Abstract
PepGen P-15 Putty comprises anorganic bovine bone matrix (ABM) coupled with a synthetic cell-binding peptide, suspended in a sodium hyaluronate carrier. The P-15 portion exhibits a similar structure and properties to the cell-binding region of type I collagen. This study was performed to evaluate ABM/P-15 putty as the sole graft in sinus augmentation. Ten patients for whom both a sinus augmentation and two implants were indicated in the posterior maxilla were enrolled. Bone cores were harvested at 8 and 16 weeks, followed by placement of one implant at 8 weeks and the second at 16 weeks. Twenty collected bone cores were evaluated histologically and by micro-computed tomography. Results showed a significant increase ( P < 0.05) in bone mineral density at 8 weeks (0.70 ± 0.13 g/cm 3 ) and 16 weeks (0.97 ± 0.08 g/cm 3 ) in the graft compared to native (control) bone (0.04 ± 0.02 g/cm 3 ). There was no significant difference ( P > 0.05) in the percentage bone volume at the two time intervals (PBV 21.14 ± 4.56 at 8 weeks and 26.33 ± 5.60 at 16 weeks). The average increase in bone height at 16 weeks was 10.55 ± 0.53 mm. It is concluded that PepGen P-15 Putty is capable of conducting and accelerating new bone formation and can successfully support dental implants.
The posterior maxillary edentulous region presents many unique and challenging conditions in implant dentistry. A few of the problematic issues for optimal implant placement include poor bone quality, three-dimensional (3D) bone loss, and a subsequent cascade of exacerbating conditions, such as increased inter-arch space, reverse root to crown ratio, and poor inter-arch relationships. Simultaneous pneumatization of the maxillary sinus and physiological bone loss secondary to tooth loss further compounds the anatomical deficiency. The magnitude of the deformity has led to a multitude of innovative reconstructive surgical techniques, including sinus grafting for implant reconstruction.
Introduced by Tatum in the mid-1970s, sinus augmentation has become a very popular and predictable procedure over the decades, with success rates in the 90th percentile. A variety of grafting materials have been utilized for sinus augmentation procedures. These include autografts, allografts, xenografts, and growth factors, used alone or in combination. While autologous bone remains the gold standard, its use can be limited due to quantity and the potential negative impact of an additional surgery and donor site morbidity.
Improvements in the conduction and induction properties of bone substitutes have progressed rapidly. Collagen, which comprises more than 90% of the spatially fixed matrix of bone, is a major regulator of cell adhesion and osteogenic differentiation. Bhatnagar et al. identified a potent cell-binding domain of type I collagen. It has been shown that a 15-residue synthetic peptide (P-15) analogous to the sequence GTPGPQGIAGQRGVV in the α1 (I) chain of type I collagen, binds cells with high affinity. The literature is replete with documentation of the osteoconductive capabilities of anorganic bone matrix (ABM), also known as hydroxyapatite.
Coupling the synthetic clone of the 15-amino acid sequence of type I collagen to the AMB particles facilitates the attachment, migration, and differentiation of cells, similar to the physiological process provided by the collagen molecule. Taking this into consideration, it is expected that bone will be regenerated in an accelerated time compared to unmodified bovine bone.
Recently, the standard powder form of many bone substitutes has been combined with various carrier materials to improve graft handling and physical properties. The effects of these carrier materials on bone formation are still unknown. Sodium hyaluronate is one type of carrier material presently used to improve ABM grafting techniques. In this study, sodium hyaluronate was used with AMB/P-15 to produce a putty form consistency (PepGen P-15 Putty).
The sodium hyaluronate used in this study is chemically identical to that naturally found in humans. Synthetically, it is produced from a bacterial fermentation process and considered a non-animal-derived semi-synthetic material. Hyaluronate is degraded by the lymphatic system. The degraded hyaluronate enters the blood and is then transported to the liver, where it is catabolized. The gel is biodegradable and fully absorbed rapidly. The gel resorption is sufficiently rapid that it does not interfere with bone apposition and formation. In a canine fenestration model, no carrier was observed after 3 weeks. This study used 3D computed tomography (CT scan) analysis to evaluate the bone formation using AMB/P-15 putty as the sole graft material in sinus augmentation.
