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Effect of recombinant human tissue inhibitor of matrix metalloproteinase-1 in rabbit mandibular distraction osteogenesis: A histological and immunohistochemical study
ARTICLE IN PRESS Journal of Cranio-Maxillofacial Surgery (2006) 34, 277–282 r 2006 European Association for Cranio-Maxillofacial Surgery doi:10.1016/j.jcms.2006.02.005, available online at http://www.sciencedirect.com
Effect of recombinant human tissue inhibitor of matrix metalloproteinase-1 in rabbit mandibular distraction osteogenesis: A histological and immunohistochemical study Li Wu ZHENG1, Li MA1, A. Bakr M. RABIE2, Lim Kwong CHEUNG1 Discipline of Oral & Maxillofacial Surgery (Chair Professor: Prof. Lim Kwong Cheung); 2Discipline of Orthodontics (Chair Professor: Prof. Urban HAGG), Faculty of Dentistry, The University of Hong Kong, China 1
Available online 13 June 2006
Background: Bone matrix metalloproteinases are capable of degrading bone matrix during the remodelling, and their degradation activities can be down regulated by the tissue inhibitors of matrix metalloproteinases. This study evaluated the influence of exogenous tissue inhibitor of matrix metalloproteinase1 on the expression of matrix metalloproteinases and endogenous tissue inhibitor of matrix metalloproteinases in mandibular distraction osteogenesis. Material and Methods: Fifteen New Zealand white rabbits were assigned to three groups: a negative control; a sham control group implanted with a collagen sheet; and an experimental group implanted with recombinant human tissue inhibitor of matrix metalloproteinase-1 impregnated in a collagen sheet. Rabbits were sacrificed at 6 weeks, 12 weeks and 24 weeks of consolidation. Results: Major expression of matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases was observed at the early stage of consolidation, only positive signals of tissue inhibitors of matrix metalloproteinases were observed at 24 weeks. The addition of recombinant human tissue inhibitor of matrix metalloproteinases-1 did not affect bone maturation and remodelling. Conclusions: An equilibrium of bone formation and resorption was reached at 24 weeks of consolidation in the rabbit mandible. No obvious influence of recombinant human tissue inhibitor of matrix metalloproteinase-1 on bone remodelling of mandibular distraction osteogenesis was noted. r 2006 European Association for Cranio-Maxillofacial Surgery SUMMARY.
Keywords: distraction osteogenesis; bone matrix metalloproteinase; tissue inhibitor of matrix metalloproteinase; immunohistochemistry; rabbit; mandible
Distraction osteogenesis is a method of inducing new bone directly from the osteotomy site by gradual distraction of the two bone fragments. Since McCarthy et al. (1992) reported on the use of distraction osteogenesis to lengthen the human mandible, it has become an accepted treatment for severe craniofacial deformities. Distraction osteogenesis involves deposition of new bone, resorption of primary trabeculae, and ultimate replacement by mature bone (McTavish et al., 2000). The remodelling process takes time. MMPs and TIMPs have demonstrated to play important roles in regulating bone remodelling in distraction osteogenesis. The expression levels of MMP-1, -2 and -3 and TIMP-1 and -2 are marked at 3 months following distraction, and decrease thereafter to the same levels as the undistracted controls by 12 months (Marucci et al., 2002). Continuous upregulation of TIMP-1 protein and mRNA can be observed in successful distraction osteogenesis (0.25 mm twice daily), whereas in an acute lengthening (3.0 mm, immediately after osteotomy) TIMP-1 expression returned to a near-normal level at 4 weeks into the consolidation period (Warren et al., 2001). Collagen membranes are mechanically malleable, adaptable, and easy to manipulate, which may be
INTRODUCTION At least two classes of proteinases are involved in bone remodelling: the cysteine proteinases (Hill et al., 1993; Gelb et al., 1996) and matrix metalloproteinases (MMPs) (Everts et al., 1992; Hill et al., 1994). MMPs are capable of degrading connective tissues and are involved in bone matrix degradation during remodelling. Tissue inhibitors of matrix metalloproteinases (TIMPs) are endogenous and are capable of downregulating the MMPs by forming non-covalent bimolecular complexes, and preventing proenzyme activation (Shingleton et al., 1996). In disorders involving bone destruction and necrosis, a high level of MMPs and a low level of TIMP were found (Bord et al., 1999). In contrast, in normal developing bone, widespread expression of TIMP was recorded (Breckon et al., 1995; Bord et al., 1999). This suggests that TIMP may play a crucial balancing role in controlling MMP activities, thus contributing to the regulation of bone remodelling and resorption. In an in vitro study it was demonstrated that a low concentration of TIMP-1 in ng/ml could stimulate osteoclastic activation in a bone marrow culture system, whereas in concentrations of mg/ml, it could inhibit bone resorption (Sobue et al., 2001). 277
ARTICLE IN PRESS 278 Journal of Cranio-Maxillofacial Surgery
beneficial in clinical application. Other advantageous properties of collagen include predictable degradation, biocompatibility, haemostatic function, facilitating early wound stabilization, semi-permeability (allowing nutrient passage, and a limited degree of inflammation; Oh et al., 2003). Type I crosslinked bovine collagen is one of the most widely used resorbable membranes in reconstructive procedures for oral and maxillofacial surgery (McGinnis et al., 1998). A computed tomographic (CT) study which was conducted showed that this kind of collagen membrane had no obvious influence on the bone regenerate of mandibular distraction osteogenesis (being published separately). It seems reasonable to hypothesize that exogenous TIMP-1 may inhibit the expression of the MMPs and ultimately reduce the resorption of the primary trabeculae. The present study aims to investigate the influence of exogenous TIMP-1 on the expression of MMPs and TIMPs during the consolidation of mandibular distraction osteogenesis.
