Combination of bone substitutes and vectors in periodontology and implantology: A systematic review

The aim of the systematic review was to analyze the use of combination of bone substitutes and vectors in periodontology and implantology among animals models and humans. Electronic databases were searched, and additional hand search was performed. The research strategy was achieved according to the PRISMA guidelines. The including criteria were: combination of bone substitutes and vectors, in vivo studies, a precise number of specimens, histological and radiographic analysis, written in English. The risk of bias was evaluated for individual studies. Thirty-two articles were selected and investigated in this systematic review. The results do not show a superiority of the use of composite biomaterial in comparison with simple biomaterial but suggest the efficacity of their utilization as a carrier of bioactive agents. Future studies need to identify the suitable association of bone substitutes and vectors and explore interest in their use such as the support of growth factors.


INTRODUCTION
Bone grafts are used in periodontology for the treatment of intrabony and furcation defects; they are used in implantology for alveolar ridge preservation, guided bone regeneration (GBR), or sinus lift. An ideal graft material should be biocompatible, safe, non-allergenic, non-toxic, and have no risk of disease transmission. Ideally, it should provide a role of space-maintaining, and have similar resorption rate, composition and porosity to human bone 1,2) . This interconnected porosity should allow the ingrowth of blood vessels and the diffusion of bone cells and nutrients. Finally, it should have a controlled biodegradability to ensure a balance between resorption and volume maintenance during bone ingrowth and a dimensional stability to allow this adaptation in the defect 3) . Bone grafts promote bone formation under three concepts: osteoconduction (material acts like a scaffold), osteoinduction (material contains proteins which lead to proliferation and differentiation of bone cells), and osteogenesis (material containing stem cells) 4,5) .
The current gold standard is still autologous graft (bone from the patient); it is the only bone graft that is osteoconductive, osteoinductive and osteogenic. This technique has several detriments, such as the necessity of a secondary operative site, which represents an augmented risk of supplementary comorbidities, or a low quantity of bone 6) . For these reasons, some alternatives have been developed. The first alternative to autologous bone is the use of allogenic graft: tissue from a human donor or cadaver. Three types of allografts exist: fresh frozen bone (FFB), freeze-dried bone allograft (FDBA), and demineralized freeze-dried bone allograft (DFDBA). The risk of transmission of bacteria, virus, or prion cannot be excluded for this type of bone substitute 7) . For these reasons, their uses are restricted, particularly in Europe 3) . The second option is xenografts: transplantation of bone tissue across species. In periodontology and implantology, deproteinized bovine bone is the most commonly used. Lastly, alloplastic bone substitutes have been developed in the form of synthetic hydroxyapatites (HA), beta-tricalcium phosphate (β-TCP), biphasic calcium phosphate (BCP), and bioglasses. HA is nonresorbable biomaterial with a low resorption rate and high space-maintaining potential contrary to β-TCP. BCP is composed of different ratios of HA and β-TCP to combine the advantages of these two families. Thus, this biomaterial can have different biodegradability and stability degrees according to the bone defect.
All of these bone substitutes are available in the form of granules or blocks, which can be difficult to manipulate and set up in some clinical situations. Therefore, combination of bone substitutes and vectors (composite biomaterials) have been elaborated; they are composed of two phases, granules of bone substitutes linked together by a vector. The vectors are polymer biomaterials, mainly represented by polyglycolic acid (PGA), hydrogel, or collagen 2) .
Combination of bone substitutes and vectors are in the form of paste or injectable material. The goal of this galenic form is to allow an augmented usability for the clinician and a better stability in chirurgical sites; these materials can also be used as a support for stem cells or growth factors 8) . However, the addition of polymer between bone particles can change the property of the biomaterial and its capacities of bone neoformation.

MATERIALS AND METHODS
The different studies concerning the use of combination of bone substitutes and vectors in periodontology and implantology on human or animal models have been collected and analyzed.

Question
Based on the PRISMA directives (Preferred Reporting Items For Systematic Reviews and Meta-Analyses) 9) , a specific question has been developed with the PICO (Participant, Interventions, Control, Outcomes) method 10) : "Do combination of bone substitutes and vectors enhance clinical results in patients treated in periodontology and implantology?"

Information sources and search strategy
The search strategy was established according to the PRISMA guidelines. Original articles were searched using electronic databases (Medline and Cochrane Library), and relevant articles were screened by hand to potentially add relevant articles. A combination of Medical Subject Heading (MeSH) terms were used to identify appropriate studies: "combination of bone substitutes and vectors", "periodontology", "implantology", "pre-clinical study", "clinical study". Only English articles were included and no publication dates or publication status restriction were imposed.

