Improvement of fracture healing by systemic administration of growth hormone and local application of insulin-like growth factor-1 and transforming growth factor-β1

doi:10.1016/S8756-3282(02)00798-6

Bone

Volume 31, Issue 1, July 2002, Pages 165-172

https://doi.org/10.1016/S8756-3282(02)00798-6 Get rights and content

Abstract

Fracture healing is influenced by numerous hormones, growth factors, and cytokines. The systemic administration of growth hormone (GH) has shown to accelerate bone regeneration. Local application of growth factors, such as insulin-like growth factor-1 (IGF-1) and transforming growth factor-β-1 (TGF-β1), are known to stimulate bone metabolism. Until now, the exact local and systemic mechanisms that lead to improved bone regeneration remain unclear. In addition, the effect of systemic administration of GH as compared with locally delivered growth factors on fracture healing in rats is not known. A midshaft fracture of the right tibia of 5-month-old female Sprague-Dawley rats (n = 80) was intramedullary stabilized with IGF-1 and TGF-β1 coated vs. uncoated titanium K-wires. The growth factors were incorporated in a poly(D,L-lactide) (PDLLA) coating and released continuously throughout the experiment. Recombinant species-specific (rat) GH was applied systemically (2 mg/kg body weight) by daily subcutaneous injection and compared with a placebo group. The healing process was radiologically monitored. Twenty-eight days after fracture biomechanical torsional testing was performed. The consolidation and callus composition, including quantification of cartilage and mineralized tissue, was traced in histomorphometrical investigations using an image analysis system. Both methods, the systemic administration of GH and the local application of growth factors, showed significant biomechanical and histological effects on fracture healing. The local growth factor application showed a stronger effect on fracture healing than the systemic GH injection. The combined application of both methods did not accelerate the effect on bone healing compared with the single application. It is therefore concluded that combining local and systemic stimulating methods does not provide further additive effects with regard to fracture healing.

Introduction

Fracture healing is regulated by a complex interaction of growth factors, hormones, cytokines, and extracellular matrix. Methods accelerating bone formation and fracture healing are the subject of the current investigation. The osteoinductive effect of different substances has been proven in several in vitro and in vivo studies. The systemic administration of growth hormone (GH) has been shown to accelerate bone formation and regeneration.2, 25 Growth factors, locally applied from a variety of local drug carriers, have shown osteoinductive effects.4, 27, 31 We therefore investigated both methods of enhancement of fracture healing in a well-established model27 in order to evaluate their combined application as compared with the single mode of application, and to assess local and systemic outcomes.

GH is known to have both a direct and indirect stimulatory effect on bone growth and fracture healing, whereas the indirect mechanisms may be mediated mainly by IGF-1. Daily injections of species-specific growth hormone have shown a significant effect on intramembranous bone formation in distraction osteogenesis and in secondary fracture healing, using a porcine animal model.23, 25 The family of insulin-like growth factors (IGFs), especially IGF-1, are mediators of the GH-related effects on target tissues,10 and have been found to stimulate the replication of osteoblasts and the synthesis of bone matrix.11 Local application of IGF-1, using devices such as pumps or catheters, have been proven to enhance fracture healing in rat models.13, 19 The systemic application of IGF-1 has been shown to result in an increased growth rate in a human study on patients with a GH-receptor deficiency.34

Transforming growth factors (TGFs) are a heterogeneous group of factors influencing tissue growth and differentiation. Transforming growth factor-β1 (TGF-β1) is known to regulate different cell types, such as mesenchymal cells, chondrocytes, osteoblasts, and osteoclasts, which are directly involved in bone remodeling and fracture healing.14, 22 In a bone defect model in rabbits, systemic application of TGF-β1 positively influenced bone matrix formation and bone remodeling.5 Local application of TGF-β has been shown to accelerate fracture healing in a dose-dependent manner.16 IGF-1 and TGF-β1 seem to have certain synergistic effects,21, 22 and may therefore have an increased stimulating effect on fracture healing, if applied in combination.

