The Combined Use of Particulate Allografts (DFDBA) and Platelet Concentrates in Oral and Maxillo-Facial Surgery

Abstract

Among the biomaterials used in the field of oral surgery, allogeneic bone is considered as a good material. In the particulate form, demineralized freeze-dried bone allograft (DFDBA) is useful in many procedures in maxillofacial surgery. Platelet concentrate (platelet-rich fibrin, PRF) is obtained by centrifugation of blood. This contains high concentrations of growth factors and could enhance healing, and possibly improve bone repair and regeneration. Although it remains unclear whether they are able to accelerate bone healing and influence bone quality, platelet concentrates accelerate dermal soft tissue and oral mucosa healing post-extraction. A combination of particulate DFDBA and platelet concentrate (PRF) is used in our department for horizontal and vertical bone grafts.

1. Introduction

Autogenous bone has long been considered the gold standard for bone reconstruction in maxillo-facial surgery. However, the current trend is to increasingly use biomaterials as an alternative to autogenous bone, with the aim of reducing post-operative morbidity related to the act of bone harvesting at the donor site, and to reduce the operating time.

2. Biomaterials used in oral and maxillo-facial surgery

Biomaterials used in oral surgery are classified into two categories: biomaterials of natural origin and biomaterials of synthetic origin. For review, see [1].

2.1 Biomaterials of synthetic origin

Biomaterials of synthetic origin are tricalcium phosphate, hydroxyapatites, two-phase ceramics, bioactive glasses, and polymers.

Tricalcium phosphates (ßTCP) Ca3(PO4)2 are produced by heating a mixture of calcium phosphate powder and naphthalene to over a thousand degrees and under pressure, which, after sublimation, leaves a porous structure that gives rise to the osteoconductive properties of the material.

The porous hydroxyapatite Ca10(PO4)6(OH)2 is obtained by the thermal transformation of calcium carbonate. Chemically, this calcium phosphate is the closest to biological apatite crystals. Several porosities are available. The higher the porosity, the better the osteoconduction.

Two-phase ceramics (biphasic calcium phosphate (BCP)) include a combination of hydroxyapatite and tricalcium phosphate in different proportions, which make it possible to combine the qualities of the two materials, in particular to obtain adequate resorption and mechanical qualities.

Bioglasses, SiO2-P2O5-CaO-NaO, are materials called “bioactive”. This “bioactivity” would take place due to surface reactions of the bioglass and ion exchanges with biological fluids. The bioactive bone/glass bond would take place through a layer of amorphous silica gel which would exert a chemotactic effect on the osteoblasts. Polymers with, in particular polymethylmethacrylate (PMMA), have excellent biocompatibility.

2.2 Biomaterials of natural origin

The biomaterials of natural origin are xenogenic bone, natural coral and allogeneic bone.

Xenogenic bone (xenograft) is most often of porcine or bovine origin. Inorganic bovine bone retains a bony spatial structure, giving it osteoconductive properties. These are products presented in freeze-dried form [2]. Natural coral is 99% calcium carbonate. After heat treatment, it retains a porous structure that gives it osteoinductive properties [2].

Allogeneic bone substitutes are produced from femoral head bone taken from human individuals (fresh corpse). Compared to autogenous bone, they have the advantage of not requiring harvesting from the patient (in fact, the harvesting of autogenous bone is accompanied by an increased risk of post-operative morbidity). There are two types of allograft depending on the treatment applied to the harvested bone: freeze-dried allograft called the Freeze Dried Bone Allograft (FDBA) and freeze-dried demineralized allograft called the Demineralized Freeze Dried Bone Allograft (DFDBA) or Demineralized Bone Matric (DBM).

To obtain the FDBA, the bone removed will undergo a series of treatments such as [3]: the elimination of residual musculo-fibrous tissue, size reduction until 5 mm particles are obtained, initial decontamination, microbial treatment with antibacterial, antifungal, and antiviral solutions, freezing in liquid nitrogen at −80°C, dehydration by freeze-drying, a second reduction in particle size, packaging in sterile packaging, and finally sterilization with gamma rays to reduce the risk of contamination. The FDBA will mainly serve as a matrix for bone regeneration: osteoconduction.