MATERIAL AND METHODS Animal care and grouping The animal experiment protocol was approved by the Committee of the Use of Live Animals for Teaching and Research of the University of Hong Kong. The rabbits were kept in a dedicated animal holding facility under veterinary supervision in the Laboratory Animal Unit of the Faculty of Medicine, the University of Hong Kong. Fifteen rabbits were randomly assigned to 3 groups of 5 animals each. In the negative control group (group A), no collagen membrane was implanted. In the sham control group (group B), collagen membrane (Biomend ExtendTM, Sulzer Calcitek Inc., Carlsbad, USA) soaked with distilled water was placed. In the experimental group (group C), a collagen membrane incorporating 30 mg rhTIMP-1 (R&D Systems Inc., Minneapolis, USA) was implanted. One rabbit from each group was sacrificed at 6 weeks and 12 weeks of consolidation respectively by an intravenous administration of sodium pentobarbitone overdose (60 mg/kg), and the remaining nine animals were sacrificed at 24 weeks. After sacrifice, mandibular samples were harvested and fixed in 4% paraformaldehyde solution. Surgical procedure and postoperative care A standardized surgical procedure was performed on all 15 rabbits: after administration of preoperative antibiotics and analgesics (30 mg/kg long acting oxytetracycline and 0.03 mg/kg buprenorphine), each rabbit was anaesthetized by intramuscular injection of ketamine (35 mg/kg), xylazine (5 mg/kg) and acepromazine (1 mg/kg). The skin was incised along the inferior border of the mandibular body with the
rabbit’s head hyperextended. The platysma was divided and the periosteum was reflected laterally from the mandible. A straight body osteotomy was performed immediately anterior to the first premolar root on one side of the mandible. A custom-designed bone-borne distractor was adapted along a plane perpendicular to the osteotomy and fixed by 2-mm diameter titanium screws. The periosteum, muscle and skin were closed in layers. After the operation, an antibiotic (long acting oxytetracycline 30 mg/kg) was administered to each rabbit intramuscularly twice per week for 2 weeks. For pain relief, buprenorphine (0.03 mg/kg) was administered subcutaneously twice daily for 10 days. Each animal remained under close observation by a veterinary technician until it regained consciousness. The clinical conditions, weight and food consumption of the animals were monitored.
Distraction procedure Following a 5-day latency period, unilateral distraction was activated at a rate of 0.9 mm once daily for 11 days. The distractors were kept in situ until 4 weeks into the consolidation period.
Implantation of the absorbable collagen membrane After 4 weeks of consolidation, the wound was exposed by the same submandibular surgical approach. The screws were removed and the distractor was detached from the mandibular body. The periosteum was reflected carefully once more from the mandibular body, and every effort was made to preserve the integrity of the periosteal flap. An implantable and resorbable collagen membrane (Biomend ExtendTM, Sulzer Calcitek Inc., Carlsbad, USA), capable of being incorporated into the surrounding tissue and being completely resorbed within 18 weeks, was used as a carrier for rhTIMP-1. The collagen sheet was cut to a size of 15 25 mm2 to cover the buccal surface of the bone regenerate formed by distraction. The trimmed collagen membrane was then submerged into rhTIMP-1 (R&D Systems Inc., Minneapolis, USA) solution (30 mg rhTIMP-1 in 100 ml distilled water). Alternatively the membrane was submerged in pure distilled water for a (sham) control. After soaking for 10 min, the collagen sheet was placed carefully over the buccal surface of the distracted regenerate so as to leave no gap between the bone surface and the sheet. Two screws were screwed back through the collagen membrane into the primary holes in order to fix the collagen sheet on the mandibular surface. The lingual side of the mandible was kept undisturbed in order to maintain the integrity of the blood supply from the lingual periosteum and the lingual soft tissues. The buccal soft tissues were repositioned and closed in layers.
ARTICLE IN PRESS Effect of recombinant human tissue inhibitor 279
Assessment methods Histology: The samples were decalcified in 14.5% ethylene diaminetetraacetic acid buffer (pH 7.2) and embedded in paraffin. Five micrometer sections were cut longitudinally in the axial plane and subsequently stained with haematoxylin and eosin (H&E). Immunohistochemistry: The sections were incubated with the primary antibodies against MMP-1, MMP-2, TIMP-1, TIMP-2 (goat polyclonal IgG, 1:100 dilution; Santa Cruz Biotechnology Inc., Santa Cruz, USA) overnight at 4 1C. For the negative controls, the primary antibodies were omitted. A kit of goat ABC staining system (Santa Cruz Biotechnology Inc., Santa Cruz, USA) was used for biotinylated second antibodies and horseradish peroxidase enzyme-avidin conjugate incubation according to the manufacturer’s instructions. The sections were counterstained with haematoxylin. The cells expressing MMPs and TIMPs within the distraction regenerate were counted by standard cellcounting technique using a computer-assisted image analysing system (Leica DC 300 V 2.0, Leica,
Wetzlar, Germany) with appropriate software (Qwin V 2.4, Leica, Cambridge, UK).
