INTRODUCTION

Bone resorption is a critical step for the integration of graft bone with host bone as well as new bone formed in bony defects. The main cell responsible for this process is the osteoclast. Resorption of bone consists of multiple steps, including osteoclast recruitment, osteoclast differentiation, activation of osteoclast function, degradation of bone matrix proteins, and bone mineral mobilization.1

Degradation of the organic matrix in bone depends on the activity of two major classes of proteolytic enzymes: the cysteine proteinase (CPs) family and the matrix metalloproteinase (MMPs) family.23 Both CPs and MMPs participate in the degradation of the collagenous bone matrix within the osteoclastic resorption lacuna.2

Furthermore, a sequential action of enzymes was proposed in osteoclastic degradation of calvarial bone: when a low pH is created to dissolve the mineral, CPs digest part of the bone matrix at a such low pH, and MMPs digest the rest of the bone matrix when the pH increases somewhat.3 MMPs is a family of at least 23 highly homologous Zn⁺⁺-endopeptidases that collectively cleave most if not all the constituents of the extracellular matrix.4 Among them, matrix metalloproteinase–9 (MMP-9), also known as gelatinase B, is a key factor involved in bone resorption.56 In the mouse embryo, MMP-9 is mainly expressed in osteoclasts.5 Tezuka et al6 reported that MMP-9 was secreted by isolated osteoclasts, and the expression of MMP-9 in vivo was also observed in osteoclasts in metacarpal bones of newborn rabbits by in situ hybridization. Furthermore, detection of MMP-9 expression in the intramembranous (IM) bones of the craniofacial region reveals that MMP-9 may play a key role in osteoclastic bone resorption.7

Availability of a broad, well-rounded alveolar ridge is essential for the success of orthodontic tooth movement. However, alveolar bony defects can be created after extraction for orthodontic treatment, and collapse of the alveolar ridge, eg, after trauma, poses a problem for implant placement and orthodontic treatment. Furthermore, patients with cleft lip and palate often require a secondary bone graft procedure to allow for eruption of anterior teeth and orthodontic treatment.

Therefore, bone grafting is essential to restore form and function of the alveolar ridge. Autogenous bone graft is hard to harvest for a large defect.8 Fresh-frozen allogenic bone grafts commercially available from bone banks have been used to treat deficient bone with some success.9 However, resorption of structural bone grafts is a problem in both autogenous and allogenic bone grafts. Limited understanding of the basic biologic principles of resorption of bone graft and subsequently the integration process has made it difficult to improve the success rate of bone graft surgery.

Therefore, we examined the localization and the expression of the MMP-9 gene at messenger RNA (mRNA) and protein level in autogenous endochondral (EC) and IM bone grafts and in allogenic IM bone grafts at the rabbit mandibular defects. The aim of this study was to examine the level of expression of MMP-9 three months after grafting and to relate that to the resorption of different types of bone grafts.

MATERIALS AND METHODS

RESULTS

Immunolocalization of MMP-9

A little space of positive staining was observed in the allogenic IM bone group (Figure 1a). Intense positive staining was localized in the bone marrow within the graft bone in the autogenous IM bone group (Figure 1b). Numerous and intense positive staining was observed among the graft bone area in the autogenous EC bone group (Figure 1c). An adjacent section stained without MMP-9 primary antibody was used as a negative control (Figure 1d. Immunoreactive MMP-9 expression was mainly localized in the cytoplasm of multinucleated osteoclasts (Figure 1e) and mononuclear preosteoclasts (Figure 1f).

View Figure

• Download as Powerpoint
• Download Figure

Expression of MMP-9 mRNA

Slightly blue signal of mRNA was localized on the surface of allogenic bone in allogenic IM bone group (Figure 2a). An adjacent section stained with TRAP recognized only mature osteoclasts involved in the bone-resorption sites (Figure 2b). Many positive signals of mRNA were shown among graft bone area in autogenous IM bone group (Figure 2c). Abundant and intensive purple/blue signals of mRNA were present around the surface of graft bone and in the bone marrow nearby in autogenous EC bone group (Figure 2d). Higher magnification showed that MMP-9 mRNA expression was mainly localized in the cytoplasm of active multinucleated osteoclasts and some mononuclear preosteoclasts in the marrow space (Figure 2e). All control sections hybridized with the sense probe were negative (Figure 2f). MMP-9 mRNA expression was associated with multinucleated osteoclasts covering the surface of graft bone and mononuclear preosteoclasts near the bone-resorbing compartment.

View Figure

• Download as Powerpoint
• Download Figure

Quantitative analysis

Figure 3 shows a close correlation between the expression of MMP-9 and bone resorption. In the graph, the level of MMP-9 was directly correlated by graft bone resorption in three experimental groups for a total of 540 tissue sections, digitized in Leica Q-win system. The percentage of autogenous EC bone graft resorption was 138% more than that of autogenous IM bone (P ;lt .01) and 206% more than that of allogenic IM bone (P ;lt .01). The percentage of autogenous IM bone graft resorption was 28% more than that of allogenic IM bone (P > .05). The area of MMP-9–positive immunohistochemical staining in defects grafted with autogenous EC bone was 145% more than that with autogenous IM bone (P ;lt .01) and 222% more than that with allogenic IM bone (P ;lt .01). The area of positive immunohistochemical staining in defects grafted with autogenous IM bone was 31% more than that with allogenic IM bone (P > .05). The positive area of MMP-9 mRNA signals in defects grafted with autogenous EC bone was 151% more than that with autogenous IM bone (P ;lt .01) and 217% more than that with allogenic IM bone (P ;lt .01). The area of mRNA signals in defects grafted with autogenous IM bone was 27% more than that with allogenic IM bone (P > .05).

