MMP-9 and CD68+ cells are required for tissue remodeling in response to natural hydroxyapatite

Abstract

Large bone defects represent major clinical problems in the practice of reconstructive orthopedic and craniofacial surgery. The aim of this study was to examine, through immunohistochemistry approach, the involvement of MMP-9 and CD68+ cells during tissue remodeling in response to natural hydroxyapatite (HA) implanted in rat subcutaneous tissue. Before experimentation, forty animals were randomly distributed into two experimental groups: Group-I (Gen-Ox™ micro-granules) and Group-II (Gen-Ox™ macro-granules). Afterwards, the biopsies were collected after 10, 20, 30, and 60 days post-implantation. Our results showed that at 10 days, a low-renewal foreign body type granuloma formation was observed in most of the cases. Macrophage- and fibroblast-like cells were the predominant type of cells positively stained for MMP-9 in both groups. Once macrophage-like cells seemed to be the major source of MMP9, antibody against pan-CD68 epitope was used to correlate these findings. In agreement, MMP-9 and CD68+ cells were distributed at the periphery and the central region of the granuloma in all experimental periods, however no staining was observed in cell contacting to material. Besides macrophages, the lysosomal glycoprotein epitope recognized by CD68 antibodies can be expressed by mast cell granules and sometimes by fibroblasts. Taken together, our results suggest that xenogenic HA promotes extracellular matrix remodeling through induction of MMP-9 activity and presence of CD68+ cells.

Introduction

Bone is an extremely complex tissue that provides many essential functions in the body. Large bone defects represent major clinical problems in the practice of reconstructive orthopedic and craniofacial surgery. Nowadays, bone tissue engineering holds great promise in providing strategies that will result in complete regeneration of bone and restoration of its function. Many bone substitutes have been used based on their osteoconductive and osteoindutive properties (van Heest and Swiontkowski 1999). While positive results using xenografts in bone loss therapies have been reported (Mellonig 1999; Artzi et al. 2001; Gonçalves et al. 2005), the molecular and cellular mechanisms involved in the tissue response to these materials need to be clarified.

Subcutaneous tissue response to both macro—(Zambuzzi et al. 2005) and micro- (Zambuzzi et al. 2006a, b) granular hydroxyapatite (HA), from bovine bone, are compatible with a low renewal foreign body type granuloma. Anatomically, these granulomatous capsules exhibit characteristics similar to the original connective tissue, with fat cells and blood vessels.

On the other hand, the Matrix Metalloproteinases (MMPs) family currently consists of 24 members characterized in humans, rodents, and amphibians (Woessner and Nagase 2000; Hannas et al. 2007). Initially classified as zinc-dependent proteinases capable of digesting the various structural components of the extracellular matrix (ECM), their specific proteolytic targets (substrates) have since expanded to many other extracellular proteins. These substrates include an array of other proteinases, proteinase inhibitors, clotting factors, chemotactic molecules, latent growth factors, growth factor binding proteins, cell surface receptors, and cell–cell and cell–matrix adhesion molecules (McCawley and Matrisian 2001). MMP transcripts are usually expressed at low levels, but these levels rise rapidly when tissues undergo remodeling, such as in inflammation, wound healing, and cancer. MMPs are synthesized as latent enzymes that can be stored in inflammatory cell granules but are more often secreted and found anchored to the cell surface or within the ECM (Coussens et al. 2002; Keibel et al. 2009).

Particularly, MMP-9 (also known as gelatinase B), is a multidomain enzyme functioning in acute and chronic inflammatory and neoplasic diseases. Macrophages and neutrophils are well-known cells to secrete MMP-9 (Muroski et al. 2008). MMP-9 is essential for initiating the osteoclastic resorption process by removing the collagenous layer from the bone surface before demineralization can start (Delaissé et al. 2000). In addition, Corotti et al. (2009) showed that MMPs -2 and -9 are required for remodeling of tissue during apical periodontitis development.

Very recently, we reported that RECK (Reversion-inducing-cysteine-rich protein with Kazal Motifs) and TIMP-2 (Tissue inhibitors of metalloproteinases-2) are involved in the control of ECM remodeling in distinct phases of osteoblast differentiation in vitro, by modulating MMP activities while a multitude of signaling proteins governs these events (Zambuzzi et al. 2009a). Also in bone tissue, we have showed that a there is an involvement of both MMPs and theirs inhibitors during alveolar bone regeneration (Accorsi-Mendonça et al. 2008a, b).