RESULTS Clinical examination All rabbits completed the experimental process successfully. No obvious complications were noted during the experimental procedure. The animals were fed regular food, and all gained weight. After unilateral mandibular lengthening, the rabbits developed a severe cross-bite and overgrowth of incisors. Histology Histological study revealed no obvious influence of the rhTIMP-1 and implanted collagen sheet on bone formation and remodelling of the distraction regenerate (Fig. 1). The morphological observations of each group were very similar when compared at the
Fig. 1 – Histological study of distraction regenerate (H&E staining, Bar ¼ 1 mm). At 6 weeks of consolidation (A ¼ group A, B ¼ group B, C ¼ group C), the trabeculae underwent active remodelling and cortical bone is not yet clearly seen. At 12 weeks (D ¼ group A, E ¼ group B, F ¼ group C), cortex formation obvious and the trabeculae still visible. At 24 weeks (G ¼ group A, H ¼ group B, I ¼ group C), complete and mature cortices in all the mandibular samples, the marrow cavity has been replaced by a more sparsely populated trabecular pattern (Group A – negative control; Group B – sham control; Group C – study group).
ARTICLE IN PRESS 280 Journal of Cranio-Maxillofacial Surgery
same time of sacrifice. At 6 weeks of consolidation, the trabeculae had undergone active remodelling and cortical bone was not yet clearly seen (Figs. 1A–C). At 12 weeks, cortex formation was obvious and the trabeculae were still visible (Figs. 1D–F). At 24 weeks, complete and mature cortices were noted in all the mandibular samples. The marrow cavity was replaced by a more sparsely trabecular pattern (Figs. 1G–I). Immunohistochemistry The expression pattern and location of the MMPs (1 and 2), TIMPs (1 and 2) were similar in each group. At 6 weeks and 12 weeks of the consolidation period, extensive positive signals of MMPs (Fig. 2) and TIMPs (Fig. 3) were observed at the osteoblasts, osteocytes and bone matrices of the trabeculae. At 24 weeks, no obvious expression of the MMPs remained apparent (Fig. 2), while positive signals of the TIMPs
Fig. 3 – Expression of the endogenous TIMPs (1 and 2) in group C, i.e. study group (counterstain: H&E). Extensive positive signals in osteoblasts, osteocytes and bone matrices of the trabeculae (A) at 6 weeks and (B) 12 weeks. (C) The expression decreased at 24 weeks but was still detectable around the osteoblasts lining the trabeculae. Bar ¼ 50 mm.
were still observed in the osteoblasts lining the trabeculae (Fig. 3). No obvious difference in the expression of MMPs and TIMPs was noted between the negative control, sham control and experimental groups at any time points.
DISCUSSION
Fig. 2 – Expression of the endogenous MMPs (1 and 2) in group C, i.e. study group (counterstain: H&E). Extensive positive signals in the osteoblasts, osteocytes and bone matrices of the trabeculae at (A) 6 weeks and (B) 12 weeks. (C) No positive signal detected at 24 weeks. Bar ¼ 50 mm.
Bone transplants undergo normal physiological creeping substitution and remodelling, and are ultimately incorporated into the recipient site (Goldberg and Stevenson, 1993). The extent of remodelling or resorption depends on the source of the graft, the stability of graft fixation, the site of the transplant, and the vascularity of the surrounding soft tissues (Goldberg and Stevenson, 1993). The stability of jaw reconstruction largely depends on the success of controlling all these factors. Physiological remodelling of the bone regenerate formed by distraction
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occurs over time as the woven bone is progressively resorbed and replaced by mature lamellar bone. Distraction osteogenesis can therefore be regarded as a good model to study the exogenous factors and their influences on the inhibition of bone remodelling. At least 16 members of the MMP family have been identified (Kahari and Saarialho-Kere, 1999; Nagase and Woessner, 1999). MMP-1 has a preference for degrading fibrillar collagen types I, II, and III. MMP2 prefers to degrade collagen types IV and V, denatured collagen (gelatin), and aggrecan core protein. TIMPs are secreted as endogenous inhibitors that downregulate the activity of MMPs. Based on the primary structure and functional analysis, four mammalian TIMPs have been cloned and characterized (Ray and Stetler-Stevenson 1994; Nagase et al., 1996). TIMP-1 and TIMP-2 have similar biochemical properties that can bind tightly to various types of active MMPs and form 1:1 non-covalent complexes (Nagase et al., 1996). MMPs and TIMPs have demonstrated their crucial role in bone formation and remodelling, but few studies of their roles in distraction osteogenesis have been reported (Warren et al., 2001; Marucci et al., 2002). The expression of the MMPs and TIMPs is supposed to be able to reflect the maturity and remodelling characteristics of the distracted bone, which may provide an index for the bone healing process following distraction osteogenesis. In the present study, the expression of MMPs and TIMPs was