Bone Vol. 29, No. 4 October 2001:368 –373
Homologous Growth Hormone Accelerates Healing of Segmental Bone Defects M. RASCHKE,1 S. KOLBECK,1 H. BAIL,1 G. SCHMIDMAIER,1 A. FLYVBJERG,2 T. LINDNER,1 M. DAHNE,1 I.-A. ROENNE,3 and N. HAAS1 1
Trauma and Reconstructive Surgery, Medical Faculty Charite´, Virchow Clinic, Humboldt University, Berlin, Germany Institute of Experimental Clinical Research and Medical Department M, University of Aarhus, Aarhus, Denmark 3 Health Care Discovery-Endocrinology, Growth Hormone Biology, Novo Nordisk A/S, Copenhagen, Denmark 2
Introduction The effect of homologous recombinant porcine growth hormone (r-pGH) on secondary fracture healing was investigated in a diaphyseal defect of the tibia in Yucatan micropigs. A 1 cm defect of the tibia was created surgically and stabilized with an AO 3.5 mm DCP plate. The treatment group (12 animals) received 100 g of r-pGH per kilogram of body weight subcutaneously once per day, whereas the control pigs (12 animals) received 1 mL of sodium chloride as placebo. For evaluation of the GH-axis, serum levels of insulin-like growth factor-I (IGF-I) were sampled every fourth day. The animals were killed 6 weeks after surgery. Quantitative computed tomography (qCT) was performed to determine bone mineral density (BMD) and bone mineral content (BMC) of the defect zone. The torsional stiffness and the torsional failure load were measured by destructive torsional testing of the defect and contralateral tibiae. qCT measurements revealed a significant increase in the BMC of the defect zone in the treatment group compared with controls (GH BMC ⴝ 2833 ⴞ 679 mg, placebo BMC ⴝ 2215 ⴞ 636 mg; p < 0.05), whereas the BMD values were similar in both groups (GH BMD ⴝ 668 ⴞ 60 mg/mm2, placebo BMD ⴝ 629 ⴞ 52 mg/mm2, p ⴝ 0.12). Torsional failure load was 70% higher and torsional stiffness 83% higher in the treatment group than in the control group (p < 0.05). The mean serum level of IGF-I in the treatment group increased to 382% of the preoperative basal level and decreased to 69% in the control group, and this difference was highly significant (p < 0.001). Our data indicate that daily administration of recombinant GH leads to an increase of serum IGF-I levels and stimulates secondary fracture healing, resulting in increased mechanical strength and stiffness of the callus. (Bone 29:368 –373; 2001) © 2001 by Elsevier Science Inc. All rights reserved.
Treatment of fractures with segmental bone loss remains one of the most challenging problems in orthopedic surgery. Multiple procedures, such as the implantation of autogenous or allogenous bone grafts, bone transport, or the application of ultrasound are frequently required to heal a severely injured limb.11,14 Some of these procedures may result in considerable donor-site morbidity37,40 and may cause transmission of infectious agents.23,35 Acceleration of fracture repair in complicated fractures through nonoperative techniques is therefore one of the most active areas in orthopedic research. Many clinicians have observed that systemic humoral mechanisms enhance bone formation. Patients having sustained a severe head injury have been reported to have skeletal fractures that heal faster than those without a head injury.5,36 In addition, it has been shown that an injury to one part of the skeleton may increase bone formation at a distal skeletal site.10 Growth hormone (GH) has been implicated as one of these humoral factors. The important role of GH and its dependent growth factors in the development and regulation of skeletal growth were first described by Evans and Long in 1921.12 The first clinical examinations and animal experiments concerning GH and bone healing under special circumstances were made by Koskinen.19,20 Due to the lack of availability of GH, only a few clinical and animal studies concerning GH and stimulation of bone healing were performed, with controversial results.16,18,21,25,27,29 Although recombinant GH is now readily available, only a few animal studies investigating GH’s effect on fracture healing have been published. These investigations have reported conflicting conclusions with regard to the ability of GH to affect fracture healing. Bak et al. described a significant increase of biomechanical parameters in a rat model by application of recombinant human GH (r-hGH).2– 4 In contrast, Carpenter et al.6 observed no such effect on fracture healing in a rabbit model. In a previous study we showed that GH stimulates bone formation in a model of intramembranous bone formation.32 Thus, the aim of the present study was to investigate the influence of r-GH in a similar animal model, which utilizes secondary bone healing, using a well-characterized defect model of the tibia in micropigs. To our knowledge, no other studies have investigated the influence of recombinant, species-specific GH on secondary fracture healing in a large animal model. The objective of the present study was therefore to determine the effect of systemic administration of homologous recombinant
Key Words: Secondary fracture healing; Bone defect; Recombinant growth hormone; Insulin-like growth factor; Mechanical testing; Micropig.
