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The Matrix Metalloproteinase-9 Concentration in Gingival Crevicular Fluid during Healing of Bone Defects treated by Guided Bone Regeneration

*Correspondence to: Dr. Ferdinand M Machibya*, Department of Orthodontics,Pedodontics and Community Dentistry, Muhimbili University of Health and Allied Sciences, Dar es salaam, Tanzania, E-mail:

Article Information

Article Type: Research

Received Date : 29/10/2018
Accepted Date : 14/11/2018
published Date : 20/11/2018



Wound healing is a complex process requiring controlled cellular and molecular activities where Matrix metalloproteinases-2 and -9 play great roles at all stages of the process.

Methods: Twenty-four standard alveolar bone defects in six male beagle dogs were treated by GBR using either Bio Oss® or beta-tricalcium phosphate (experimental) whereas the control defects were left empty.

The Enzyme Linked Immunosorbent Assay was used to examine the concentration of matrix metalloproteinases-9 in gingival crevicular fluid for any potential influence of bone regeneration materials on matrix metalloproteinases-9 concentration.

Results: Negligible amount of matrix metalloproteinases-9 was detected in GCF of all groups of regeneration materials on 3rd day post operative. However, there was a steep increase of matrix metalloproteinases-9 concentration in beta-tricalcium phosphate and limited increase for Bio Oss on 7th day. The concentration in all groups remained relatively constant in all groups from 7th to 14th day post operative with beta-tricalcium phosphate recording the highest concentration while Control group registered the lowest amount than all groups. 

Conclusion: Matrix metalloproteinases-9 is up regulated during wound healing of bone defects treated by guided bone regeneration and bone regeneration materials have significant impact on matrix metalloproteinases-9 concentration with higher concentration in beta-tricalcium phosphate and Control than Bio Oss group.

Keywords: Bone regeneration; Bone substitutes; Dentistry; Biomarkers; Matrix metalloproteinase.




The recruitment of skeletal stem/progenitor cells and their differentiation into osteoblasts and chondrocytes is key to the success of bone repair. Many factors can influence skeletal progenitors during the early stages of repair, including mechanical stimuli and inflammatory mediators [1]. The mechanical environment is crucial in determining healing via endochondral versus intramembranous ossification [2]. A stabilized environment favours osteogenic differentiation, whereas the loss of stabilization favours chondrogenic differentiation at the fracture site. These cell fate decisions occur during the inflammatory phase of fracture repair [3, 4], however the role of inflammatory signals in skeletal cell fate is not well characterized. The inflammatory phase of bone repair is marked by the infiltration of inflammatory cells that contribute to formation of the hematoma and removal of damaged tissue. While a controlled inflammatory response is necessary for stimulating tissue regeneration, prolonged inflammation can hinder the completion of the repair process [5, 6, 7]. Inflammatory mediators such as tumour necrosis factor-α (TNFα) are required for bone formation but can also impair later stages of repair by stimulating cartilage degradation [8,9]. Likewise, apparent opposite results have been reported on the role of nonsteroidal anti-inflammatory drugs (NSAIDs), which target various cell types at different stages of repair including osteoblasts, chondrocytes and osteoclasts [10,11]. 

Matrix metalloproteinases (MMPs) play important roles in bone development and repair and these enzymes may participate in the interaction between inflammatory cells and skeletal progenitors [3, 12]. MMP-9, along with other MMPs, is expressed in inflammatory cells and regulates inflammation in other tissues and diseases [13,14]. MMP-9 is a known mediator of inflammation and plays a role in fracture repair [3]. The profile of inflammatory cell recruitment was affected by MMP-9 in mice and changes were mostly observed for macrophages and CD4 T cells between genotypes in a study which assessed the role of inflammation on skeletal cell differentiation during fracture healing. Thus, the absence of MMP-9 affected the recruitment of inflammatory cells in the callus of stabilized and no stabilized fractures [1]. The first developmental phenotype reported in an MMP-knockout mouse was a defect in endochondral ossification of long bones and concluded that deletion of MMP-9 results in expansion of the zone of hypertrophic chondrocytes in the growth plate because of a failure of apoptosis [15].