evaluated during the consolidation period of mandibular distraction osteogenesis. At 6 weeks and 12 weeks, pronounced expression was observed in osteoblasts, osteocytes and bone matrices. This corresponds to the active remodelling of the primary trabeculae at these stages. At 24 weeks of consolidation, histological examination demonstrated mature cortical bone formation with most primary trabeculae having been resorbed. MMPs were not demonstrated, while a positive signal of TIMPs was still observed in the surface osteoblasts. The decreased expression of MMPs confirmed that the bone remodelling activity was close to completion along with the formation of cortical bone. Compared with the negative expression of the MMPs, the positive expression of TIMPs at 24 weeks demonstrated that the bony regenerate was stable at the late stage of distraction osteogenesis. In vitro studies have demonstrated the potential of using TIMP-1 to inhibit bone resorption (Shimizu et al., 1990; Everts et al., 1992; Ellis et al., 1994). Osteoclastic bone resorption was stimulated by low concentrations (ng/ml) of TIMP-1, whereas the resorption process was inhibited by higher concentration (mg/ml; Sobue et al., 2001). The present study assessed the influence of rhTIMP-1 in mandibular distraction osteogenesis at a high concentration (300 mg/ml). In order to slow down the speed of release and maintain the long-term effectiveness of the recombinant TIMP-1, an resorbable collagen membrane, which can be incorporated into the surrounding tissue and is normally completely
degraded within 18 weeks, was used as a carrier. In order to minimize damage to the blood supply, the collagen membrane was implanted subperiosteally only on the buccal surface of the distraction regenerate, and the lingual surface was kept undisturbed. The histological analyses showed no obvious morphological differences between the non-collagen sheet control, collagen sheet control and the experimental groups. A CT and micro-CT study also showed that the collagen membrane had no obvious influence on the distraction regenerate morphologically and quantitatively (being published separately). The expression of the MMPs had not been affected by the application of rhTIMP-1, which was different from the hypothesis that exogenous TIMP-1 inhibits the expression of the MMPs and ultimately reduces resorption of the primary trabeculae. Many factors may affect the in vivo function of TIMP-1. Although a relatively high concentration of rhTIMP-1 was used in the present study, it was difficult to assess the effective concentration of rhTIMP-1 in the impregnated collagen membrane. Furthermore, the total dosage and effective exposure time of the rhTIMP-1 may not have been sufficient to induce any obvious differences on the expression of the MMPs. TIMP-1 inactivates MMPs by forming 1:1 non-covalent bimolecular complexes and preventing proenzyme activation (Nagase et al., 1996). So a long exposure time of TIMP at a high concentration may be an important consideration. Although the collagen membrane is absorbed gradually and can stay in vivo for a relatively long time, it was impossible to evaluate the concentration and persistence of the effective rhTIMP-1 in the animals.
CONCLUSION MMPs and their natural inhibitors, TIMPs, reflect the metabolic activities involved in bone remodelling and demonstrate that an equilibrium of bone formation and resorption is normally reached at 24 weeks following distraction osteogenesis in the rabbit mandible. No obvious influence of rhTIMP-1 applied in a collagen sheet carrier was noted on the expression of MMPs.
ACKNOWLEDGMENTS
This investigation was supported by the Seed Funding Programme for Basic Research from the University of Hong Kong, and a Competitive Earmarked Research Grant from the Hong Kong Research Grants Council (Reference Code: HKU/7389/03 M). The authors also would like to acknowledge the valuable technical assistance provided by the Laboratory Animal Unit, the Hard Tissue and the Dental Materials Science Laboratories of the University of Hong Kong.
ARTICLE IN PRESS 282 Journal of Cranio-Maxillofacial Surgery
References Bord S, Horner A, Beeton CA, Hembry RM, Compston JE: Tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) distribution in normal and pathological human bone. Bone 24: 229–235, 1999 Breckon JJ, Hembry RM, Reynolds JJ, Meikle MC: Matrix metalloproteinases and TIMP-1 localization at sites of osteogenesis in the craniofacial region of the rabbit embryo. Anat Rec 242: 177–187, 1995 Ellis AJ, Curry VA, Powell EK, Cawston TE: The prevention of collagen breakdown in bovine nasal cartilage by TIMP, TIMP2 and a low molecular weight synthetic inhibitor. Biochem Biophys Res Commun 201: 94–101, 1994 Everts V, Delaisse JM, Korper W, Niehof A, Vaes G, Beertsen W: Degradation of collagen in the bone-resorbing compartment underlying the osteoclast involves both cysteine-proteinases and matrix metalloproteinases. J Cell Physiol 150: 221–231, 1992 Gelb BD, Shi GP, Chapman HA, Desnick RJ: Pycnodysostosis, a lysosomal disease caused by cathepsin K