
Samples received
S.-T. Tzeng et al. / The Spine Journal 10 (2010) e17–e20 Table 2 Sample results Sample no.
BMP-2 concentration (pg/mL)*
Optical density value
Preoperative 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
78.629y 78.629y 78.629y 78.629y 78.629y 78.629y 78.629y 78.629y 55.444y 78.629y 78.629y 55.444y 78.629y 55.444y 55.444y 32.258y 14.113y 55.444y
0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.003 0.002 0.002 0.003 0.002 0.003 0.003 0.004 0.006 0.003
BMP-2, bone morphogenetic protein-2. * Dose range for the standard curve: 62.5 to 500 pg/mL. y Value is outside of standard curve.
with the enzyme-linked immunosorbent assay kits. The linear regression analysis was performed. This calculation used four standards, and the correlation coefficient (r) for the resulting line was 0.9998, indicating an acceptable degree of statistical reliability. The optical density values and the extrapolated BMP-2 concentrations of the samples were measured and listed in Table 2. The dose ranges for the standard curve were 62.5 to 500 pg/mL for the BMP-2 concentration and 0.008 to 0.026 for the optical density value. The optical density values of the samples ranged from 0.002 to 0.006; all of them are less than that of the lowest standard (‘‘limit of detection’’). Using this precise standard curve, 17 of the 18 samples gave negative BMP-2 concentrations (78.629 pg/mL in 11 samples, 55.444 pg/mL in 5 samples, and 32.258 pg/mL in 1 sample), that is, none was detected. One sample gave a positive result (14.113 pg/mL), which was less than one quarter that of the lowest standard (62.5 pg/mL). However, this is a statistical artifact. This cannot be regarded as an accurate measurement and is regarded as below the level of detection for the assay. Therefore, if we took into consideration of this statistical artifact, the BMP-2 was not detected in all the milk samples with a limit of detection of 62.5 pg/mL.
Discussion There has been growing concern regarding the safety of using rhBMP-2 for spine surgery. A single report was located indicating that animal milk proteins enhance bone
e19
growth [8]. To our knowledge, there is no literature focusing on the presence of BMP-2 within human milk. If BMP-2 exists in breast milk, it is possible that it could be transmitted to the baby through breast-feeding. We do not know yet whether BMP-2 could have positive or negative effects on child development. This is a basic fact we must ascertain to avoid potential risks to child development. In our study, BMP-2 was not detected in human milk with a limit of detection of 62.5 pg/mL. Although some statistical artifact was inevitably generated during data analysis, confidence in this negative result is increased by the fact that two assays using different reagents were performed and gave the same results. Pharmacokinetic studies using rat and nonhuman primates have shown rapid systemic clearance of rhBMP-2 after intravenous administration. The use of rhBMP-2 in combination with absorbable collagen sponges has resulted in retention of rhBMP-2 at the site of application and low levels of detectable rhBMP-2 within the systemic circulation. The slow release of the rhBMP-2 from the implant site coupled with the rapid clearance of rhBMP-2 results in low systemic exposure [5]. Besides, these processes still could be dose related. If we use larger amount of rhBMP-2 during surgery, it would increase the chance of BMP-2 to be detected in the assay. For this study, the time between rhBMP-2 implantation and the collection of the first postoperative sample is unknown. Therefore, we cannot rule out the possibility that rhBMP-2 might have been detected shortly after implantation. It is possible that because of rapid clearance of rhBMP-2, systemic concentrations (such as in the serum or breast milk) are virtually undetectable. Other factors must be taken into consideration when interpreting the results of this study. For example, the protein may have been denatured during the process of sample collection and storage. This may have caused false negative results to be obtained. In addition, animal studies using rhBMP-2 have demonstrated that rhBMP-2 does not have teratogenic properties. Furthermore, rhBMP-2 has not demonstrated systemic toxicity in human studies [4,5]. Finally, this study is only a single case report; a stronger conclusion could be generated through completion of a more comprehensive study involving a larger number of patients.
Conclusion In this single case report, rhBMP-2 was not detected in human milk before or after the use of the growth protein in a spine surgery. Although this result seemed promising, it did not eliminate the concerns regarding the potential side effects of transmission of rhBMP-2 on child development. We still need further studies including larger case numbers to verify this conclusion.
e20
S.-T. Tzeng et al. / The Spine Journal 10 (2010) e17–e20
Acknowledgments Key points
Bone morphogenetic protein-2 has been used extensively in recent years to enhance the process of spinal fusion. However, numerous side effects have been reported. This raises concerns regarding the safety of using this material in human beings. It is possible that because of rapid clearance of recombinant human bone morphogenetic protein-2 (rhBMP-2), systemic concentrations (such as in the serum or breast milk) are virtually undetectable. The protein may have been denatured during the process of sample collection and storage. This may have caused false negative results to be obtained. No rhBMP-2 was detected in human milk in this single case report. This promising result did not mean that we do not need to concern about the potential risk of rhBMP-2 on child development. Further studies including more cases are needed to verify this conclusion.
The authors thank Dr Shireen Guide for her contributions in this report. References [1] Carlisle E, Fischgrund JS. Bone morphogenetic proteins for spinal fusion. Spine J 2005;5:240S–9S. [2] Sandhu HS. Bone morphogenetic proteins and spinal surgery. Spine 2003;28:S64–73. [3] Gautschi OP, Frey SP, Zellweger R. Bone morphogenetic proteins in clinical applications. ANZ J Surg 2007;77:626–31. [4] Poynton AR, Lane JM. Safety profile for the clinical use of bone morphogenetic proteins in the spine. Spine 2002;27:S40–8. [5] Benglis D, Wang MY, Levi AD. A comprehensive review of the safety profile of bone morphogenetic protein in spine surgery. Neurosurgery 2008;62(5 Suppl 2):ONS423–31. [6] FDA public health notification: life-threatening complications associated with recombinant human bone morphogenetic protein in cervical spine fusion. 2008 July. [7] Oslislo A, Czuba Z, Slawska H, et al. Decreased human milk concentration of epidermal growth factor after preterm delivery of intrauterine growth-restricted newborns. J Pediatr Gastroenterol Nutr 2007;44: 464–7. [8] Aoe S, Toba Y, Yamamura J, et al. Controlled trial of the effects of milk basic protein (MBP) supplementation on bone metabolism in healthy adult women. Biosci Biotechnol Biochem 2001;65:913–8.