Improved Alveolar Ridge Augmentation Using Synthetic Monetite Based Onlay Grafts

Once teeth are lost due to trauma and/or disease, such as advanced periodontitis and not replaced, the alveolar ridge resorption begins rapidly. 1 An average alveolar bone loss of 1.5-2 mm (vertically) and 40-50% (horizontally) occurs within first six months after tooth loss. 2 If no treatment is provided, then continued bone loss with up to 60% of total ridge volume lost occurs in the first three years. 3 The loss of vertical bone height leads to great challenges to dental implant placement due to anatomical and bone volume limitations, 4 as sufficient bone volume and ridge height is imperative for achieving long-term success of osseointegrated dental implants (Fig. 1). 5 Various surgical techniques such as distraction osteogenesis, guided bone regeneration (GBR), and onlay block grafting have been investigated to enable dental implant placement in severely resorbed alveolar bone. 6,7 Distraction osteogenesis produces greater bone height than GBR and onlay block grafting, but it has a higher rate of complication associated with it. 8,9 Although the results of GBR for vertical ridge augmentation are promising, clinical success is limited due to the procedure being highly technique-sensitive, 10 and often failing due to wound dehiscence. 11 Onlay block grafting to increase the vertical height of the mandible and maxilla usually requires extraction of an autologous bone block from donor site (intra-oral or extra-oral origin) and its fixation with screws onto the recipient site. 12 Autologous onlay grafting is associated with complications such as donor site morbidity. 13 In addition, autologous onlay grafts are associated with rapid resorption in sites that receive mechanical load and soft tissue tension. 13 Hence, research in the past few years has been focused upon development of biomaterial graft options that could successfully replace autologous onlay bone grafts and allow for more predictable vertical ridge augmentation. 7,14

Fig. 1
(A & B) Clinical photographs showing resorbed maxillary and mandibular alveolar ridges (red arrows) in a completely edentulous patient, (C) Panoramic radiograph showing resorbed and insufficient alveolar ridge.

Calcium Phosphates for Bone Regeneration
Calcium phosphates have similar composition to bone and several synthetic calcium phosphate based bone replacement graft biomaterials have been investigated over the years as autograft alternatives. 6,14 However, one of the main problems with these materials such as hydroxyapatite (HA) is the variable integration rate and inadequate total amount of resorption resulting in limited new bone formation and infiltration into the graft area. 15 Ideally, resorption of graft material should be concurrent with new bone formation, in order to obtain a stabilized repair and eventually a fully healed bone defect with no or negligible remnants of the graft material. 6 Dicalcium phosphate cements, brushite and monetite, show osteoconductive, osteoinductive and in vivo resorption potential. 16,17 Dicalcium phosphate dihydrate (DCPD), also known as brushite is a biomaterial that can be prepared in the form of hydraulic cements with a wide range of applications. 18 However, upon implantation brushite shows re-precipitation to insoluble HA that slows resorption and limits replacement by new bone. The anhydrous form of dicalcium phosphate (DCPA), also known as monetite, has been shown not to convert to HA and demonstrates greater resorption than brushite. 19-23 Monetite biomaterials have already shown the ability to regenerate bone in animal and human bone defects and to also stimulate vertical bone augmentation. 16,24,25

Fabrication Of Monetite Onlay Grafts
The main objective was to fabricate monetite grafts and to evaluate their osteoconductive potential as a new therapy for augmenting resorbed alveolar ridges. These grafts in the future would be expected to shorten time to implant placement and improve implant to bone integration which would improve the long-term success of implant therapy. Monetite grafts were prepared by wet heat conversion (autoclaving) of preset brushite cements. Brushite was prepared with a mixture of ß-TCP and commercially available monocalcium phosphate hydrate. The cements were produced at powder-to-liquid (P/L) mixing ratio of 1.4 and cement pastes were cast into moulds with a diameter of ~9.5 mm and height of ~4 mm (Fig. 2). The discs were allowed to set for 24 hours to form hardened brushite. Monetite grafts were subsequently prepared by autoclaving the brushite discs at autoclaving conditions (121°C, 100% humidity and 15 psi, for 30 minutes). The phase purity of monetite grafts was confirmed using X-ray diffraction (XRD). Microstructural morphology of the prepared monetite grafts conjugate was examined with a scanning electron microscope (SEM) and the micrographs obtained from the surface of the monetite grafts appeared to have a fine microstructural appearance (100-200 nm crystals in length) (Fig. 2F). The monetite grafts had similar density to pure monetite (2.92 g/cm3) 18 and the compressive strength of prepared monetite grafts (Fig. 2G) was lower than that of cancellous bone (9-25 MPa). 26 There is a need for optimization of these monetite grafts in future to improve the mechanical properties.