For better understanding, the process of alveolar bone remodelling can be divided into three stages: exudative, proliferative, and reparative. In the exudative stage (from 1 to 7 days after extraction), the dental alveolus at the cervical, medium, and apical thirds is filled with blood clot, and shows weak immunostaining for MMPs. The alveolar bone at the apical region is internally covered with a layer of osteoblast-like cells immunolabeled for MMPs [16].

During the proliferative stage (from 7 to 14 days after extraction), the remaining blood clot is gradually replaced by connective tissue and bone trabeculae from the alveolar bone surface. 

At 14 days, the advance in osteogenesis from the periphery to the centre is evident

In the reparative stage (21–42 days after extraction), the MMPs and RECK are located in the areas of tissue remodelling, being expressed at the cervical region by fibroblast-like cells of the connective tissue and at the apical region by osteoblast-like cells close to the newly formed bone [16]. The current study’s objective was to assess the pattern of Matrix metalloproteinase-9 concentration in Gingival Crevicular Fluid during healing of bone defects treated by Guided Bone Regeneration using Bio Oss and Beta-TCP.


Ethical considerations

The study was approved by the Ethics Committee of Fujian Medical University. All animal handling and surgical procedures were conducted according to the Institutional Review Board (IRB) guide lines for the use and care of laboratory animals.

Animal experiments

The animal experiment procedure was previously described by [17]. The study included six male beagle dogs aged 18 months with a mean weight of 11.8 Kg. 

The data were collected by intraoral clinical examination and immunoassay analyses of gingival crevicular fluid (GCF). Twenty-four alveolar bone defects were created by extending the first pre-molar extraction socket. The experimental defects were treated by GBR using synthetic β-TCP (Bio-lu Biomaterials Co., Ltd. Shanghai, China) or xenograft Bio- Oss® (Geistlich, Wolhusen, Switzerland) regeneration materials, whereas the control defects were left empty.

Resorbable collagen membranes Bio-Gide® (Geistlich, Wolhusen, Switzerland) were used in both experimental and control defects. The regeneration materials were equally allocated to the maxillary right and left (UR and UL) as well as to the mandibular right and left (LR and LL) defects by randomizing three pre-determined sets of defect managements to the six experimental animals (i.e. set 1: UR- β-TCP, UL-Bio Oss, LR-Control and LL- β-TCP; set 2: UR-Bio Oss, UL- β-TCP, LR-Bio Oss and LL Control; set 3: UR-Control, LR-β- TCP, UL-Control and LL Bio Oss). Every set was randomly assigned to two dogs; consequently, the three GBR groups (β-TCP, Bio Oss and Control) were equally distributed to the right and left of maxillary and mandibular jaws. The set randomization also allowed for every GBR group to be assigned to eight defects.

Surgical procedure

Under general anaesthesia, the maxillary and mandibular first premolar extraction sockets were extended medially from the second premolar using cylindrical tungsten bur to create standardized artificial defects measuring 5 mm deep, 7 mm long (mesial-distal) and 5 mm wide on each quadrant of the animal’s jaws. Depending on the GBR group allocation, the defects were filled with β-TCP or Bio Oss mixed with animal’s blood collected during defect preparation. The mixture was packed into the artificial defects to the natural alveolar height level whereas; the control defects were left empty. The filled experimental and the empty control defects were all covered by resorbable collagen membranes Bio Gide® followed by wound closure using 3/0 nylon sutures which remained in the site for two weeks.

Gingival crevicular fluid (GCF) collection

The gingival clavicular fluid samples were collected from all defects on third, fifth, seventh, tenth and fourteenth day post-operatively. Prior to GCF collection, the animals were anaesthetized cleaned in the mouth and washed with normal saline. A methylcellulose paper strip was gently inserted in the gingival sulcus on the mesial aspect of second premolar and left in for 30 seconds. Afterwards, the paper strips were placed into Eppendorf tubes and preserved at -80 °C. To quantify the GCF collected, the Eppendorf tubes with strips and those with paper points were weighed before and after sampling.