For DFDBA, the treatment sequence is similar but with a demineralization phase in a hydrochloric acid bath, which is added following the second reduction in particle size.

Demineralized freeze-dried bone allograft (DFDBA) has osteoconductive properties, that is, it serves as a scaffold for the colonization of the recipient site by different cell types and growth factors, and some authors have shown that it has osteoinductive properties; that is to say that it would allow the neoformation of bone. This would take place due to the presence of Bone Morphogenetic Proteins (BMPs) within the bone matrix [4, 5]. Indeed, as it resorbs, the bone releases growth factors, including BMPs belonging to the transforming growth factor beta (TGF β) superfamily and isolated for the first time in the 1960s by Marshall Urist, an American orthopedic surgeon. BMPs and, in particular, isoforms 2, 3, 4, and 7 play a crucial role in bone healing by stimulating the differentiation of mesenchymal stem cells into bone cells [5].

The ability of DFDBA to be osteoconductive and osteoinductive would be influenced by various factors, such as the age of the donor (best between 41 and 50 years old for a man and 51 and 60 years old for a woman), the size of the particles (which must be between 500 and 710 μm to ensure an optimal effect), the residual calcium level (osteo-induction is optimal when the percentage of residual calcium is 2%) [6], as well as the methods of preparation, sterilization, and conservation [7]. It was shown that there is a large variation in the amounts of proteins extracted within different batches of DFDBA, and the authors hypothesized that proteins could be degraded within certain DFDBA batches or present in too small quantity within the bone matrix and therefore remain undetectable with their method [5].

3. Adjuvants to surgery: autologous platelet concentrates

The purpose of using autologous platelet concentrates (PRP and PRF) in oral and implant surgery is to accelerate and improve the phenomena leading to healing, and in particular during surgical procedures aimed at bone regeneration (for review, see [8]).

Platelet-Rich Plasma (PRP), introduced by Marx et al. in 1998, is obtained following two successive centrifugations in tubes with the anticoagulant citrate dextrose A (avoiding platelet activation and degranulation). Gelation of the platelet concentrate is achieved instantly by adding bovine thrombin, recombinant human thrombin, or recombinant human tissue factor, which triggers platelet activation and fibrin polymerization [9].

Authors have developed a simplified protocol to obtain an autologous platelet concentrate without the use of anticoagulants or thrombin called PRF (Platelet-Rich Fibrin) [10]. Venous blood collected without the addition of anticoagulant is immediately centrifuged for 10–12 minutes at 2700–3000 rpm. The authors obtained three layers successively from the bottom to the surface of the tube: the red blood cells, the PRF clot rich in platelets, and on the surface the acellular plasma rich in fibrin.

The natural coagulation mechanism is triggered when blood comes into contact with the surface of the glass tube and allows the production of a fibrin clot rich in platelets and white blood cells, without biochemical changes, that is without the addition of anticoagulant, thrombin or calcium chloride. PRF can be used in the form of gel or membranes.

Platelet-rich fibrin (PRF) has the advantage over PRP of being completely autologous and has been shown to release platelet growth factors over a period of at least one week [11], unlike PRP that releases them within 1 hour following its preparation [12]. Due to the ease of obtaining it, its purely autologous nature, and the fact that the coagulation cascade takes place physiologically without the addition of bovine thrombin, PRF is currently tending to replace PRP.

Platelet concentrates contain fibrinogen, cell adhesion molecules (fibrin, fibronectin, and vitronectin) playing a role in cell migration and osteoconduction, and also growth factors such as PDGF (Platelet-Derived Growth Factor), TGF-β, EGF (Epithelial Growth Factor), IGF (Insulin-like Growth Factor), and VEGF (Vascular Endothelial Growth Factor) [13].