Study selection and inclusion/exclusion criteria
Selection was based on the inclusion and exclusion criteria defined so as to include only the most valuable articles ( Table 1).
The selection process was recorded in detail to a PRISMA 2009 flow diagram (Fig. 1).

Risk of bias in individual studies
To ascertain the risk of bias in eligible articles, their methodology was evaluated by SYRCLE's Risk of Bias tool for animal intervention studies 11) , or by Risk Of Bias Non-randomized Studies of Interventions (ROBINS-I) tool 12) , or by the Cochrane Collaboration's tool for human randomized trials 13) .

Data synthesis
A meta-analysis was not performed; we conducted a descriptive and systematic analysis of the studies. CT analysis: NB area significantly wider in group b than in group a. These results suggested that application of HAP/Col increased the outgrowth of NB much more prominently than did collagen. Micro-CT Histological Vertical distance between buccal and lingual crest: no significant difference between C and T1; and between T1 and T2. Distance significantly smaller in T2 than in C. The amount of mineralized bone was significantly lower in T1 group than in C group. No difference between T1 and T2; C and T2. When grafts were used in the socket, quantity of mineralized bone tented to be less. ) was significant.
The addition of BMP2 to PCBB or CM did not render statistically significant improvement of their performance for horizontal ridge augmentation. Radiographic Histological Histomorphometric Radiographic analysis: total augmented volume did not differ significantly between IAB and OAB groups but was significantly lower in SBC group.
Histological and histomorphometric analysis: Residual bone material and NB significantly greater in groups b and c than in group a.
Median bone-to-implant contact significantly higher in group c than in group a. Median mineralized tissue area not significantly different between the three groups.  Clinical Histological The total amount of bone volume showed no statistically significant augmentation in control group. In test groups 1 and 2: relevant ridge augmentation was observed.
Significant bone formation was histologically observed in all test areas. The association of a membrane seemed to enhance the process of bone formation.

Study selection
The MEDLINE literature search resulted in 516 potentially relevant articles. After the first selection step, based on the title and/or the abstract, 54 publications were included for further analysis. Based upon the full-text screening, 33 articles were included in the systematic review.

Study characteristics
The studies were ranked in a comparative table (Tables 2 and 3

Risk of bias within studies
The results of the risk of bias assessment are viewable in Fig. 2. The adapted methodology was applied for each subgroup of studies: pre-clinical animal studies ( Fig. 2.a), non-randomized human trials (Fig. 2.b), and randomized human trials (Fig. 2.c).

Pre-clinical studies
Only 45% and 23% of the studies mention randomization or blinding, respectively. Our data show a high score of unclear risk of bias for the performance and detection items (59% and 77%, respectively). The majority of the studies were free from selective outcome reporting (54%).

Non-randomized human trials
Only two studies were included in this category. Our results show that 100% of the studies were free from pre-intervention bias, and 50% were marked for unclear risk of bias for at-intervention items and 100% for postintervention items.

Randomized human trials
As expected, our results did not show a high risk of bias. Regarding selection and attrition items, 75% of the studies were free from risk of bias. Our data show that 50% and 37.5% of performance and detection items, respectively, show an unclear risk of bias. Finally, 75% of the included studies were marked for an unclear risk of bias for their reporting.

Synthesis of results
For each selected study, the significant results are shown in Table 2 for pre-clinical animal models and Table 3 for human studies.