Address for correspondence and reprints: Michael Raschke, M.D., Unfall- und Wiederherstellungschirurgie Charite´, Campus Virchow Clinic, Augustenburger Platz 1, 13353 Berlin, Germany. E-mail: michael.
[email protected] Parts of this study have been presented at the meeting of the Orthopaedic Research Society, Anaheim, CA, 1999 © 2001 by Elsevier Science Inc. All rights reserved.
368
8756-3282/01/$20.00 PII S8756-3282(01)00587-7
Bone Vol. 29, No. 4 October 2001:368 –373
M. Raschke et al. Growth hormone accelerates fracture healing
369
GH on bone regenerate consolidation in secondary fracture healing. For this purpose, we measured bone mineral density (BMD) and bone mineral content (BMC) of the defect zone and final bone regenerate strength in two groups of micropigs, one serving as a control and the other treated with GH. Materials and Methods Animals All procedures were undertaken in compliance with the “Guidelines for the Care and Use of Animals,” as described in the American Journal of Physiology. Study protocols were reviewed and approved by the local governmental animal rights protection authorities and supervised by the local animal protection officer. Twenty-four mature female Yucatan micropigs, mean age 19 months (12–27 months) and mean body weight 41 kg (33– 49 kg), were used. The animals were matched by age and weight prior to the experiment. Animals were housed in groups of six during the experimental period and fed a diet of standard pellet cereal (300 g/day) and water ad libitum. Surgery Prior to surgery all animals received a subcutaneous implantable port system (Vascular Access Port, Access Technologies, Skokie, IL) to provide easier access for blood sampling and intravenous injections. Animals received 2 mg of metomidate hydrochloride intravenously as preanesthetic sedation and were then induced intravenously with sodium thiopental. The micropigs were intubated and muscle relaxation was achieved with pancuronium bromide. General anesthesia was maintained with sodium thiopental and fentanyl dihydrogencitrate for the duration of surgery. The right hind-limb was prepared in the usual sterile fashion and a 6 cm skin incision was made in the middle of the tibia using an anteromedial approach. The subcutaneous tissue was incised and the periosteum carefully preserved. A nine or ten hole 3.5 mm dynamic compression plate (Synthes, Bochum, Germany) was contoured to the medial surface of the intact tibia under fluoroscopic control. The plates were provisionally held to the tibia and all holes were predrilled with a 2.5 mm drill bit. The plate was then removed and the periosteum was incised longitudinally for a length of 4.0 cm carefully loosened and elevated. Using an oscillating saw a 1.0 cm cylindrical segment was cut and removed. After suturing the periosteum with absorbable suture the plate was reattached and fixed with 3.5 mm cortical screws. The subcutaneous tissue was closed with absorbable suture, the skin with non-absorbable suture. A sterile dressing was then applied. Posteroanterior X-rays were performed after the operation (Figure 1) and the animals were returned to their cages. Perioperative antimicrobial prophylactics, consisting of amoxicilline, clavulanate, and depot benzylpenicilline, were administered. Full weight bearing was allowed immediately after surgery. Analgesia was maintained by intravenous administration of flunixine meglumine for three postoperative days. The animals were visited twice daily. Wound inspections, temperature measurements, and radiographic examinations were performed throughout the entire study period until killing (Figure 2).