In this present work, we hypothesized that MMP-9 could be involved in the tissue remodeling in response to both-size xenogenic HAs implantation, due to its role in the breakdown of ECM components. We also investigated the presence of CD68+ cells in response to natural HA.

Material and methods

Biomaterials

Two commercial biomaterials from inorganic cancellous bovine bone were used in this study: Gen-Ox™ micro-granules (with particles from 200 to 800 μm) and Gen-Ox™ macro-granules (with particles from 1,000 to 2,000 μm). The morphology of the material studied is presented in Fig. 1.

Fig. 1
figure1

Electron micrographs of the natural HA—Images of hydroxyapatites are shown as a representative example: a Macrogranules and b Microgranules, both from bovine cancellous bone. These xenografts are particularly interesting due to their biologically designed porous on their structure, which enhance both cellular and vascular invasion, favoring graft integration

Antibodies

Polyclonal goat antibodies anti-MMP-9 (#sc-6841) and anti-CD68 (#sc-7084) were purchased from Santa Cruz Biotechnology Inc. (California, USA). Biotinylated rabbit anti-goat secondary antibodies were purchased from Pierce Co. (ImmunoPure®, 31732, Pierce, Rockford, IL, USA). Antibodies were used at the following dilutions: MMP-9 (1.0 μg/mL), CD68 (1.0 μg/mL), and secondary antibody (6.0 μg/mL).

Methods

Acquisition of biomaterials from bovine bone

Briefly, bovine bones were dissected out and gently washed to eliminate blood, fat, and other organic impurities. Thereafter the bones were chemically treated with sodium hydroxide and organic solvents. The resulting biomaterial is a highly crystalline natural HA with low degradability (Zambuzzi et al. 2005, 2006a, b; Accorsi-Mendonça et al. 2008a, b). Both materials (macro- and micro-granular) were deproteinized at 950–1,000°C, according to the manufacturer’s protocol (Baumer SA, Mogi Mirim, São Paulo, Brazil, under registration at the Ministry of Health No. 10345500001).

Animals and surgical procedures

After proper IRB approval by the Ethics Committee of Bauru Dental School, University of Sao Paulo, and under the principles of the Brazilian College of Animal Research (COBEA), forty male Wistar rats (Rattus norvergicus, 90-days old) were randomly distributed into two experimental groups: Group-I (Gen-Ox™ micro-granules) and Group-II (Gen-Ox™ macro-granules). Four different experimental periods (10, 20, 30, and 60 days) were used based on the day that the material was implanted (n = 5/period/group). Before surgical procedures, the animals were anesthetized with ketamine hydrochloride and xylazine (Dopalen and Anasedan, Paulínia, São Paulo, Brazil) in the ratio of 0.14 mL/100 g of body weight. Trichotomy and antisepsis with iodated alcohol were performed and a straight incision was made in the animal dorsal region (Fig. 2a). One hundred milligrams of each biomaterial were implanted in the subcutaneous pocket (Fig. 2b). After suture (Fig. 2c), antisepsis was repeated and the animals received 0.5 mL of antibiotic (Garamycin, 0.52 mg/mL) and anti-inflammatory medication (Brexin, 0.14 mg/mL). The animals were killed by lethal anesthesia. The animals received food and water “ad libitum throughout the all experimental periods.

Fig. 2
figure2

Surgical Procedures. In short, the images bring out the sequence of surgical procedures carried during biomaterial implantation in rat dorsal region

Histological procedures

Tissue collection

The animals were killed at each experimental period, and the surrounding tissues collected and immediately fixed in 10% buffered formalin (phosphate buffer, pH 7.2) for 24 h, demineralized in 0.05 M EDTA (pH 7.2), dehydrated in an ethanol-graded series, clarified in xylol and embedded in synthetic paraffin wax Histosec™ (Merck, Darmstadt, Germany). Serial sections of 5 μm in thickness were obtained and used for microscopic and immunohistochemical analysis.