DISCUSSION
Literature searches retrieved 32 studies. After a careful analysis, our results revealed that it was not possible to perform direct head-to-head comparisons of these studies as a result of variations between studies, in terms of bone substitute, polymer vectors, the defect type and size, and the healing time. Not surprisingly, no meta-analysis of the data could be carried out.
The majority of the selected articles in this systematic review are pre-clinical animal studies (22/32 studies) with a larger panel of biomaterials and type defects tested than in clinical studies. Indeed, the selected human studies employed biomaterials exclusively in alveolar ridge preservation and sinus augmentation; none of the studies were about intrabony periodontal defect. Consequently, the use of combination of bone substitutes and vectors in periodontal defects is based only on pre-clinical studies that have a lot of risk of bias, with randomization and blinding infrequently described. This failure makes it difficult to draw conclusions from pre-clinical studies. Nevertheless, the combined analysis of the different included studies affords the retrieval of valuable information.
The combination of bone substitutes and vectors are composed of granules bound together by vectors, which can be coated with growth factors. This discussion focuses on the different combinations used in pre-clinical and clinical studies and the utilization of growth factors.
The most used bone substitute in pre-clinical studies was β-TCP, mainly in combination with PGLA, a scaffold commonly used for tissue repair. Two studies (Leventis et al. 14) and Naenni et al. 15) did not show relevant results for the use of β-TCP/PLGA combination. Okada et al. 16) showed that β-TCP/PLGA seems to be more effective than conventional β-TCP for alveolar ridge preservation. The results show that this injected and moldable biomaterial maintains its shape, secures the regenerative space and enlarges the osteoconductive area.
Two studies used β-TCP in gelatin sponges incorporating growth factors (rh-FGF). Hoshi et al. 17) showed that the combined use of rh-FGF and gelatin sponge/β-TCP is effective for alveolar ridge augmentation. Fukuba et al. 18) concluded that the controlled release of rh-FGF in time induces notably more alveolar bone regeneration than short-term application of this growth factor. Here, the use of a combination of bone substitutes and vectors seemed to be essential to control 11 Dent Mater J 2021; : the propagation of the growth factor and optimize the bone regeneration.
Only one clinical randomized trial tested β-TCP/ autogenous combination and rhGDF-5 coated β-TCP 19) and concluded in the absence of significant difference.
The relevant results obtained in animal studies with β-TCP and polymer suggest that these combinations should be tested in clinical study in human models to attest their effectiveness.
BCP is used in five animal studies combined with Si-HPMC, hydrogel and collagen. Two studies of Struillou et al. 20,21) tested BCP with hydrogel in intrabony defects and peri-implant defects. They showed that hydrogel/ BCP can promote new bone formation in large defect and implant sites, the viscosity of hydrogel, allowing for increased retention capacity and mechanical strength. Three studies employed BCP in combination with collagen (CBCP) loaded with growth factor BMP2 (bone morphogenic protein) and rh-BMP2 (recombinant human bone morphogenic protein). Two of these studies 22,23) concluded that the combination of BCP/ Collagen and BMP2 was favorable for the new bone formation. It is supposed that the addition of BMP2 induced post-operative swelling at the origin of an early bone formation.
The use of BMP-2 in combination with biomaterial to promote bone regeneration has been studied in numerous pre-clinical and clinical studies 24) . This growth factor has the highest evidence of a positive effect on bone formation in comparison with other agents. However, plenty of growth factors act on the bone-healing process 25) , which suggests that only one factor in a biomaterial may be insufficient to stimulate the regeneration. Future studies might be directed toward the combination of factors and the use of combination of bone substitutes and vectors like a delivery system for these bioactive agents.
Mainly randomized clinical trials were realized with DBBM or DFDBA in combination with collagen (5 studies), and one non-randomized clinical trial used synthetic oligopeptide as a vector. All these studies concluded that DBBM-Collagen is effective in the alveolar ridge preservation procedure, but no significant differences were observed with the control group.
One randomized clinical trial 26) employed autogenous bone (AB) in combination with melatonin in immediate implant placement. The result showed a significant benefit for AB/melatonin, and the author suggested that the addition of melatonin has a positive role in new bone formation around the implant and could protect and recover the gingival tissue integrity. However, only radiological analysis was performed in this study; further clinical trials with histological and histomorphometric analysis will be necessary to attest the efficacity of AB/melatonin combination in new bone formation.
One non-randomized study 27 in a human model used anorganic bovine-derived HA combined with putty P15. Cell-binding peptide was also used in one pre-clinical study 28) . The good results suggested that HA/P15 has greater compatibility with host bone than HA alone for alveolar ridge preservation.
None of the selected clinical trials studied combination of bone substitutes and vectors in periodontal defects in contrast with pre-clinical studies. Some relevant results in the use of these bone substitutes in periodontal bony defect in animal models suggested the necessity to realize these studies in human models. Particularly for the use of combination of bone substitutes and vectors as growth factors vectors, this could be interesting to promote periodontal regeneration. Indeed, a plethora of publications brings to light the valuable role of growth factors and stem cells in the bone healing process 29,24,25) and the necessity of developing sophisticated delivery systems to lead them in the defect. In that way, combination of bone substitutes and vectors may be valuable. Indeed, some studies included in this systematic review suggest this, but additional research is necessary to develop an optimal biomaterial able to support specific molecules to promote bone and periodontal system regeneration in specific clinical situations 30) .

CONCLUSION
Several combinations of bone substitutes and vectors are studied with various clinical applications. The corresponding studies have heterogenous results concerning their applications in periodontology and implantology. For these reasons, a systematic approach appears essential to serve as a guide for future studies and provide data that can be generalized. The results of our systematic review indicate that combination of bone substitutes and vectors do not enhance clinical results in comparison with classical bone substitutes, but they may provide beneficial effects in combination with growth factors. The present review supports important information for the evolution of research concerning the use of growth factor in bone graft for periodontal regeneration and implantology in the future. Future studies should focus on the use of combination of bone substitutes and vectors as stem cells and bioactive molecules carrier and explore their use in complex periodontal defects to find a combination more effective than simple bone substitute.