Materials and methods
A prospective clinical trial was designed and implemented. The study sample comprised a population of 10 patients (eight females and two males; age range 29–59 years), who presented to the Oral and Maxillofacial Surgery Department, Faculty of Oral and Dental Medicine, Cairo University, Egypt. Each of these patients required posterior maxillary rehabilitation with a treatment plan to fabricate a fixed partial denture using two implants. A total of 10 operated sinuses were selected using the following criteria: posterior maxillary alveolar bone height 4 mm or less, naturally opposing teeth or prostheses, medical history void of acute or chronic medical diseases, and non-smoker. All patients enrolled participated in the study from beginning to end.
Consent was obtained after explaining the procedure and possible risks and complications. The graduate advisory committee on human research of the Faculty of Oral and Dental Medicine, Cairo University, Egypt approved the study in accordance with the Declaration of Helsinki.
Preoperative evaluation
Following careful extraoral and intraoral examination of all patients, preoperative CT scans and panoramic radiographs were used to measure the height of the atrophic alveolar ridge and rule out any sinus pathology prior to surgery ( Fig. 1 ). Surgical stents with guided drill holes were fabricated on patient dental models to accurately identify the exact location for grafting and implant placement. This step was also important regarding implant positioning for optimum prosthetic fabrication. Preoperatively, each patient was given a standard dose of 3 g ampicillin/sulbactam (Unasyn) and 8 mg of dexamethasone intravenously. All patients underwent treatment under local anaesthesia (2% lidocaine/1:100,000 epinephrine).
Sinus augmentation procedure
A lateral window approach was used to access all sinus cavities. After sinus membrane elevation, the trap door (bone window) was positioned superior and medially to create and maintain a space for graft placement. Sinus perforations were inadvertently created in two patients. These perforations were managed by applying a collagen membrane to seal the openings and ensure confinement of the graft material. A bone calliper was used to measure the bone height between the sinus floor (after membrane reflection) and the alveolar crest in order to confirm the accuracy of the preoperative CT scan radiographic bone height measurements ( Fig. 2 ). The graft material (PepGen P-15 Putty; DENTSPLY Friadent CeraMed, Lakewood, CO, USA) was mixed according to the manufacturer’s instructions and placed below the sinus membrane against the alveolar recess at the planned implant sites ( Fig. 3 ). Primary closure was accomplished using 4–0 polyglactin 910 (Vicryl).
Postoperative care following sinus augmentation
The standard postoperative protocol for sinus surgery was followed. Instructions to avoid prosthesis placement for 2 weeks post-surgery were given to the patient. Patients were also instructed to avoid any sinus pressure-inducing actions (e.g., use of straws, nose blowing). Nasal decongestant was prescribed for 3 days, while an appropriate antibiotic and analgesic were prescribed for 1 week.
A CT scan was done at 8 and 16 weeks postoperatively to measure the bone height at these time intervals.
Core bone biopsy and implant placement
A core bone biopsy (trephination) and simultaneous implant placement were planned at 8 weeks for the first implant and 16 weeks for the second. The 8- and 16-week time-frame was chosen based on the expected accelerated bone regeneration properties of the PepGen P-15 Putty. A surgical stent created from a prefabricated model was placed for the anatomical accuracy of bone biopsy and implant placement. A 2.0-mm (22 mm long barrel) internally irrigated trephine bur was used to obtain core bone biopsies with identical axial inclination guided by the surgical stent ( Fig. 4 ). Biopsy cores were followed by immediate implant placement using the recommended implant drilling sequence. The height of the initial core bone biopsy was determined with the aid of the postoperative CT scans.