The effect of growth factors on tissue depends on: (a) the choice of growth factors and their combination, respectively; (b) the dose applied; and (c) the mode of application. Opposite effects at high or low doses may even be observed.8, 30 The modes of administration of stimulating factors investigated so far include: (a) systemic application;25 (b) local injection into the fracture gap;8 (c) the use of pump systems;16 (d) and the use of various carrier systems such as minipellets,12 collagen sponges,33 polylactide blocks,36 or deorganified bovine bone.35

Potential disadvantages are known for each of these application modes: (a) systemic side effects due to stimulation of tissues other than the target tissue; (b) the risk of infection following injection; (c) difficult, clinically irrelevant techniques with high complication rates in using pump systems; and (d) uncontrolled release due to nonstandardized kinetics of different carrier systems with possible undesired tissue reactions.

With the exception of systemic application, all approaches of local growth factor application require direct access to the fracture site. The use of surgical implants, which are coated with growth factors, is another possible route of application. In vivo experiments in rats and pigs revealed an accelerating effect on bone healing of this bioactive coating.24, 27

Polymers of lactic acid (PLA) or glycolic acid (PGA) and their copolymers are used in orthopedic surgery and have been tested as drug-delivery systems.9, 32 The mechanical stability of the coating may be essential, especially when used with surgical implants. Damage or displacement of the coating with possible action of the incorporated drug in undesired regions or alteration of the release kinetics should not occur during implantation. The characteristics of a biomechanically stabile, approximately 10-μm-thin poly(D,L-lactide) (PDLLA) coating of implants, which was used in this study, have been tested in detail and described elsewhere.28

The purpose of this study was to investigate the effect of the local application of growth factors and the systemic administration of growth hormone on fracture healing and to determine a possible additive effect using both methods in combination.

Section snippets

Animals and fracture model

A well-established fracture model was used, which has been described previously.27 A closed midshaft fracture of the right tibia of 5-month-old female Sprague-Dawley rats (Harlan-Winkelmann, Borchen, Germany) (n = 80) was produced using a fracture device, which produces a standardized fracture. The animals were sedated with isoflurane (Forene) and intraperitoneal anesthesia with ketamine hydrochloride (100 mg/mL; 80 mg/kg body weight) and xylazine 2% (12 mg/kg body weight). After closed

Failure parameters

Three animals were excluded from the study: two animals due to death during anesthesia and one animal because of a complex tibial fracture at day 0. The dropout animals were replaced. No infections, no further implant failures, and no failures during biomechanical and histological measurements occurred.

X-ray examinations

The X-ray examinations of all treatment groups — local application of growth factors and systemic administration of GH — showed an enhanced consolidation of the fractures compared with the

Discussion

The aim of the present study was to evaluate the local, systemic, and combined effects of different modes of acceleration of fracture healing in a well-established animal model, and to assess local and systemic outcomes.

Growth factors, IGF-1 and TGF-β1, were locally applied using poly(D,L-lactide)-coated implants as the drug carrier and GH was injected daily subcutaneously throughout the entire experiment. Both methods, the local application of growth factors and the systemic administration of

Acknowledgements

The authors thank Dr. Ch. Müller, Department of Pathobiochemistry, Charité, Humboldt University of Berlin, Germany, for analyzing the blood and serum parameters, and Novo-Nordisk, Bagsvaerd, Denmark, for providing the recombinant rat growth hormone. This study was supported by Deutsche Forschungsgemeinschaft DFG (Schm 1436 1-1).

References (36)