deficiency. Science 273: 1236–1238, 1996 Goldberg VM, Stevenson S: The biology of bone grafts. Semin Arthroplasty 4: 58–63, 1993 Hill PA, Reynolds JJ, Meikle MC: Inhibition of stimulated bone resorption in vitro by TIMP-1 and TIMP-2. Biochem Biophys Acta 1177: 71–74, 1993 Hill PA, Murphy G, Docherty AJ, Hembry RM, Millican TA, Reynolds JJ: The effects of selective inhibitors of matrix metalloproteinases (MMPs) on bone resorption and the identification of MMPs and TIMP-1 in isolated osteoclasts. J Cell Sci 107: 3055–3064, 1994 Kahari VM, Saarialho-Kere U: Matrix metalloproteinases and their inhibitors in tumour growth and invasion. Ann Med 31: 34–45, 1999 Marucci DD, Yu Y, McTavish J, Fiona Bonar S, Poole MD, Walsh WR: Matrix metalloproteinases and their inhibitors in bone remodelling following distraction osteogenesis of the sheep mandible. J Craniomaxillofac Surg 30: 208–212, 2002 McCarthy JG, Schreiber J, Karp N, Thorne CH, Grayson BH: Lengthening the human mandible by gradual distraction. Plast Reconstr Surg 89: 1–8, 1992 McGinnis M, Larsen P, Miloro M, Beck FM: Comparison of resorbable and nonresorbable guided bone regeneration materials: a preliminary study. Int J Oral Maxillofac Implants 13: 30–35, 1998
McTavish J, Marucci DD, Bonar SF, Walsh WR, Poole MD: Does the sheep mandible relapse following lengthening by distraction osteogenesis?. J Cranio-maxillofac Surg 28: 251–257, 2000 Nagase H, Woessner Jr JF: Matrix metalloproteinases. J Biol Chem 274: 21491–21494, 1999 Nagase H, Suzuki K, Itoh Y, Kan CC, Gehring MR, Huang W: Involvement of tissue inhibitors of metalloproteinases (TIMPS) during matrix metalloproteinase activation. Adv Exp Med Biol 389: 23–31, 1996 Oh TJ, Meraw SJ, Lee EJ, Giannobile WV, Wang HL: Comparative analysis of collagen membranes for the treatment of implant dehiscence defects. Clin Oral Implants Res 14: 80–90, 2003 Ray JM, Stetler-Stevenson WG: The role of matrix metalloproteases and their inhibitors in tumour invasion, metastasis and angiogenesis. Eur Respir J 7: 2062–2072, 1994 Shimizu H, Sakamoto M, Sakamoto S: Bone resorption by isolated osteoclasts in living versus devitalized bone: differences in mode and extent and the effects of human recombinant tissue inhibitor of metalloproteinases. J Bone Miner Res 5: 411–418, 1990 Shingleton WD, Hodges DJ, Brick P, Cawston TE: Collagenase: a key enzyme in collagen turnover. Biochem Cell Biol 74: 759–775, 1996 Sobue T, Hakeda Y, Kobayashi Y, Hayakawa H, Yamashita K, Aoki T: Tissue inhibitor of metalloproteinases 1 and 2 directly stimulate the bone-resorbing activity of isolated mature osteoclasts. J Bone Miner Res 16: 2205–2214, 2001 Warren SM, Mehrara BJ, Steinbrech DS, Paccione MF, Greenwald JA, Spector JA: Rat mandibular distraction osteogenesis: part III. Gradual distraction versus acute lengthening. Plast Reconstr Surg 107: 441–453, 2001
Prof. Lim Kwong CHEUNG Prince Philip Dental Hospital, 34 Hospital Road, Hong Kong SAR China Tel.:+852 2859 0267 Fax: +852 2559 9014 E-mail: [email protected] Paper received 22 December 2004 Accepted 14 February 2006
Effect of recombinant human tissue inhibitor of matrix metalloproteinase-1 in rabbit mandibular distraction osteogenesis: A histological and immunohistochemical study Li Wu ZHENG1, Li MA1, A. Bakr M. RABIE2, Lim Kwong CHEUNG1 Discipline of Oral & Maxillofacial Surgery (Chair Professor: Prof. Lim Kwong Cheung); 2Discipline of Orthodontics (Chair Professor: Prof. Urban HAGG), Faculty of Dentistry, The University of Hong Kong, China 1
Available online 13 June 2006
Background: Bone matrix metalloproteinases are capable of degrading bone matrix during the remodelling, and their degradation activities can be down regulated by the tissue inhibitors of matrix metalloproteinases. This study evaluated the influence of exogenous tissue inhibitor of matrix metalloproteinase1 on the expression of matrix metalloproteinases and endogenous tissue inhibitor of matrix metalloproteinases in mandibular distraction osteogenesis. Material and Methods: Fifteen New Zealand white rabbits were assigned to three groups: a negative control; a sham control group implanted with a collagen sheet; and an experimental group implanted with recombinant human tissue inhibitor of matrix metalloproteinase-1 impregnated in a collagen sheet. Rabbits were sacrificed at 6 weeks, 12 weeks and 24 weeks of consolidation. Results: Major expression of matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases was observed at the early stage of consolidation, only positive signals of tissue inhibitors of matrix metalloproteinases were observed at 24 weeks. The addition of recombinant human tissue inhibitor of matrix metalloproteinases-1 did not affect bone maturation and remodelling. Conclusions: An equilibrium of bone formation and resorption was reached at 24 weeks of consolidation in the rabbit mandible. No obvious influence of recombinant human tissue inhibitor of matrix metalloproteinase-1 on bone remodelling of mandibular distraction osteogenesis was noted. r 2006 European Association for Cranio-Maxillofacial Surgery SUMMARY.