In oral implant surgery, platelet concentrates are used as adjuvants to bone reconstruction procedures. The purpose of their use is to accelerate and improve the phenomena leading to healing, and in particular to the integration of bone grafts and bone regeneration [13].

They are used in many clinical applications: maxillary sinus filling, alveolar bone crest augmentation, mandibular reconstructions, treatment of periodontal pockets, filling of post-extraction dental sockets, and osseointegration of dental implants [14, 15, 16, 17]. Studies have been able to show the beneficial effects of platelet concentrates such as improved soft tissue healing [18, 19]. However, the improvement of bone regeneration thanks to the contribution of platelet concentrates is still controversial [20, 21]. Lack of standardization in procurement protocols of these platelet concentrates may explain the lack of concordance in the various studies, and in addition, the kinetics of delivery of growth factors is a mechanism that is still poorly understood, and which may show large variations between patients and in the same patient according to the time of day when blood collection takes place (there are circadian variations in platelet concentration) [10, 11]. In addition, the contradictory results noted in the literature concerning the benefits provided by the use of platelet coagulation factors can be partly explained by the fact that these results come from clinical studies for some and from animal studies for others, and that it is difficult to extrapolate the results obtained from one species to another [22, 23].

On the other hand, the minimum concentration of platelets necessary to obtain a blood clot entering into the criteria defining the PRF is not well specified by the authors. However, they agree on the fact that a clinical benefit can be obtained for a platelet concentration of 1 million/μl of plasma (4 to 7 times the basal level) [24].

4. Use of a combination of particulate allograft (DFDBA) and platelet concentrates (PRF) in oral and maxillo-facial surgery

In our department, we routinely use a combination of particulate allograft (DFDBA 300–500 μm) and platelet concentrates (PRF) for the preservation of alveolar bone volume after dental extractions, for sinus grafts (sinus lifts), and for horizontal and vertical bone grafts.

The DFDBA comes from the cortical part of the long bones of the lower limbs of deceased tissue donors in the Erasmus Hospital Bone and Tissue Bank. It is collected by the Erasmus Bone and Tissue Bank team. The samples are then demineralized in HCl to obtain a residual calcium value <10%. Sterilization is then performed using two chemical baths of NaOH and H₂0₂ validated by the Institut Pasteur for the destruction of bacteria and viruses and finally undergoes Gamma 25 kGy irradiation at the end of the process. The different particle sizes are obtained by sieving.

To assess whether this DFDBA and PRF mixture was favorable for post-extraction bone filling, we conducted clinical and histological/histomorphometric research.

First, a retrospective radiological clinical study was conducted on 56 patients for whom 95 extractions had been done, to evaluate vertical alveolar bone loss 3 months after tooth extraction, to find out if a combination of particulate allograft (300–500 μm) and PRF could be beneficial for the patient, with predictable results, to preserve alveolar bone volume. Three months after tooth extraction, the mean of vertical loss of the midbuccal bone wall was 0.72 (SD 0.71) mm (5.53%), indicating a good potential for the technique using DFDBA 300–500 μm and platelet concentrates in alveolar bone preservation [25].

In a second study [26], a retrospective clinical study was performed. A total of 84 patients were included with 247 dental implants, to compare peri-implant bone loss at implants placed in alveolar sockets filled with a particulate allogenous bone graft (DFDBA 300–500 μm) and platelet concentrates versus at implants placed in the native bone. At 6 and 12 months, the peri-implant bone loss in sockets preserved with DFDBA and platelet concentrates was similar to the peri-implant bone loss in native bone.