H. Green et al.
A dual effector theory of growth hormone action
Differentiation
(1985)
M. Raschke et al.
Homologous growth hormone accelerates healing of segmental bone defects
Bone
(2001)
M. Raschke et al.
Insulin-like growth factor-1 and transforming growth factor-beta1 accelerates osteotomy healing using polylactide-coated implants as a delivery systemA biomechanical and histological study in minipigs
Bone
(2002)
M.J. Raschke et al.
Recombinant growth hormone accelerates bone regenerate consolidation in distraction osteogenesis
Bone
(1999)
G. Schmidmaier et al.
Local application of growth factors (insulin-like growth factor-1 and transforming growth factor-β1) from a biodegradable poly(D,L-lactide) coating of osteosynthetic implants accelerates fracture healing in rats
Bone
(2001)
T.G. Terrell et al.
Pathology of recombinant human transforming growth factor-beta 1 in rats and rabbits
Int Rev Exp Pathol
(1993)
L. Tielinen et al.
Combining transforming growth factor-beta(1) to a bioabsorbable self-reinforced polylactide pin for osteotomy healingAn experimental study on rats
J Orthop Sci
(1999)
R. Abs et al.
GH replacement in 1034 growth hormone deficient hypopituitary adultsDemographic and clinical characteristics, dosing and safety
Clin Endocrinol (Oxf)
(1999)
T.T. Andreassen et al.
Growth hormone stimulates bone formation and strength of cortical bone in aged rats
J Bone Miner Res
(1995)
B. Bak et al.
Dose response of growth hormone on fracture healing in the rat
Acta Orthop Scand
(1990)

B.E. Bax et al.

Bone morphogenetic protein-2 increases the rate of callus formation after fracture of the rabbit tibia

Calcif Tissue Int

(1999)

L. Beck et al.

TGF-beta1 induces bone closure of skull defects—temporal dynamics of bone formation in defects exposed to rhTGF-beta1

J Bone Miner Res

(1993)

C. Clanget et al.

Effects of 6 years of growth hormone (GH) treatment on bone mineral density in GH-deficient adults

Clin Endocrinol

(2001)

A. Flyvbjerg et al.

Inhibitory effect of a growth hormone receptor antagonist (G120K-PEG) on renal enlargement, glomerular hypertrophy, and urinary albumin excretion in experimental diabetes in mice

Diabetes

(1999)

R. Fujimoto et al.

Local effects of transforming growth factor-beta1 on rat calvariaChanges depending on the dose and the injection site

J Bone Miner Metab

(1999)

A. Göpferich

Bioresorbable polymers as drug delivery systems

J. Hock et al.

Insulin like growth factor I has independent effects on bone matrix formation and cell replication

Endocrinology

(1988)

K. Inui et al.

Local application of basic fibroblast growth factor minipellet induces the healing of segmental bony defects in rabbits

Calcif Tissue Int

(1998)

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Original articleImprovement of fracture healing by systemic administration of growth hormone and local application of insulin-like growth factor-1 and transforming growth factor-β1

Abstract

Introduction

Section snippets

Animals and fracture model

Failure parameters

X-ray examinations

Discussion

Acknowledgements

Differentiation

Bone

Bone

Bone

Bone

Int Rev Exp Pathol

J Orthop Sci

GH replacement in 1034 growth hormone deficient hypopituitary adultsDemographic and clinical characteristics, dosing and safety

Clin Endocrinol (Oxf)

Growth hormone stimulates bone formation and strength of cortical bone in aged rats

J Bone Miner Res

Dose response of growth hormone on fracture healing in the rat

Acta Orthop Scand

Bone morphogenetic protein-2 increases the rate of callus formation after fracture of the rabbit tibia

Calcif Tissue Int

TGF-beta1 induces bone closure of skull defects—temporal dynamics of bone formation in defects exposed to rhTGF-beta1

J Bone Miner Res

Effects of 6 years of growth hormone (GH) treatment on bone mineral density in GH-deficient adults

Clin Endocrinol

Inhibitory effect of a growth hormone receptor antagonist (G120K-PEG) on renal enlargement, glomerular hypertrophy, and urinary albumin excretion in experimental diabetes in mice

Diabetes

Local effects of transforming growth factor-beta1 on rat calvariaChanges depending on the dose and the injection site

J Bone Miner Metab

Bioresorbable polymers as drug delivery systems

Insulin like growth factor I has independent effects on bone matrix formation and cell replication

Endocrinology

Local application of basic fibroblast growth factor minipellet induces the healing of segmental bony defects in rabbits

Calcif Tissue Int

Original article
Improvement of fracture healing by systemic administration of growth hormone and local application of insulin-like growth factor-1 and transforming growth factor-β1