Figure 1. Anteroposterior radiograph after surgery showing the 1 cm defect and the plate on the medial side of the tibia.
neck skinfold every day between 8 and 10 A.M., starting on the day of surgery and continuing until the day of killing. Measurement of Serum IGF-I After sedation and intubation of the animals, but before implantation of the port system, 20 mL of blood was drawn from an ear vein to obtain initial values of serum IGF-I. The animals were sedated every fourth day (days 4, 8, 12, up to 40) with intravenous metomidate for blood drawing and radiography. The blood was immediately centrifuged and the serum frozen at ⫺80°C. Acid ethanol was used to remove the IGF-binding proteins (IGFBPs) from thawed serum.9 Serum extracts were diluted in assay buffer (final dilution 1:1000) and total IGF-I serum levels were determined with a noncompetitive time-resolved immunofluorometric assay (TR-IFMA) as previously de-
Recombinant Porcine Growth Hormone Micropigs in the treatment group received a single daily subcutaneous injection of 100 g of recombinant porcine GH (r-pGH) per kg bodyweight (met-pGH, Brese Gen. Ltd., Adelaide, Australia), while micropigs in the control group received sodium chloride as a placebo. The injections were given in a marked
Figure 2. Conventional X-ray 6 weeks post operatively. (a) Control animal: the defect is only partially filled with new callus; (b) r-pGHtreated animal: the 1 cm defect is fully bridged by new callus.
370
M. Raschke et al. Growth hormone accelerates fracture healing
Bone Vol. 29, No. 4 October 2001:368 –373
to quantify BMD in equivalent mineral density milligrams per milliliter. The bone mineral content of the defect zone was then calculated by integrating the BMD of the nine 1 mm slices within the defect. Biomechanical Testing The tibiae were embedded in polymethylmethacrylate, keeping a constant distance of 7 cm between the embedded bone ends. Then they were mounted in a materials testing machine (Zwick 1455, Zwick GmbH, Ulm, Germany). Each specimen was kept moist throughout biomechanical testing. A preload of 1 Nm was applied and the construct was then loaded in torsion under displacement control of 10°/min until failure. Torsional failure load (yield load) and torsional stiffness were determined from the load-displacement curve. Statistical Analysis
Figure 3. Sagittal CT scan after killing. (a) Control animal: the defect is only partially filled with new callus; (b) r-pGH-treated animal: the 1 cm defect is fully bridged by new callus.
scribed.13 Briefly, two sets of monoclonal IGF-I antibodies were used: the first was immobilized on microtestplate wells (IGF-I mAb from Novo-Nordisk A/S, Denmark), the second was labeled with europium (Eu3⫹, Diagnostic System Laboratories, Inc., Webster, TX). Biosynthetic human IGF-I (hIGF-I, Amgen Biologicals, Thousand Oaks, CA, distributed by Amersham International, Amersham, Bucks, UK) served as standard. Detection limit was 0.0025 g/L and the operating range was 0.005– 2.5 g/L. The calibration curve was linear in this interval. Intraand interassay coefficients of variation were less than 5% and 10%, respectively. qCT Measurements Animals were anesthetized with intravenous sodium thiopental and killed with potassium chloride 42 days after surgery. Both tibiae were harvested, all soft tissue was dissected, and the plate carefully removed. The tibiae were placed in a physiological salt solution and qCT was performed (Somatom Plus, Siemens, Germany). The scanning parameters were 210 mA at 120 kV. First, the tibiae were imaged with a scout view parallel to the long axis of the tibia and sagittal scans were performed to determine the total amount of callus in the defect (Figure 3). The tibiae were then placed in a perpendicular manner and crosssectional scans were made of the defect zone. Nine consecutive horizontal sections with a slice distance of 1 mm were obtained within the defect zone at a thickness of 1 mm and a matrix size of 1024 ⫻ 1024 pixels. The CT scanner was used to measure the cross-sectional area of callus. Callus was defined as tissue density ⬎250 Hounsfield units (HU). This value was determined by measuring the radiodensity of various tissues in the tibia of the micropig. It was found that, at ⬎250 HU, only mineralized tissue was present. The manufacturer’s software package was used for image processing and data evaluation. A lucid calibration phantom with varying concentrations of K2HPO4 was used