Fig. 2
(A) ß-Tricalcium phosphate (ß-TCP) and Monoclacium phosphate monohydrate (MCPM) powders for fabricating disc grafts, (B) Fabricated monetite grafts, (C) Screw-hole being drilled in the monetite graft, (D) Fabricated monetite graft with screw hole, (E) Schematic representation of graft disc geometry and dimensions, (F) SEM images obtained from the monetite graft surface (Scale bar in the images represent 2 μm), and (G) Table showing characterization of fabricated disc grafts. Values are presented as mean ± standard deviation (n=10).

Surgical Procedure
Both the calvarial bone and the mandible originate from intra-membranous type of bone. 27 The calvarial bone also has low bone marrow content and limited vascular supply and hence resembles an atrophic mandible. This suggests that calvaria can be considered a reliable site for testing bone augmentation procedures. 28 Briefly, calvarial bone was exposed through a skin incision approximately 4 cm in length over the linea media. The periostium was also incised in the same place and a periosteal elevator was used for separating the periosteum from the bone surface. The monetite onlay grafts were stabilised by utilising titanium osteosynthesis screws (1.5 mm screw diameter and 7 mm screw length) on either side of the midline (Fig. 3). In each animal two grafts were placed either side of midline of the skull and were firmly in contact with the bone surface with no micromovement. The wound was next closed with 3-0 running subcuticular monocryl sutures and interupted skin sutures (Fig. 3). Post mortem, bone blocks containing the stabilised monetite graft discs were retrieved from the animal calvaria and processed for further characterisation and analysis (Fig. 3).

Fig. 3
(A) Surgical incision being made to expose the calvarial bone, (B) Retraction of tissues after incision, (C) Monetite disc graft prior to implantation, (D) Disc graft being fixed onto the calvaria with osteosynthesis screws, (E) Screw stabilized monetite grafts, (F) Interrupted sutures close the incision site, (G) The appearance of the grafts exposed after 12 weeks of implantation, (H) The titanium screws removed from the grafts, and (I) The graft with calvarial bone block cut and retrieved.

Post-Implantation Observations
No complications were noted during the surgical phase of the onlay grafting. Healing progressed uneventfully for all surgical sites during the 12 weeks post implantation without any signs of rejection. Surgical re-entry revealed that the shape of the grafts had been preserved (Fig. 3G). The blocks appeared to be stable and fused to the calvarial bone upon retrieval (Figs. 3H & 3I). Upon histological observation, the grafts appeared to be well integrated with the calvarial bone after 12 weeks (Fig. 4). At higher magnification of the augmented area, the remaining material of monetite grafts appeared to be surrounded by new bone (Fig. 5).

Fig. 4
Histological micrographs of a central coronal section from the calvarial bone/onlay explant sites of monetite onlay graft after 12 weeks (Scale bar represent 2000 ßm).

Fig. 5
Higher magnification histological micrograph of a central coronal section from the calvarial bone/onlay explant sites of monetite onlay grafts after 12 weeks. The section show remaining monetite graft material (*) mixed with bone tissue infiltration (+). (X) is the original calvarial bone tissue below the red line. (Scale bar represent 500 μm)

The Back scatter-SEM cross-section micrographs of the implanted monetite grafts revealed a dense micro-porous structure where the material remained un-resorbed (Fig. 6). The monetite grafts appeared to have resorbed with new bone infiltrating into the graft area (Figs. 6 & 7). After 12 weeks of implantation, the remaining un-resorbed monetite graft material could be easily differentiated from the original calvarial surface and the newly formed bone within the original implant area (Fig. 7). Isolated sites of new bone formation were observed away from the original bone surface in the monetite grafts which indicated good osteoconductive properties (Figs. 6 & 7).