View Figure

• Download as Powerpoint
• Download Figure

DISCUSSION

Fresh autogenous bone of EC or IM origin is used clinically8 as well as experimentally12 to regenerate bone in osseous defects. However, clinical practice and literature concur that bone grafts from the calvaria and facial sites, which are IM bone, have a superior volumetric maintenance and survival over the grafts from rib, tibia, or iliac crest, which are EC bone.1314

In this study, we confirmed the above results by revealing the different percentage of graft bone resorption between the two different origins of bone grafts when grafted into mandibular defects for 3 months. The percentage of autogenous EC bone graft resorption in defects grafted with was 138% more than that of autogenous IM bone (P < .01) (Figure 3).

Interestingly, the amount of MMP-9 expression in the grafted EC bone was found to be significantly higher than that expressed by IM bone grafts (protein 145% and mRNA 151%; P < .01) (Figure 3), pointing to a close correlation between graft resorption and levels of expression of MMP-9. It is well-known that expression of MMP-9 leads to bone resorption. It cleaves various components of the extracellular matrix, such as collagens IV and V, proteoglycans, elastin, and denatured collagen (gelatin).6 It was concluded that MMP-9 is one of the major proteases constitutively produced by osteoclasts and may be involved in degradation of bone matrix proteins during osteoclastic bone resorption.6 Therefore, the higher levels of expression of MMP-9 by the EC bone grafts 3 months after grafting could be a contributing factor to the loss of volume of bone grafts from EC origin over bone grafts from IM origin. Because EC bone grafts heal through the EC ossification route in which cartilage intermediate stage is formed first and later is replaced by bone,15 it is conceivable that the level of expression of MMP-9 is maintained at a higher level 3 months after grafting because the cartilage matrix has to be vascularized to trigger the onset of EC ossification.

The invasion of new blood vessels requires removal of the cartilage matrix followed by migration of new blood vessels into the hypertrophic cartilage matrix.16 These blood vessels deliver mesenchymal cells and osteoprogenitor cells, which engage in osteogenesis that later replaces the cartilage. This process requires the expression of MMP-9 for the breakdown of the matrix to allow for the invasion of the new blood vessels, and that could explain the high level of MMP-9 expressed by the grafted EC bone. Such a high level of MMP-9 eventually leads to graft bone resorption and that explains why EC bone grafts undergo more resorption.

On the other hand, IM bone grafts expressed much less MMP-9 three months after grafting (Figure 3), which could be because of the fact that IM bone grafts heal directly through bone bypassing a cartilage intermediate stage.15 Also, the fact that IM bone grafts revascularize earlier than EC bone grafts.17 Such higher levels of MMP-9 required for the breakdown of the matrix to allow earlier revascularization must have been reached soon after grafting. The levels expressed at 3 months after grafting in the IM bone graft could be lower. Furthermore, the levels of MMP-9 detected 3 months after grafting could be expressed by the osteoclasts for the purpose of integration of the grafted bone with the newly formed bone, which is a process that continues to occur until the grafted bone is replaced by new bone.

Allogenic IM bone grafts were found to undergo much less resorption than autogenous EC bone grafts (206%) and slightly less than autogenous IM bone grafts (28%). This is because of the fact that osteoclasts-expressing MMP-9 are absent from the allogenic bone (Figures 1a and 2a) but actively engaged in resorption in autogenous bone (Figures 1c and 2d). Because there are signs of resorption of the allogenic bone grafts, one must wonder in the absence of such osteoclasts in the allogenic bone, where the MMP-9 comes from and how such a process of resorption of allogenic bone is carried out? In this study, we found that mononuclear cells present in perivascular sites of new blood vessels recruited from the host tissues expressed MMP-9 (Figure 1f). This MMP-9 could be the factor needed for the resorption of the allogenic bone graft to allow for the invasion of new blood vessels into the grafted allogenic bone and help with its integration with the newly formed bone as well as the host bone.

The above-mentioned data clearly show that allogenic bone showed less resorption and subsequently better volume maintenance compared with autogenous EC bone grafts. These data have to be considered in light of the ability of the bone graft to integrate and amalgamate with the host bone. Without the required resorption for the blood vessels to invade the allogenic bone graft, the chances of integration are minimal. The reason is that the invading blood vessels recruited from the host bone carry the osteoprogenitor cells required for the process of osteogenesis. As these blood vessels invade the allogenic bone graft, they deliver the osteoprogenitor cells, which will differentiate into bone-making cells and start forming bone using the allogenic bone as a scaffold.10 Although both IM autogenous and allogenic bone grafts show similar levels of resorption or volume maintenance, the autogenous bone graft was able to integrate and fuse with the host bone and participate in the process of osteogenesis through bone induction,18 whereas the allogenic bone failed to resorb, which led to failure of blood vessel invasion within 3 months after grafting, and it only contributed to the process of osteogenesis through acting as a scaffold.

CONCLUSIONS

• MMP-9 plays an important role in graft bone resorption.

• Autogenous EC bone grafts expressed higher levels of MMP-9, leading to resorption and subsequently volume loss, whereas IM bone grafts expressed less MMP-9 and subsequently showed better volume maintenance.

• In defects in the oral cavity, such as alveolar clefts, cleft palate, or narrow alveolar ridge, it would be more appropriate to use bone grafts of IM origin whenever possible.