The Kolmogoroff–Smirnov goodness-of-fit test was used to ensure that data were normally distributed. A repeated-measures analysis of variance (ANOVA) was performed between IGF-I data of both groups. Covariates included drugs applied, age, and preoperative weight. An independent-sample t-test was used to determine differences between groups in BMC, BMD, torsional stiffness, and maximum torsional failure load. Data are expressed as the mean ⫾ standard deviation (SD). Statistical differences were analyzed using Student’s t-test and balanced with the Bernoulli’s test if more than two groups were compared. p ⬍ 0.05 was considered statistically significant. All statistical analyses were carried out using the SPSS software package (Statistical Package for Social Sciences, SPSS, Inc.). Results Clinical Data Two animals had to be excluded from the study (one in the treatment group and one in the control group) due to infection in the defect zone, leaving 11 animals in each group. Both animals had high body temperatures after the third week of the experiment. The X-ray controls for both animals demonstrated hypertrophic ossifications after week 4 of the experiment. After skin incision at the time of killing, pus was present in the fracture gap of these two animals. Smear tests indicated Escherichia coli upon culturing. All other pigs appeared healthy throughout the experiment. The mean temperature and the mean white blood cell count were not statistically different between the groups. Serum IGF-I Levels The mean level of serum IGF-I in relation to the pretreatment level increased in the GH-treated animals after a time period of approximately 20 days to 382 ⫾ 86%, whereas IGF-I levels in the control group decreased to 69 ⫾ 14%. The difference between groups was statistically highly significant (p ⬍ 0.001). The mean level of serum IGF-I increased steadily up to day 16 and then remained unchanged until killing for the treatment group (Figure 4). In the control group, IGF-I levels decreased up to day 8 of the experiment, recovered between days 8 and 12 to the preoperative level, and then remained nearly unchanged until killing. Repeated-measures ANOVA showed a significant difference between groups starting at day 4 at every examination timepoint (p ⬍ 0.001; Figure 4).
Bone Vol. 29, No. 4 October 2001:368 –373
Figure 4. IGF-I measurements. Statistical analysis showed significant differences (p ⬍ 0.001, ANOVA model for repeated measurements) between the r-pGH-treated and the control groups (mean ⫾ SEM).
M. Raschke et al. Growth hormone accelerates fracture healing
371
Figure 6. Bone mineral density of the defect zone. Statistical analysis showed no significant differences (p ⫽ 0.12; independent-sample t-test) between the r-pGH-treated and control groups.
for torsional stiffness were 134.8 ⫾ 67.7% for the GH group and 64.9 ⫾ 30.37% for the control animals (p ⫽ 0.005; Figure 7).
qCT Measurements Discussion The sagittal CT scans (CT scan parallel to axis of the tibia) demonstrated a visible difference between the treatment and control groups. In the control animals, the defect was only partially filled with callus, whereas, in most of the GH-treated animals, the defect was fully bridged by new callus (Figure 3). This qualitative impression was confirmed by qCT analysis of the cross-sectional CT scans. These measurements revealed a significant difference in the BMC of the defect zone between the GH group and the placebo group (GH BMC ⫽ 2833 ⫾ 679 mg, placebo BMC ⫽ 2215 ⫾ 636 mg; p ⬍ 0.05). The BMD in the defect zone was comparable in both groups (GH BMD ⫽ 668 ⫾ 60 mg/mm2, placebo BMD ⫽ 629 ⫾ 52 mg/mm2; p ⫽ 0.12, Figures 5 and 6). Final Biomechanical Testing The tibiae of the r-pGH-treated group exhibited a 70% higher mean torsional failure load than the tibiae of the placebo group (GH 18.06 ⫾ 5.91 Nm, placebo 10.62 ⫾ 5.2 Nm; p ⫽ 0.005). The mean torsional stiffness was 83% higher in the treatment group (1.74 ⫾ 0.92 Nm/°) compared with the control group (0.95 ⫾ 0.40 Nm/°; p ⬍ 0.05; Table 1). In relation to the intact contralateral tibia, the defect tibia in the treatment group and the control group measured 75.21 ⫾ 30.5% and 40.57 ⫾ 22.1% of the torsional failure load, respectively (p ⫽ 0.006). The values
Figure 5. Bone mineral content (BMC) of the defect zone. Statistical analysis showed a significant difference (p ⬍ 0.05; independent-sample t-test) between the r-pGH-treated and control groups.