In a recent study [27] to evaluate the bone quality of sinus and alveolar grafts following filling with particulate allogenous bone (DFDBA 300–500 μm) and platelet concentrate (PRF), we realized a prospective interventional clinical and histomorphometric study. A total of 40 bone cores, 2 mm in diameter, were taken from 21 patients: 22 from grafted alveoli, 7 from grafted sinus sites, and 11 from native bone used as a control. Fixed, paraffin-embedded samples were subjected to histological staining with hematoxylin–eosin and Masson’s trichrome. Bone maturity of the samples was evaluated by two independent operators using histomorphometric analysis. There existed a greater proportion of lamellar neoformed bone than woven neoformed bone as the healing time increased. Moreover, there was also an increasing proportion of newly formed bone in the grafted sockets as a function of healing time (average: 41.22% ≤ 5 months, 55.89% ˃ 5 months). Resorption of DFDBA particles also appears to be correlated with healing time in the grafted socket (average: 15.43% ≤ 5 months, 13.72% ˃ 5 months). We were able to conclude that performing sinus lift and alveolar socket preservation techniques using DFDBA and PRF results in high-quality, mature bone tissue according to histological criteria.

Our studies show that the use of particulate DFDBA mixed with platelet concentrate (PRF) leads to good clinical results with regard to post-extraction bone preservation and success rate of implants placed in the grafted bone. Moreover, a histomorphometric study shows that this technique results in a high-quality mature bone according to histological criteria.

4.1 Horizontal and vertical bone grafting

For bone grafting of the maxillas, autologous bone grafting has long been considered the gold standard owing to its osteogenic, osteoconductive, and osteoinductive properties [28]. However, this graft, which is taken from intra- or extraoral donor sites, limits the amount of bone available and requires a second invasive surgical procedure, thus increasing the risk of post-operative morbidity [29]. Moreover, it features a mean resorption rate of 50% [28].

Demineralized freeze-dried bone allograft (DFDBA) in the particulate form has been used successfully for many years in the Stomatology and Maxillofacial Surgery Department of the Erasmus Hospital for bone grafting procedures in maxillo-facial and implant surgery.

Its particulate character (particles whose diameter is between 300 and 500 μm and 500–1000 μm) allows its easy adaptation to the recipient bone bed. Nevertheless, the forces applied by the soft tissues can compromise its stability and the maintenance of the volume of the graft, and it is necessary to add a mechanical means of maintenance of volume during the integration period of the bone graft.

The principle of guided bone regeneration (GBR) consists in placing the graft material under a support acting as a space maintainer, to prevent the non-osteogenic tissues from interfering with bone regeneration [30].

To this end, the use of titanium osteosynthesis screws arranged as a “tent peg” is currently considered an effective therapeutic procedure in the treatment of significant vertical bone deficits (Figure 1) [31].

Figure 1.

The placement of osteosynthesis screws arranged in a “tent peg” to counter the forces exerted by the soft tissues. After placement of the demineralized freeze-dried bone allograft (DFDBA) and platelet-rich fibrin (PRF) mixture against the recipient bone bed, a collagenic membrane is positioned to cover the graft. The mucosal flap can be put back in place with sutures.

We recently performed a 2-year clinical prospective study [submitted] to quantify the study of horizontal oral bone ridge augmentation using GBR with an association of particulate allografts mixed with platelet-rich fibrin, collagen membrane, and tenting screws. This study aimed to radiologically evaluate the horizontal bone gain. A total of 42 patients with an insufficient alveolar bone width for dental implant placement were treated with a GBR technique using a mixture of particulate allograft (demineralized freeze-dried bone allograft 300–500 and 500–1000 μm), advanced platelet-rich fibrin (A-PRF), resorbable collagen membranes, and screw tents (1.2 mm in diameter). Bone gains were measured by cone-beam computed tomography (CBCT) at 9.1 ± 2.0 months post-operatively. A significant mean increase (P < 0.001) of 3.2 ± 0.9 mm was observed regardless of bone defect location and without complications during the entire post-operative follow-up. All patients benefited from implant placement following the bone augmentation protocol.