Fig. 6
Back scatter-SEM image of monetite graft after 12 weeks (Scale bar represent 2000 μm).

Fig. 7
Higher magnifications back scatter SEM images of monetite onlay graft. The image shows the remaining monetite graft material (*), bone augmented (+), and the original bone calvarium (X) bellow the red line. The radiopaque white material is the graft material surrounded by new bone (Scale bar represent 500 μm).

Vertical Height Gain & Bone Volume Augmented Within Graft Area
The maximum bone height gained in the monetite onlay grafts was on average 2.6 ± 0.7 mm (Fig. 8A). It was observed that the height of bone gained decreased progressively to both the medial and lateral sides from the central screw area (Fig. 8A). The anatomical contouring of the rabbit calvarial bone along with differences in blood supply is thought to be responsible for this observation. 29 The bone thickness of calvarium is usually around 2.5 mm, this when added to the 2.6 mm of vertical height gained with monetite grafts results in a total height of ~5.1 mm. This amount of bone height would be expected to be sufficient for the placement of short implants (5.0-6.0 mm). 30,31 It has been reported that that dental implants can be successfully placed into regenerated bone having a volume of 30-40%. 32,33 Since the volume of new bone formed within the augmented area of brushite monetite with C3 conjugate graft materials was ~36% (Fig. 8B), this indicates that the volume of bone augmented would be sufficient for placement of titanium dental implants as has been reported in other bone augmentation procedures.

Fig. 8
(A) Mapping of average bone height augmented with monetite onlay grafts, and (B) The percentage of graft material remaining and the bone tissue formed within the graft area after 12 weeks of implantation.

Monetite Onlay Graft Resorption
Monetite grafts implanted for 12 weeks showed that ~52% of the original graft material had resorbed (Fig. 8B). The resorption of monetite biomaterial grafts is mainly regulated by passive dissolution and/or by cellular activity. 34,35 The total porosity present in the grafts also greatly influences the total resorption by providing access to resorptive cellular components and also allow for passive disintegration of the grafts. This in turn creates the required space for the new bone tissue to infiltrate into the graft area. The ~26% total porosity of monetite grafts (Fig. 2G) can be attributed to the enhanced biological behavior observed in terms of resorption and bone volume augmented. In addition, the results obtained after grafting are dependent on the site of implantation. 22,36 Areas with higher vascularity, such as cancellous bone of the femoral condyle and metaphysis in the proximal humerus are known to enhance resorption when used as sites for cement implantation. 37,38 It has been previously reported that monetite grafts undergo resorption at a rate of ~1.12% per day when implanted in calvaria, ~1.1% per day in femoral condyle, ~2.65% per day in the proximal tibial plateau and ~0.2-0.3% per day subcutaneously. 23 The graft resorption rates are also heavily dependent upon variations between graft physico-chemical properties.

Conclusion & Clinical Relevance
It is shown here that monetite onlay grafts were successfully fabricated and implanted in a rabbit calvarial implantation model to evaluate the ability to integrate and grow bone vertically into the graft area. Monetite graft materials resulted in de novo bone formation within the graft area and this will be clinically very relevant and important if reproduced in humans. The bioresorbable monetite grafts have shown the potential to achieve rapid, enhanced and clinically significant bone regeneration in the vertical dimension and that the newly formed bone is physiologically active. There is a need for future studies with optimized monetite grafts that have improved mechanical properties for vertical ridge augmentation applications. OH

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About the Authors
Dr. Michael Glogauer is a Professor at the University of Toronto. His research and clinical interests focuses on developing novel bone grafting approaches prior to implant placement and the role of the oral innate immune system in maintenance of health. He is currently focusing on using oral innate immune biomarkers to detect early stages of periodontal diseases through his role as Scientific Director at the Mt. Sinai Hospital’s Centre for Advanced Dental Research and Care. He is a periodontist at OMGPerio.ca.

Dr. Zeeshan Sheikh works at the Faculty of Dentistry/Faculty of Medicine, University of Toronto and at Mt. Sinai Hospital. He was trained as a clinician scientist from institutions like Queen Mary University of London, McGill University and University of Toronto. His expertise lies in developing novel biomaterials for bone grafting for dental and orthopaedic applications.