The mechanisms of GH action on skeletal tissue were first described by Salmon and Daughaday in 1957, who found that the effect of GH in cultured cartilage was mediated by GH-dependent peptides in serum.34 Subsequently a group of peptides, initially named somatomedines and later insulin-like growth factors (IGFs), were identified as responsible for GH action.8 After the purification of two distinct somatomedines by Rinderknecht and Humbel in 1978, subsequent chemical and molecular analyses identified peptides similar to IGFs. Physiological studies showed that only IGF-I was directly regulated by GH.33 Two theories have been brought forth to explain the role of the GH–IGF-I axis in mesenchymal cell response to GH. The somatomedine theory postulates that GH produced in the pituitary is released into the bloodstream and transported to nonskeletal tissues (principally the liver) and skeletal tissues (such as the growth plate). There, it stimulates the production of IGF-I, which is released into the circulation and acts in an endocrine fashion on its target tissues.24 The second theory is the “dual effector theory,” introduced by Green and Isaakson.15 This theory postulates that GH induces the differentiation of precursor cells to an IGF-I responsive state. These immature cells are then susceptible to IGF-I and able to proliferate when IGF-I is released by GH action in the liver. Regardless of the pending validation of either theory, it is clear that an intact GH–IGF-I axis is necessary for mesenchymal tissue response to GH. Conflicting results concerning the influence of GH on fracture healing have been reported in different animal models. Although several studies have demonstrated the beneficial effect of GH on fracture healing,30,41 other studies have reported that GH has no stimulatory effect on the repair of fractures.6,16,29,39 In reviewing these studies there are differences with regard to animal species, fracture models used, types and dosages of GH, and methods of testing. The studies published earlier are particularly characterized by inhomogeneity of the treatment and control groups, different sources of GH, and insufficient control for animal gender and age. More recent experiments using recombinant GH in fracture models have focused on differences in biomechanical parameters of different treatment groups. Bak and coworkers found a significant increase of torsional stiffness and strength in different fracture models using recombinant human GH in rats.1,3,28 Carpenter et al. found no effect of
372
M. Raschke et al. Growth hormone accelerates fracture healing
Bone Vol. 29, No. 4 October 2001:368 –373
Table 1. Results of final biomechanical testing (mean ⫾ SD) using an independent sample t-test
Torsional failure load (Nm) of defect tibia Torsional failure load (Nm) of contralateral tibia Torsional failure load (Nm) of defect tibia in percentage of contralateral tibia Torsional stiffness (Nm/°) of defect tibia Torsional stiffness (Nm/°) of contralateral tibia Torsional stiffness (Nm/°) of defect tibia in percentage of contralateral tibia
r-pGH (n ⫽ 11)
Placebo (n ⫽ 11)
Significance level
18.06 ⫾ 5.91 25.33 ⫾ 6.89 75.21 ⫾ 30.49
10.62 ⫾ 5.20 27.39 ⫾ 6.14 40.57 ⫾ 22.14
p ⫽ 0.005 n.s. p ⫽ 0.005
1.74 ⫾ 0.92 1.30 ⫾ 0.29 134.80 ⫾ 67.74
0.95 ⫾ 0.40 1.57 ⫾ 0.53 64.94 ⫾ 30.37
p ⬍ 0.05 n.s. p ⫽ 0.005
KEY: n.s., not significant; r-pGH, recombinant porcine growth hormone.