Keywords: distraction osteogenesis; bone matrix metalloproteinase; tissue inhibitor of matrix metalloproteinase; immunohistochemistry; rabbit; mandible
Distraction osteogenesis is a method of inducing new bone directly from the osteotomy site by gradual distraction of the two bone fragments. Since McCarthy et al. (1992) reported on the use of distraction osteogenesis to lengthen the human mandible, it has become an accepted treatment for severe craniofacial deformities. Distraction osteogenesis involves deposition of new bone, resorption of primary trabeculae, and ultimate replacement by mature bone (McTavish et al., 2000). The remodelling process takes time. MMPs and TIMPs have demonstrated to play important roles in regulating bone remodelling in distraction osteogenesis. The expression levels of MMP-1, -2 and -3 and TIMP-1 and -2 are marked at 3 months following distraction, and decrease thereafter to the same levels as the undistracted controls by 12 months (Marucci et al., 2002). Continuous upregulation of TIMP-1 protein and mRNA can be observed in successful distraction osteogenesis (0.25 mm twice daily), whereas in an acute lengthening (3.0 mm, immediately after osteotomy) TIMP-1 expression returned to a near-normal level at 4 weeks into the consolidation period (Warren et al., 2001). Collagen membranes are mechanically malleable, adaptable, and easy to manipulate, which may be
INTRODUCTION At least two classes of proteinases are involved in bone remodelling: the cysteine proteinases (Hill et al., 1993; Gelb et al., 1996) and matrix metalloproteinases (MMPs) (Everts et al., 1992; Hill et al., 1994). MMPs are capable of degrading connective tissues and are involved in bone matrix degradation during remodelling. Tissue inhibitors of matrix metalloproteinases (TIMPs) are endogenous and are capable of downregulating the MMPs by forming non-covalent bimolecular complexes, and preventing proenzyme activation (Shingleton et al., 1996). In disorders involving bone destruction and necrosis, a high level of MMPs and a low level of TIMP were found (Bord et al., 1999). In contrast, in normal developing bone, widespread expression of TIMP was recorded (Breckon et al., 1995; Bord et al., 1999). This suggests that TIMP may play a crucial balancing role in controlling MMP activities, thus contributing to the regulation of bone remodelling and resorption. In an in vitro study it was demonstrated that a low concentration of TIMP-1 in ng/ml could stimulate osteoclastic activation in a bone marrow culture system, whereas in concentrations of mg/ml, it could inhibit bone resorption (Sobue et al., 2001). 277
ARTICLE IN PRESS 278 Journal of Cranio-Maxillofacial Surgery
beneficial in clinical application. Other advantageous properties of collagen include predictable degradation, biocompatibility, haemostatic function, facilitating early wound stabilization, semi-permeability (allowing nutrient passage, and a limited degree of inflammation; Oh et al., 2003). Type I crosslinked bovine collagen is one of the most widely used resorbable membranes in reconstructive procedures for oral and maxillofacial surgery (McGinnis et al., 1998). A computed tomographic (CT) study which was conducted showed that this kind of collagen membrane had no obvious influence on the bone regenerate of mandibular distraction osteogenesis (being published separately). It seems reasonable to hypothesize that exogenous TIMP-1 may inhibit the expression of the MMPs and ultimately reduce the resorption of the primary trabeculae. The present study aims to investigate the influence of exogenous TIMP-1 on the expression of MMPs and TIMPs during the consolidation of mandibular distraction osteogenesis.
MATERIAL AND METHODS Animal care and grouping The animal experiment protocol was approved by the Committee of the Use of Live Animals for Teaching and Research of the University of Hong Kong. The rabbits were kept in a dedicated animal holding facility under veterinary supervision in the Laboratory Animal Unit of the Faculty of Medicine, the University of Hong Kong. Fifteen rabbits were randomly assigned to 3 groups of 5 animals each. In the negative control group (group A), no collagen membrane was implanted. In the sham control group (group B), collagen membrane (Biomend ExtendTM, Sulzer Calcitek Inc., Carlsbad, USA) soaked with distilled water was placed. In the experimental group (group C), a collagen membrane incorporating 30 mg rhTIMP-1 (R&D Systems Inc., Minneapolis, USA) was implanted. One rabbit from each group was sacrificed at 6 weeks and 12 weeks of consolidation respectively by an intravenous administration of sodium pentobarbitone overdose (60 mg/kg), and the remaining nine animals were sacrificed at 24 weeks. After sacrifice, mandibular samples were harvested and fixed in 4% paraformaldehyde solution. Surgical procedure and postoperative care A standardized surgical procedure was performed on all 15 rabbits: after administration of preoperative antibiotics and analgesics (30 mg/kg long acting oxytetracycline and 0.03 mg/kg buprenorphine), each rabbit was anaesthetized by intramuscular injection of ketamine (35 mg/kg), xylazine (5 mg/kg) and acepromazine (1 mg/kg). The skin was incised along the inferior border of the mandibular body with the
rabbit’s head hyperextended. The platysma was divided and the periosteum was reflected laterally from the mandible. A straight body osteotomy was performed immediately anterior to the first premolar root on one side of the mandible. A custom-designed bone-borne distractor was adapted along a plane perpendicular to the osteotomy and fixed by 2-mm diameter titanium screws. The periosteum, muscle and skin were closed in layers. After the operation, an antibiotic (long acting oxytetracycline 30 mg/kg) was administered to each rabbit intramuscularly twice per week for 2 weeks. For pain relief, buprenorphine (0.03 mg/kg) was administered subcutaneously twice daily for 10 days. Each animal remained under close observation by a veterinary technician until it regained consciousness. The clinical conditions, weight and food consumption of the animals were monitored.
Distraction procedure Following a 5-day latency period, unilateral distraction was activated at a rate of 0.9 mm once daily for 11 days. The distractors were kept in situ until 4 weeks into the consolidation period.