Immunohistochemistry

Immunohistochemical procedures were performed as described elsewhere (Menezes et al. 2006; Zambuzzi et al. 2009b; Paiva et al. 2009) with slight modifications: sections were deparaffinized and rehydrated in ethanol-graded series. Endogenous peroxidase was blocked using 3% hydrogen peroxide solution for 45 min. Antigen retrieval was obtained with phosphate-citrate buffer (pH 6.0, Sigma–Aldrich, Germany) at 96°C for 20 min, and non-specific binding was blocked by incubation in 4% non-fat milk solution for 20 min at room temperature. Sections were incubated with primary antibodies in a humid chamber at 4°C overnight followed by incubation with biotinylated rabbit anti-goat secondary antibody at room temperature for 1 h. Sections were then incubated with StreptABComplex/HRP duet kit mouse/rabbit (Dakocytomation, Carpenteria, USA) for 45 min at room temperature and stained with DAB-chromogen (3,3–Diaminobenzidine-HCl, Dakocytomation, Carpenteria, USA). Afterwards, the sections were counterstained with Harris’s haematoxylin (nuclear staining). All rinses were performed 3 × with 0.1% PBS/Triton-X. All antibodies were diluted in 0.1% PBS/BSA solution. We used human breast cancer as positive control to MMP9 and rat’s lung for CD68. As negative control, we replaced primary antibodies for 0.1% PBS/BSA solution.

Semi-quantitative image analysis

Two calibrated investigators analyzed the intensity of the immuno-staining for MMP-9 and CD68 under 40× magnification according to the following criteria: (0) absence of staining, (1) light, (2) moderate staining and (3) intense staining. The mean and standard deviation of the samples (n = 5/period/group) were analyzed by using the Kruskal–Wallis Test and the Dunn post-test if p < 0.05 (Instat, Graph Pad Software). Spearman Rank Correlation Test was used for correlation analysis.

Results

The architecture of the granules was observed by Scanning Electron Microscopy (SEM), as shown in the Fig. 1. Xenogenic grafts of bovine cancellous bone are particularly interesting due to their biologically designed porous structure that enhance both cellular and vascular invasion. We did not observe any significant effect on immunological host response to macro- and micro-granular natural HA.

At 10 days, a moderate to intense inflammatory infiltrate characterized by the presence of a large number of macrophages and inflammatory multinucleated giant cells (GC) was observed around the grafted particles in addition to a discrete presence of polymorphonuclear leukocytes and lymphocytes. These GCs were not immuno-stained against MMP-9 neither CD68 for both macro- and micro-granules. A fibrous tissue developed surrounding the granules and some flat-shape cells were positives to MMP-9. Apparently, those cells were suggestive of fibroblast (Fig. 3a) and macrophage (Fig. 4a). Overall, cells immunostained for CD68 antigen are shown at the Figs. 3e and 4e.

Fig. 3
figure3

MMP-9 and CD68 expressions in response to microgranular particles. Immunhistochemical assay is able to detect proteins in tissues using specific antibodies. Here, the samples from different periods were collected and immuno-processed using antibodies against MMP-9 and CD68 proteins. a 10 days after surgery, fibroblastlike (➩) and mononuclear cells ( ) expressed MMP-9, but only mononuclear cells were CD68+. At the period of 20 days, we found mononuclear cells MMP-9+ (c) and CD68+ (d) Same profile was found for the periods of 30 and 60 days after surgery (eh). Magnification: ×40

Fig. 4
figure4

MMP-9 and CD68 expressions in response to macrogranular particles. Here, the samples from different periods were collected and immuno-processed using antibodies against MMP-9 (➩) and CD68 ( ) proteins. 10-days after surgery, mononuclear cells were immuno-stained for MMP-9 (a) and CD68 (e). At the period of 20 days, we found mononuclear cells MMP-9+ (b) and CD68+ (f). Same profile was found for the periods of 30 (c and g) and 60 days (d and h) after surgery (eh). Magnification: ×40

At 20 days, there was a decrease in the intensity of the inflammatory infiltrate (moderate to mild) and an increased fibrosis around the graft particles in the both test groups. A fibrous capsule was formed in the outer limit exhibiting similar characteristics to the original connective tissue, presenting fat cells and blood vessels presence (angiogeniesis). It was found less inflammatory cells around both the biomaterials implanted and the same profile was found for macrophage-like cells immuno-stained for MMP-9 and CD68 (Figs. 3b–f and 4b–f).