recombinant human GH in a rabbit fracture model.6 In a defect model of membrane-covered, transosseous defects in rat mandibles, augmented callus formation was found when GH was administered, both systemically and locally. In this model, the mandibular defect was bridged by primary bone healing due to osteogenic cells derived from endosteal sources. A periosteal cell contribution could be excluded due to the placement of a membrane, which was only permeable to tissue fluid and macromolecules.17 In a model of intramembranous bone formation, which occurs predominantly in distraction osteogenesis, systemic administration of recombinant homologous GH greatly accelerated ossification of the bone regenerate.32 In the present study we have been able to show an intact GH–IGF-I axis with the dosage used (100 g r-pGH/kg body weight per day). Daily subcutaneous application of r-pGH in pigs resulted in a marked increase in IGF-I serum levels. The IGF-I levels of the GH-treated animals increased steadily during the first 2 weeks of the observation period and remained constant at a four to fivefold level relative to preoperative baseline. This is in contrast to the the data from Carpenter et al., who found that serum IGF-I levels were unchanged in response to GH administration.6 This may explain the failure of GH treatment in this model. Antibody formation against the allogenous GH could be yet another explanation. This problem has been described in several studies.22,38,42,43 Homologous porcine GH was therefore used in the present study, which makes the formation of antibodies unlikely. We also observed that the biomechanical properties of the healing bone for secondary fracture healing were significantly affected by daily subcutaneous application of r-pGH. The torsional stiffness (TS) and the torsional failure load (TFL) of the defect tibia in the r-pGH-treated group were 83% (TS) and 70% (TFL) higher, respectively, than in the placebo group. We found
that the mean torsional stiffness of the fractured tibia in the r-pGH-treated group was 135% higher than the contralateral tibia (placebo group 65%). The torsional failure load in the r-pGHtreated group was 71% in comparison to 39% in the placebo group. The fact that the torsional failure load was lower than the torsional stiffness may be explained by the rigidity of the fracture in weight-bearing bones returning more rapidly than strength. This has been described previously by Connolly et al.7 The qCT analysis strengthens the different biomechanical results in both groups. The BMC values of the r-pGH-treated animals were significantly higher than in the placebo animals. This indicates that an increased amount of newly formed callus was present in the defect area following the 6 week observation period. The BMD values for the r-pGH-treated and placebo animals were similar. This may indicate that the structure of the callus tissue in both groups was comparable. These data require further histological evaluation. Our results are in contrast to the histological data of Mosekilde et al., who found, in a fracture model using rats, a greater amount of callus after GH application, but the newly formed callus presented a looser structure compared with placebo animals.26 This may be due to the purported greater amount of cartilaginous callus formation in rodents than in pigs.31 The results presented here are consistent with our previous study in which r-pGH administration resulted in a pronounced acceleration of bone regenerate consolidation in a distraction osteogenesis micropig model.32 Thus, the effect of GH is not limited to intramembranous bone formation, which occurs predominantly during distraction osteogenesis, but also secondary bone healing. Our findings further suggest that GH action takes place via an endocrine pathway, accelerating the basic events of bone formation through the GH–IGF-I axis. The present data further suggest an important role for recombinant GH in bone healing. Compared with currently used methods for enhancement of fracture healing, including the use of autografts and allografts or locally active growth factors (such as bone morphogenetic proteins), open access to the fracture is required. This makes further operation necessary, may result in donor-site morbidity, and may also lead to an increased risk of infection. Systemic administration of recombinant GH might therefore be a viable alternative. Although future studies are necessary to discover the local interactions between GH, IGF-I, and mesenchymal tissues during the fracture repair process, our findings demonstrate the potential of GH application in accelerating bone repair.
Figure 7. Results of the biomechanical testing. Statistical analysis showed significant differences (p ⬍ 0.05; independent-sample t-test) between the r-pGH-treated and control groups.
Acknowledgments: This study was supported by Novo Nordisk, Copenhagen, Denmark.