Implantation of the absorbable collagen membrane After 4 weeks of consolidation, the wound was exposed by the same submandibular surgical approach. The screws were removed and the distractor was detached from the mandibular body. The periosteum was reflected carefully once more from the mandibular body, and every effort was made to preserve the integrity of the periosteal flap. An implantable and resorbable collagen membrane (Biomend ExtendTM, Sulzer Calcitek Inc., Carlsbad, USA), capable of being incorporated into the surrounding tissue and being completely resorbed within 18 weeks, was used as a carrier for rhTIMP-1. The collagen sheet was cut to a size of 15 25 mm2 to cover the buccal surface of the bone regenerate formed by distraction. The trimmed collagen membrane was then submerged into rhTIMP-1 (R&D Systems Inc., Minneapolis, USA) solution (30 mg rhTIMP-1 in 100 ml distilled water). Alternatively the membrane was submerged in pure distilled water for a (sham) control. After soaking for 10 min, the collagen sheet was placed carefully over the buccal surface of the distracted regenerate so as to leave no gap between the bone surface and the sheet. Two screws were screwed back through the collagen membrane into the primary holes in order to fix the collagen sheet on the mandibular surface. The lingual side of the mandible was kept undisturbed in order to maintain the integrity of the blood supply from the lingual periosteum and the lingual soft tissues. The buccal soft tissues were repositioned and closed in layers.
ARTICLE IN PRESS Effect of recombinant human tissue inhibitor 279
Assessment methods Histology: The samples were decalcified in 14.5% ethylene diaminetetraacetic acid buffer (pH 7.2) and embedded in paraffin. Five micrometer sections were cut longitudinally in the axial plane and subsequently stained with haematoxylin and eosin (H&E). Immunohistochemistry: The sections were incubated with the primary antibodies against MMP-1, MMP-2, TIMP-1, TIMP-2 (goat polyclonal IgG, 1:100 dilution; Santa Cruz Biotechnology Inc., Santa Cruz, USA) overnight at 4 1C. For the negative controls, the primary antibodies were omitted. A kit of goat ABC staining system (Santa Cruz Biotechnology Inc., Santa Cruz, USA) was used for biotinylated second antibodies and horseradish peroxidase enzyme-avidin conjugate incubation according to the manufacturer’s instructions. The sections were counterstained with haematoxylin. The cells expressing MMPs and TIMPs within the distraction regenerate were counted by standard cellcounting technique using a computer-assisted image analysing system (Leica DC 300 V 2.0, Leica,
Wetzlar, Germany) with appropriate software (Qwin V 2.4, Leica, Cambridge, UK).
RESULTS Clinical examination All rabbits completed the experimental process successfully. No obvious complications were noted during the experimental procedure. The animals were fed regular food, and all gained weight. After unilateral mandibular lengthening, the rabbits developed a severe cross-bite and overgrowth of incisors. Histology Histological study revealed no obvious influence of the rhTIMP-1 and implanted collagen sheet on bone formation and remodelling of the distraction regenerate (Fig. 1). The morphological observations of each group were very similar when compared at the
Fig. 1 – Histological study of distraction regenerate (H&E staining, Bar ¼ 1 mm). At 6 weeks of consolidation (A ¼ group A, B ¼ group B, C ¼ group C), the trabeculae underwent active remodelling and cortical bone is not yet clearly seen. At 12 weeks (D ¼ group A, E ¼ group B, F ¼ group C), cortex formation obvious and the trabeculae still visible. At 24 weeks (G ¼ group A, H ¼ group B, I ¼ group C), complete and mature cortices in all the mandibular samples, the marrow cavity has been replaced by a more sparsely populated trabecular pattern (Group A – negative control; Group B – sham control; Group C – study group).
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same time of sacrifice. At 6 weeks of consolidation, the trabeculae had undergone active remodelling and cortical bone was not yet clearly seen (Figs. 1A–C). At 12 weeks, cortex formation was obvious and the trabeculae were still visible (Figs. 1D–F). At 24 weeks, complete and mature cortices were noted in all the mandibular samples. The marrow cavity was replaced by a more sparsely trabecular pattern (Figs. 1G–I). Immunohistochemistry The expression pattern and location of the MMPs (1 and 2), TIMPs (1 and 2) were similar in each group. At 6 weeks and 12 weeks of the consolidation period, extensive positive signals of MMPs (Fig. 2) and TIMPs (Fig. 3) were observed at the osteoblasts, osteocytes and bone matrices of the trabeculae. At 24 weeks, no obvious expression of the MMPs remained apparent (Fig. 2), while positive signals of the TIMPs
Fig. 3 – Expression of the endogenous TIMPs (1 and 2) in group C, i.e. study group (counterstain: H&E). Extensive positive signals in osteoblasts, osteocytes and bone matrices of the trabeculae (A) at 6 weeks and (B) 12 weeks. (C) The expression decreased at 24 weeks but was still detectable around the osteoblasts lining the trabeculae. Bar ¼ 50 mm.
were still observed in the osteoblasts lining the trabeculae (Fig. 3). No obvious difference in the expression of MMPs and TIMPs was noted between the negative control, sham control and experimental groups at any time points.
DISCUSSION
Fig. 2 – Expression of the endogenous MMPs (1 and 2) in group C, i.e. study group (counterstain: H&E). Extensive positive signals in the osteoblasts, osteocytes and bone matrices of the trabeculae at (A) 6 weeks and (B) 12 weeks. (C) No positive signal detected at 24 weeks. Bar ¼ 50 mm.