At 30 (Figs. 3c–g, 4c–g) and 60 days (Figs. 3d–h, 4d–h), histological and molecular aspects were very similar for both biomaterials evaluated. GCs were the most abundant cell-type in contact with the surface of the particles despite the reduction in the number of macrophages and the increase in the number of blood vessels. Intense fibrosis around the HA particles was observed. Interestingly, both biomaterials were involved by a dense fibrous capsule and new blood vessels were detected surrounding it. The MMP-9 and CD68+ cells were present in the connective tissue, but not in the fibrous capsule. No immuno-stained cells were noted in contact to the implanted particles.

Positive controls were used to validate the immuno-staining findings: Human breast cancer (MMP-9+) and rat lung (CD68+) sections. Negative controls were obtained by replacing the primary antibodies by non-immune murine serum (both cases data not shown).

Summarizing, semi-quantitative analysis of the immuno-staining intensity for both MMP-9 and CD68 are shown in Table 1. To note, no statistically significant difference was found between particles-size. Spearman correlation analysis between CD68 and MMP-9 was significant (P < 0.001) and positive (r = 0.6396) in the macrogranular group, but no significance was observed in the microgranular group.

Table 1 Temporal and particle size effect on the intensity of CD68 and MMP-9-immunopositive cells in response to xenograft

Discussion

Despite autologous bone grafts are still considered the gold-standard for reconstruction of extended bone defects (Coelho et al. 2009; Kneser et al. 2006), xenograft materials continue to arise as interesting bone substitutes. The current paradigm in designing biomaterials is to optimize material chemical and physical parameters based on correlations between these parameters and downstream biological responses, whether in vitro or in vivo.

Acellular and deproteinized bovine bone grafts have shown interesting results in vivo (Zambuzzi et al. 2005, 2006a, b) and in clinical trials (Artzi et al. 2001). In general lines, material obtained from bovine bone (xenografts) can be processed by different approaches resulting in distinct biomaterials, which present specific structural and biological properties such as: inorganic/deproteinized (Zambuzzi et al. 2005, 2006a, b; Cestari et al. 2009) or demineralized bone (Accorsi-Mendonça et al. 2005; Oliveira et al. 2006; Zambuzzi et al. 2006b). Further, the sintering temperature used in the processing of the particles modifies the material crystalinity, leading to it biodegradability.

At the time of implantation, the ingredients of the body fluid only recognize the surface of the scaffold through their migration into the macropore channels. For this reason, the surface of the scaffold needs first to be tailored so as to have favorable initial responses to adhesive molecules and progenitor cells. HA has long been recognized as an important material for creating a biologically active interface, because of its specific affinity to adhesive proteins and consequently stimulating roles in the series of cellular processes.

Physical and chemical materials properties and biological responses can be characterized rigorously using a variety of available methods. The implantation of biomaterials in the animal subcutaneous tissue could be considered an essential tool to evaluate the inflammatory process, ECM remodeling (Accorsi-Mendonça et al. 2005), tissue repair (Oliveira et al. 2004; Zambuzzi et al. 2005, 2006a, b), and ectopic bone formation (Urist 1970; Rittenberg et al. 2005).

In general, the implantation of biomaterials (organic or inorganic) in the rat subcutaneous tissue leads to a foreign body reaction, where macrophages and multinucleated giant cells are present. During this process, the development of a fibrous tissue around the implanted material occurs simultaneously to the connective tissue remodeling and angiogenesis (Stavropoulos et al. 2003).