Bone Vol. 29, No. 4 October 2001:368 –373
References 1. Bak, B., and Andreassen, T. T. The effect of growth hormone on fracture healing in old rats. Bone 12:151–154; 1991. 2. Bak, B., Jorgensen, P. H., and Andreassen, T. T. Dose response of growth hormone on fracture healing in the rat. Acta Orthop Scand 61:54 –57; 1990. 3. Bak, B., Jorgensen, P. H., and Andreassen, T. T. Increased mechanical strength of healing rat tibial fractures treated with biosynthetic human growth hormone. Bone 11:233–239; 1990. 4. Bak, B., Jorgensen, P. H., and Andreassen, T. T. The stimulating effect of growth hormone on fracture healing is dependent on onset and duration of administration. Clin Orthop 295–301; 1991. 5. Bidner, S. M., Rubins, I. M., Desjardins, J. V., Zukor, D. J., and Goltzman, D. Evidence for a humoral mechanism for enhanced osteogenesis after head injury. J Bone Jt Surg [Am] 72:1144 –1149; 1990. 6. Carpenter, J. E., Hipp, J. A., Gerhart, T. N., Rudman, C. G., Hayes, W. C., and Trippel, S. B. Failure of growth hormone to alter the biomechanics of fracturehealing in a rabbit model. J Bone Jt Surg [Am] 74:359 –367; 1992. 7. Connolly, J. F. and Hayes, W. C. The compressive behavior of bone as a two-phase porous structure. J Bone Jt Surg 59-A:954 –962; 1977. 8. Daughaday, W. H., Hall, K., Raben, M. S., Salmon, W. D., Jr., Van der Brande J. L. and van Wyk, J. J. Somatomedin: Proposed designation for sulphation factor. Nature 235:107; 1972. 9. Daughaday, W. H., Mariz, I. K., and Blethen, S. L. Inhibition of access of bound somatomedin to membrane receptor and immunobinding sites: A comparison of radioreceptor and radioimmunoassay of somatomedin in native and acid-ethanol-extracted serum. J Clin Endocrinol Metab 51:781–788; 1980. 10. Einhorn, T. A., Simon, G., Devlin, V. J., Warman, J., Sidhu, S. P., and Vigorita, V. J. The osteogenic response to distant skeletal injury. J Bone Jt Surg [Am] 72:1374 –1378; 1990. 11. Enneking, W. F., Eady, J. L., and Burchardt, H. Autogenous cortical bone grafts in the reconstruction of segmental skeletal defects. J Bone Jt Surg [Am] 62:1039 –1058; 1980. 12. Evans, H. M., and Jong, J. A. The effect of anterior lobe administered intraperitoneally on growth maturity and oestrus cycles of the rat. Anat Rec 21:62; 1921. 13. Frystyk, J., Dinesen, B., and Orskov, H. Non-competitive time-resolved immunofluorometric assays for determination of human insulin-like growth factor I and II. Growth Regul 5:169 –176; 1995. 14. Goldstrohm, G. L., Mears, D. C., and Swartz, W. M. The results of 39 fractures complicated by major segmental bone loss and/or leg length discrepancy. J Trauma 24:50 –58; 1984. 15. Green, H., Morikawa, M., and Nixon, T. A dual effector theory of growthhormone action. Differentiation 29:195–198; 1985. 16. Harris, J. M. D., Bean, D. A., and Banks, H. H. Effect of phosphate supplementation, thyrocalcitonin, and growth hormone on strength of fracture healing. Surg Forum 26:519 –521; 1975. 17. Hedner, E., Linde, A., and Nilsson, A. Systemically and locally administered growth hormone stimulates bone healing in combination with osteopromotive membranes: An experimental study in rats. J Bone Miner Res 11:1952–1960; 1996. 18. Herold, G. H. Z., Hurvitz, A., and Tadmor, A. The effect of growth hormone on the healing of experimental bone defects. Acta Orthop Scand 42:377–384; 1971. 19. Koskinen, E. The repair of experimental fractures under the action of growth hormone, thyreotropin and cortisone. A tissue analytic, roentgenologic and autoradiographic study. Ann Chir Gynaecol Fenn 90(Suppl.):1– 48; 1959. 20. Koskinen, E. V., Lindholm, R. V., Nieminen, R. A., Puranen, J., and Atila, U. Human growth hormone in fractures of long bones with delayed fracture healing. Fractures of tibial diaphysis and other bone injuries. Med Welt 26:1905–1910; 1975. 21. Lindholm, R., Lindholm, S., and Paasimaki, J. Fracture healing and mast cells. 3. Action of combined growth hormone and thyrotropin. Acta Orthop Scand 38:129 –132; 1967.