Bone transplants undergo normal physiological creeping substitution and remodelling, and are ultimately incorporated into the recipient site (Goldberg and Stevenson, 1993). The extent of remodelling or resorption depends on the source of the graft, the stability of graft fixation, the site of the transplant, and the vascularity of the surrounding soft tissues (Goldberg and Stevenson, 1993). The stability of jaw reconstruction largely depends on the success of controlling all these factors. Physiological remodelling of the bone regenerate formed by distraction
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occurs over time as the woven bone is progressively resorbed and replaced by mature lamellar bone. Distraction osteogenesis can therefore be regarded as a good model to study the exogenous factors and their influences on the inhibition of bone remodelling. At least 16 members of the MMP family have been identified (Kahari and Saarialho-Kere, 1999; Nagase and Woessner, 1999). MMP-1 has a preference for degrading fibrillar collagen types I, II, and III. MMP2 prefers to degrade collagen types IV and V, denatured collagen (gelatin), and aggrecan core protein. TIMPs are secreted as endogenous inhibitors that downregulate the activity of MMPs. Based on the primary structure and functional analysis, four mammalian TIMPs have been cloned and characterized (Ray and Stetler-Stevenson 1994; Nagase et al., 1996). TIMP-1 and TIMP-2 have similar biochemical properties that can bind tightly to various types of active MMPs and form 1:1 non-covalent complexes (Nagase et al., 1996). MMPs and TIMPs have demonstrated their crucial role in bone formation and remodelling, but few studies of their roles in distraction osteogenesis have been reported (Warren et al., 2001; Marucci et al., 2002). The expression of the MMPs and TIMPs is supposed to be able to reflect the maturity and remodelling characteristics of the distracted bone, which may provide an index for the bone healing process following distraction osteogenesis. In the present study, the expression of MMPs and TIMPs was evaluated during the consolidation period of mandibular distraction osteogenesis. At 6 weeks and 12 weeks, pronounced expression was observed in osteoblasts, osteocytes and bone matrices. This corresponds to the active remodelling of the primary trabeculae at these stages. At 24 weeks of consolidation, histological examination demonstrated mature cortical bone formation with most primary trabeculae having been resorbed. MMPs were not demonstrated, while a positive signal of TIMPs was still observed in the surface osteoblasts. The decreased expression of MMPs confirmed that the bone remodelling activity was close to completion along with the formation of cortical bone. Compared with the negative expression of the MMPs, the positive expression of TIMPs at 24 weeks demonstrated that the bony regenerate was stable at the late stage of distraction osteogenesis. In vitro studies have demonstrated the potential of using TIMP-1 to inhibit bone resorption (Shimizu et al., 1990; Everts et al., 1992; Ellis et al., 1994). Osteoclastic bone resorption was stimulated by low concentrations (ng/ml) of TIMP-1, whereas the resorption process was inhibited by higher concentration (mg/ml; Sobue et al., 2001). The present study assessed the influence of rhTIMP-1 in mandibular distraction osteogenesis at a high concentration (300 mg/ml). In order to slow down the speed of release and maintain the long-term effectiveness of the recombinant TIMP-1, an resorbable collagen membrane, which can be incorporated into the surrounding tissue and is normally completely
degraded within 18 weeks, was used as a carrier. In order to minimize damage to the blood supply, the collagen membrane was implanted subperiosteally only on the buccal surface of the distraction regenerate, and the lingual surface was kept undisturbed. The histological analyses showed no obvious morphological differences between the non-collagen sheet control, collagen sheet control and the experimental groups. A CT and micro-CT study also showed that the collagen membrane had no obvious influence on the distraction regenerate morphologically and quantitatively (being published separately). The expression of the MMPs had not been affected by the application of rhTIMP-1, which was different from the hypothesis that exogenous TIMP-1 inhibits the expression of the MMPs and ultimately reduces resorption of the primary trabeculae. Many factors may affect the in vivo function of TIMP-1. Although a relatively high concentration of rhTIMP-1 was used in the present study, it was difficult to assess the effective concentration of rhTIMP-1 in the impregnated collagen membrane. Furthermore, the total dosage and effective exposure time of the rhTIMP-1 may not have been sufficient to induce any obvious differences on the expression of the MMPs. TIMP-1 inactivates MMPs by forming 1:1 non-covalent bimolecular complexes and preventing proenzyme activation (Nagase et al., 1996). So a long exposure time of TIMP at a high concentration may be an important consideration. Although the collagen membrane is absorbed gradually and can stay in vivo for a relatively long time, it was impossible to evaluate the concentration and persistence of the effective rhTIMP-1 in the animals.
CONCLUSION MMPs and their natural inhibitors, TIMPs, reflect the metabolic activities involved in bone remodelling and demonstrate that an equilibrium of bone formation and resorption is normally reached at 24 weeks following distraction osteogenesis in the rabbit mandible. No obvious influence of rhTIMP-1 applied in a collagen sheet carrier was noted on the expression of MMPs.
ACKNOWLEDGMENTS
This investigation was supported by the Seed Funding Programme for Basic Research from the University of Hong Kong, and a Competitive Earmarked Research Grant from the Hong Kong Research Grants Council (Reference Code: HKU/7389/03 M). The authors also would like to acknowledge the valuable technical assistance provided by the Laboratory Animal Unit, the Hard Tissue and the Dental Materials Science Laboratories of the University of Hong Kong.
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Prof. Lim Kwong CHEUNG Prince Philip Dental Hospital, 34 Hospital Road, Hong Kong SAR China Tel.:+852 2859 0267 Fax: +852 2559 9014 E-mail: [email protected] Paper received 22 December 2004 Accepted 14 February 2006