We were able to conclude that maxillomandibular bone augmentation in the horizontal plane by the guided bone regeneration technique using a mixture of particulate allograft, platelet concentrates (A-PRF), and screw-tent in combination with a flap closure technique tension-free mucoperiosteal was a procedure that provides reliable, predictable, and reproducible clinical results. This guided bone regeneration technique differs from previous studies on the subject by its qualitative aspects. Indeed, it is easy to use, considerably reducing operating time, with a low complication rate and an absence of morbidity associated with the donor site in the context of autologous bone harvesting. In addition, all the materials and methods used in the context of this technique are reimbursed for the patient.

4.1.1 Clinical case: Horizontal bone grafting

This 51-year-old woman, totally edentulous at the maxilla, presents with significant horizontal bone resorption of the upper jaw (Class IV by Cawood et al. [32]). She wants an implant-supported rehabilitation solution, and it is necessary to perform bone reconstruction before placing implants (Figure 2).

Figure 2.

Intrabuccal clinical view (A) showing horizontal bone resorption of the maxilla.

Computed tomography (CT) scan in left (B) and right (C) premolar areas shows important horizontal loss of the maxillary bone (Figures 3–6).

Figure 3.

Surgical procedure. A mucoperiosteal incision is made (A) and a mucoperiosteal flap is released. Screws (1.2 mm diameter) are placed (B), to allow guided bone regeneration and four temporary implants are placed avoid prosthesis pressure on the grafted site. A mix of particulate allograft (demineralized freeze-dried bone allograft (DFDBA) 300–500 μm) and platelet concentrate (platelet-rich fibrin (PRF)) is applied between the screws, in tied contact with bone, and covered with a collagen membrane then by PRF membranes (C). Flap is sutured in place without any tension (D).

Figure 4.

Radiological assessment 9 months later. The panoramic X-ray (A) and the computed tomography (CT) scan in right premolar (B), left premolar (C), and anterior (D) areas show a bone gain of between 4 and 5 mm over the entire area that has been grafted.

Figure 5.

The placement of six implants 9 months after the grafting procedure, together with removal of tenting screws.

Figure 6.

The final prosthetic result. Six months after the placement of implants, the patient can be provided with a fixed upper bridge screwed on implants.

4.1.2 Clinical case: Vertical bone grafting

This 31-year-old woman underwent exeresis of a myxoma of the anterior part of the maxilla 9 years ago. Consecutively, she had lost a part of anterior maxilla, as well as teeth 11, 12, and 13. Clinically, there is a vertical bone loss, together with retracted scar gingiva (Figures 7–11).

Figure 7.

Clinical view showing vertical bone loss in the area where a myxoma of the anterior part of the maxilla had been removed 9 years ago.

Figure 8.

Panoramic X-ray (A) and computed tomography (CT)-scan axial (B) and sagittal (C) views show the voluminous loss of bone in the anterior region of the maxilla.

Figure 9.

The day of bone reconstruction, a mucoperiosteal flap is raised, then 1.2 mm screws are placed (A), then a mixing of particulate demineralized freeze-dried bone allograft (DFDBA) 300–500 and 500–1000 (2/1)/platelet-rich fibrin (PRF) are compacted against the recipient bone bed (B), the PRF membranes are placed to cover the graft (C), and the site is sutured together with anterior shift of gingival papillas (D).

Figure 10.

Nine months later, X-ray assessment: Panoramic X-ray (A), cephalometric R-ray (B), and computed tomography (CT) scan in axial (C) and sagittal (D) views show a good bone reconstruction in the grafted area.

Figure 11.

Two implants could be placed into the bone graft, and the patient was provided with a fixed bridge screwed on implants.

5. Conclusion

Particulate allografts are commonly used in oral and maxillo-facial surgery.

Particulate Demineralized Freeze Dried Bone Allograft (DFDBA) is mainly used in a granulometry 300–500 μm. Mixed with platelet concentrates, it is a material that is easy to handle and to mold in place on bone beds.

Clinical and histomorphometric studies carried out by our team show that the use of this DFDBA and PRF combination gives good results for post-extraction bone volume conservation, maxillary sinus grafts, horizontal and vertical maxillary augmentations.

Conflict of interest

The author declares no conflict of interest.