M. Raschke et al. Growth hormone accelerates fracture healing
373
22. Madon, R. J., Panton, D. M., and Flint, D. J. Long-term effect of a sheep antiserum to rat growth hormone in vivo in rats is explained by the rat anti-sheep immunoglobulin response. J Endocrinol 128:229 –237; 1991. 23. Marthy, S., and Richter, M. Human immunodeficiency virus activity in rib allografts. J Oral Maxillofac Surg 56:474 – 476; 1998. 24. McConaghey, P., and Sledge, C. B. Production of “sulphation factor” by the perfused liver. Nature 225:1249 –1250; 1970. 25. Misol, S., Samaan, N., and Ponseti, I. V. Growth hormone in delayed fracture union. Clin Orthop 74:206 –208; 1971. 26. Mosekilde, L., and Bak, B. The effects of growth hormone on fracture healing in rats: A histological description. Bone 14:19 –27; 1993. 27. Muhlbach, R., Tarsoly, E., Trzenschik, K., and Hirthe, D. Effect of growth hormone on the metabolism of bones and the healing of fractures. Acta Chir Acad Sci Hung 13:51– 63; 1972. 28. Nielsen, H. M., Bak, B., Jorgensen, P. H., and Andreassen, T. T. Growth hormone promotes healing of tibial fractures in the rat. Acta Orthop Scand 62:244 –247; 1991. 29. Northmore-Ball, M. D., Wood, M. R., and Meggitt, B. F. A biomechanical study of the effects of growth hormone in experimental fracture healing. J Bone Jt Surg [Br] 62:391–396; 1980. 30. Pankratiew, P. Zum Problem der biologischen Behandlung der Knochenbru¨ che. Arch Orthop Chir 32:424 – 457; 1932. 31. Pritchard, J. and Ruzicka, A. Comparison of fracture repair in the frog, lizard and rat. J Anat 84:236 –261; 1950. 32. Raschke, M. J., Bail, H., Windhagen, H. J., Kolbeck, S. F., Weiler, A., Raun, K., Kappelgard, A., Skiaerbaek, C., and Haas, N. P. Recombinant growth hormone accelerates bone regenerate consolidation in distraction osteogenesis. Bone 24:81– 88; 1999. 33. Rinderknecht, E. and Humbel, R. E. The amino acid sequence of human insulin-like growth factor I and its structural homology with proinsulin. J Biol Chem 253:2769 –2776; 1978. 34. Salmon, W. D., Jr. and Daughaday, W. H. A hormonally controlled serum factor which stimulates sulfate incorporation by cartilage in vitro. J Lab Clin Med 116:408 – 419; 1990. 35. Schratt, H. E., Regel, G., Kiesewetter, B., and Tscherne, H. HIV infection caused by cold preserved bone transplants. Unfallchirurg 99:679 – 684; 1996. 36. Smith, R. Head injury, fracture healing and callus. J Bone Jt Surg [Br] 69:518 –520; 1987. 37. Summers, B. N. and Eisenstein, S. M. Donor site pain from the ilium. A complication of lumbar spine fusion. J Bone Jt Surg [Br] 71:677– 680; 1989. 38. van Herpen, H., Rijnberk, A., and Mol, J. A. Production of antibodies to biosynthetic human growth hormone in the dog. Vet Rec 134:171; 1994. 39. Wray, B. and Goldstein, J. The effect of the pituitary gland and growth hormone upon the strength of the healing fracture in the rat. J Bone Jt Surg Am 48-A:815– 816; 1966. 40. Younger, E. M. and Chapman, M. W. Morbidity at bone graft donor sites. J Orthop Trauma 3:192–195; 1989. 41. Zadek, R. and Robinson, R. The effect of growth hormone on experimental long-bone defects. J Bone Jt Surg [Am] 43-A:1261–1261; 1961. 42. Zeisel, H. J., Lutz, A. and von Petrykowski, W. Immunogenicity of a mammalian cell-derived recombinant human growth hormone preparation during long-term treatment. Horm Res 37:47–55; 1992. 43. Zwickl, C. M., Cocke, K. S., Tamura, R. N., Holzhausen, L. M., Brophy, G. T., Bick, P. H. and Wierda, D. Comparison of the immunogenicity of recombinant and pituitary human growth hormone in rhesus monkeys. Fund Appl Toxicol 16:275–287; 1991.
Date Received: January 29, 2001 Date Revised: April 12, 2001 Date Accepted: April 18, 2001