MMP-9 enzyme linked immunosorbent assay

For immunoassay analysis, the samples were sent to Shanghai Biotechnologies, Inc. for protein extraction and immune assay process. Before analysis, the frozen GCF samples were thawed at room temperature for 1 hour, followed by addition of 200 μl Phosphate-buffered saline (PBS) and centrifuge at 10000 RPM for 15 minutes at 4 °C. Further 150 μl PBS buffer was added to the supernatant followed by centrifugation. The procedure was repeated three times to obtain the supernatant aliquots for immunoassay analysis.

The MMP-9 concentrations were determined using canine MMP-9 enzyme linked immunosorbent assays (ELISA) kit (My BioSource, CA, USA.) according to the manufacturers’ instructions and the optical densities were determined at 450 nm using Tecan® Infinite F50 microplate reader (Tecan, Austria). Finally, the concentrations of MMP-9 in each of the samples were then determined by comparing the average sample optical density readings with the concentrations from the assay standard curves and the data were reported as concentrations of biomarkers in ng/ml.


Negligible concentration of MMP-9 was detected in GCF of all groups of regeneration materials on 3rd day post operative. On the 5th day, the Beta-TCP registered the highest concentration of all groups followed by the Control group. There was steep increase of MMP9 concentration in Beta-TCP with limited increase for Bio Oss on 7th day. The concentration in all groups remained relatively constant in all groups from 7th to 14th day post operative with Beta-TCP recording the highest concentration while Control group registered the lowest amount than all groups. On 7th, 10th and 14th days post operative Beta-TCP group registered significantly higher MMP-9 concentration than Bio Oss (p<0.01) and Control (p<0.05)



In general, the MMP-9 was almost undetectable in GCF on the third day post GBR in all groups but the concentration increased with time except for Bio Oss group which displayed relatively constant low concentration of MMP-9 throughout 14 experiment days. [1] Reported that MMP-9 along with other inflammatory gene were indiscernible in stabilized and non-stabilized fractures at day 2 after injury but were significantly up-regulated at day 7 after injury in the stabilized fractures. In the current study, there was steep increase of MMP9 concentration in Beta-TCP with limited increase for Bio Oss on 7th day. The concentration in all groups remained relatively constant in all groups from 7th to 14th day post. MMPs are involved in ECM degradation and deposition that is essential for wound reepithelialisation bone healing [18]. The presence of active MMP-2 (gelatinase A) and MMP-9 (gelatinase B) in wound fluids initially identified a role for these MMPs in wound healing [19, 20].

According to [20], Wound healing requires the controlled activity of MMPs at all stages of the wound healing process. The loss of MMP regulation is a characteristic of chronic wounds and contributes to the failure to heal. The clinical appearance and pattern of biomarkers expression can be inferred to the known three phases of bone healing, namely inflammation, repair and remodelling [30]. Hence, the limited amount of MMP-9 in Bio Oss group may be indicative of impaired healing process. In fact, our earlier publications (Zhuang et al) reported of relatively high incidences of defects healing complications (prolonged bleeding and swelling) for Bio Oss group than Beta-TCP and control groups.

MMPs play a crucial role in all stages of wound healing by modifying the wound matrix, allowing for cell migration and tissue remodelling [20]. Therefore, the previous reported differences in the pattern of bone defects healing in different BRMs (Zhuang et al, Machibya et al) can be reflected by the differences of MMP-9 concentration among different BRMs (Figure 1 and Table 1).

MMPs are now known to carry out a range of diverse functions in addition to degrading or remodeling the ECM. MMPs have been shown to regulate cell–cell and cell–matrix signaling through the release of cytokines and growth factors sequestered in the ECM [20, 21].

In the previous report by [17], OPG decreased immediately following while MMP-2 and VEGF gradually increased following GBR: The MMP-2 and VEGF increase was probably associated with ECM degradation and angiogenesis processes respectively, as per authors’ speculation. The trend is relatively similar to the MMP-9 pattern during healing in the current study. The expression of MMPs is important in the initial healing stages. Since, they play active roles in the migration of inflammatory cells, degradation and remodelling of extracellular matrix proteins as well as angiogenesis processes [22, 23], essential for bone healing.


MMP-9 is up regulated during wound healing of bone defects treated by guided bone regeneration and BRMs have significant impact on MMP-9 concentration with higher concentration in Beta-TCP and Control than Bio Oss group.




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