Micro-CT
The 20 bone cores collected (taken at 8 and 16 weeks) were scanned by micro-computed radiography (SkyScan 1174 and version 1.7.0.5 software; Bruker, Kontich, Belgium) to evaluate the bone mineral density (BMD) and percentage bone volume (PBV) in a 3D pattern. Each core biopsy was scanned using a 360° angle of rotation with a 0.6° rotation step and a 15 μm pixel camera size and 1 mm aluminium filter. Digital high-resolution micro-radiographic images were acquired at 50 kV/800 μA with an exposure time of 1.8 s. 3D measurements were obtained based on the volume of interest (VOI) in the form of a circle, with a fixed diameter used for all cores ( Figs. 5 and 6 Figs 5 and 6 ). A standardized area in the form of 10 cuts (15 μm) at the centre of each core biopsy was selected using the multi-slice and region-of-interest properties. Measurements were made at the crestal end of the cores (native bone), which was considered the control, and at the grafted apical part of the cores (sinus end). This determinant between the native and grafted bone was determined visually by a line of demarcation viewed on the micro-computed radiography scan. In addition, the preoperative measurement done clinically with the calliper was measured on the micro-CT scan and identified as native bone. Mean values were obtained for comparison between bone cores collected at 8 weeks and those at 16 weeks postoperatively.
Histological evaluation
The core bone biopsies were fixed in 10% buffered formalin and decalcified using Morse’s solution (formic acid and sodium citrate). Once decalcified, routine histological processing and paraffin-embedding were done, and 6-μm sections were then stained with haematoxylin and eosin (H&E) and Goldner’s stain. The same areas analyzed by micro-CT were identified for histological analysis.
Histomorphometric analysis
Digital images were acquired with a digital spot camera attached to a stereo Zeiss dissecting microscope (Carl Zeiss, Wetzlar, Germany). These images were loaded into BIOQUANT Nova Prime image analysis software. All measurements were done to calculate the volume fraction (Vv) of the vital and non-vital (graft particles) components and this was presented as a percentage of the total bone. The original crestal bone was not included in the measurement.
Statistical analysis
The Student two-tailed t -test is an analysis tool that assumes that the variances of both ranges of data are unequal. This t -test is used when the groups being analyzed are distinct and to determine whether two sample means are equal. Statistical significance was set at P < 0.05.
Results
Postoperative healing was uneventful in all patients. Minimal swelling was observed at the surgical sites postoperatively and had subsided by the end of the first week. No clinical signs of infection or dehiscence were seen in any patient. Clinical and radiographic assessment showed that all implants were successfully osseointegrated, with no reported complications after a 3-year follow-up period.
It should be mentioned that the patient’s native bone was used as the control (i.e., the patient served as their own control). This was done for several reasons. It excludes the variability in the healing capacity amongst different patients, thus allowing an accurate comparison of the analysis outcome of bone quality (BMD) after the augmentation procedure. Regarding bone height, there are numerous articles in the literature addressing the optimum height needed to achieve primary implant stability in the maxillary region, which ranges from 3 to 5 mm. Therefore a separate control group was not needed, as the patients in the study met these criteria. Lastly, by using the native bone as the control, we were also able to compare the bone heights at two different time intervals (8 and 16 weeks) following the augmentation procedure. This was done to check the capability of the graft for bone build-up to adequately support implant placement and in addition to see if there were any significant differences in bone height at these time intervals.
Radiographic follow-up
The postoperative CT scans done at 8 and 16 weeks showed dense mineralized material in the sinus floor ( Fig. 6 ). The measured basal bone height before augmentation ranged from 1 to 3 mm, with a mean of 2.20 ± 0.32 mm. All sites showed a significant increase in ridge height after sinus augmentation, sufficient to support dental implants, with an average gain of 10.55 ± 0.53 mm ( P < 0.0001 at 8 and 16 weeks). There was no significant difference ( P = 0.43) in the postoperative bone height measurement (mean) between the 8th and 16th week (12.50 ± 0.32 mm at 8 weeks and 12.75 ± 0.40 mm at 16 weeks) ( Fig. 7 ).