It is clear to us that these events support ECM turnover governed mainly by MMPs activity. However, there is a lack of information concerning about the expression of MMPs in response to biomaterials implantation. In order to clear this issue, the present study supports the evidence of the involvement of inactive and/or active MMP-9 in the tissue response to the inorganic bovine bone xenograft (different sizes). In fact, MMP-9 appears in two distinct regions being expressed by mononuclear cells present in the granuloma surrounding HA particles, such as those ones resident in both fibrous tissue and slack connective tissue. Also, we investigated the presence of CD68+ cells in response to HA particles. The lysosomal glycoprotein epitopes recognized by CD68 antibodies are expressed by macrophage (Pilling et al. 2009) and in mast cell granules (Welker et al. 2000). Interestingly, fibroblasts can sometimes express CD68 (Kunisch et al. 2004) and exhibit phagocytic process as seen in Monsel’s phenomenon following use of Monsel’s solution on excisions of melanoma lesions (Elenitsas and Schuchter 1998; Garrett et al. 2002). More importantly, factor XIIIa positive dendritic cells, also called collagen associated dendrophages of Nickoloff (Nickoloff and Griffiths 1989), have an elongate dendritic fibroblast-like morphology and tend to accumulate in large numbers at the site of wound healing and tissue remodeling, including many mesenchymal tumors. Nickoloff and Griffiths (1989) concluded that the factor XIIIa-positive dermal dendrocyte is a common cellular denominator among diverse clinical entities that share some histological features.

It is interesting to point out that immuno-staining for MMP-9 or CD68 was not observed to inflammatory GC. Recently, we reported that organic bovine bone matrix implanted in rat muscle induced the recruitment of CD68+ cells. We suggested that those cells were able to express MMP-2 in close contact to particles during connective tissue remodeling (Accorsi-Mendonça et al. 2005). Since MMP-9 is unable to breakdown the inorganic xenograft, it would be involved in the ECM remodeling of the peri-implant connective tissue, favoring angiogenesis around implanted granules. The angiogenic potential of a biomaterial is a critical factor for successful graft intake in tissue engineering. The regulation of angiogenesis is based on numerous growth factors, proteolytic enzymes, ECM components, cell adhesion molecules, and vasoactive factors.

In addition, previous studies have shown that fibroblasts in contact with granular implants secrete cytokines, collagenases and stromelysins (Stavropoulos et al. 2003; Makowiski and Ramsby 2003), however, we did not observe immuno-stained cells in contact to both implanted natural HAs.

Similarly to our results, Laquerrier et al. (2004) demonstrated that non-phagocytable particles (170–300 nm) of HA increased the production of MMP-9 by monocytes in vitro, emphasizing the role of particle shape (and size) on cell response (Laquerrier et al. 2004). Specifically, the anorganic HAs tested here were 3- to 6-fold bigger than the Laquerrier’s study, but no significant difference was found in the level of MMP-9 and CD68 expression in the rat subcutaneous tissue between our groups. In agreement, Morgan et al. (2001) reported that HA crystals were found to up-regulate the production of a variety of MMPs, including MMP-2, -9, and -13 in MCF-7 (Morgan et al. 2001).

Physiologically, MMPs play a pivotal role in the bone homeostasis. The breakdown of this equilibrium is apparent in some disorders such as osteoporosis. Currently, there are a number of specific synthetic inhibitors of MMPs, namely tetracyclines and their chemically modified derivatives, e.g. doxycycline (Dormán et al. 2007); hydroxamic acids, e.g. GM6001 (Nakata et al. 2009); and bisphosphonates, e.g. clodronate (Valleala et al. 2003). Particularly, the bisphosphonates have been shown to inhibit MMP-1, -2, -3, -8, -9, -12, -13 and -20 at both therapeutically obtainable and, most importantly, at non-cytotoxic concentrations (Heikkilä et al. 2002). Current clinical applications generally include the management of calcium and bone metabolism disorders, e.g. osteoporosis, Paget’s disease, hypercalcaemia and metastatic cancer (Vasikaran 2001).

Taken together, our results suggest that tissue remodeling in response to both micro- and macro-granular natural HA induces the expression of inactive and/or active MMP-9 and recruitment of CD68+ cells. These mechanisms could be exploited to engineer materials in order to enhance the integration of biomaterials and improve their performance into whole bone.

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Acknowledgments

We would like to thank Tania M. Cestari and Danielle S. Ceolin for exceptional technician support. This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (W.F.Z., 08/53003-9).

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Correspondence to José M. Granjeiro.

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Zambuzzi, W.F., Paiva, K.B.S., Menezes, R. et al. MMP-9 and CD68+ cells are required for tissue remodeling in response to natural hydroxyapatite. J Mol Hist 40, 301–309 (2009). https://doi.org/10.1007/s10735-009-9241-2

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Keywords

  • Biomaterials
  • Xenografts
  • Hydroxyapatite
  • MMP-9
  • CD68 antigen