Long-Term Outcomes of Implants Placed in Maxillary Sinus Floor Augmentation with Porous Fluorohydroxyapatite (Algipore^® FRIOS^®) in Comparison with Anorganic Bovine Bone (Bio-Oss^®) and Platelet Rich Plasma (PRP): A Retrospective Study

Round 1

Reviewer 1 Report

Manuscript ID: jcm-1659022

Title: Long-Term Outcomes of Implants Placed in Maxillary Sinus Floor Augmentation with Porous Fluorohydroxyapatite (Algipore® FRIOS®) in Comparison with Anorganic Bovine Bone (Bio-Oss®) and platelet rich plasma (PRP)

1.What is the main question addressed by the research?

To evaluate the long-term clinical outcome of using algae-derived plant hydroxyapatite as a bone grafting material for maxillary sinus floor augmentation vs. demineralized anorganic bovine bone.

2.Is it relevant and interesting?

The article is relevant and interesting.

3.How original is the topic?

The topic is current.

4.What does it add to the subject area compared with other published material?

The authors have collected and analyzed a great deal of original data.

5.Is the paper well written?

Yes, the article is well written.

6.Is the text clear and easy to read?

Yes, but minor English editing is required.

7.Are the conclusions consistent with the evidence and arguments presented?

Yes, the conclusions consistent with the evidence and arguments presented but further studies are needed to confirm Authors’ hypothesis.

8.Do they address the main question posed?

Yes, the Authors addressed the main question posed.

Other comments:

• English language: Minor spell check required
• Summary of abbreviations required.
• Introduction: This section needs few improvements. For example, Authors may include a brief sentence on osseointegration and factors that can affect it based on the following reference: <<Both macroscopic and microscopic characteristics of dental implants could influence osseointegration and the success of implant-prosthetic procedures [PMID: 32475099]>>. The Authors may improve this section on the theme of alternative instruments for crestal sinus augmentation, implant site preparation, and placement. Allow me to suggest a relevant references to include: “PMID: 35055423”.

Regarding platelet concentrates Authors may include a brief sentence based on the following reference: "Anyway, the use of platelet concentrate is at the center of a recent academic debate [PMID: 31116189]."

• Materials and methods: This section has been properly prepared, anyway Authors should follow CONSORT or STROBE guidelines to improve this section.
• Results: This section has been properly prepared, anyway Authors should follow CONSORT or STROBE guidelines to improve this section.
• Discussion: What is the main theme that emerges from the authors' analysis?Is the study design a limitation? Please improve.
• Conclusion: This section has been properly prepared.
• Figures and Tables: Please improve figures and tables quality if possible.

After making the indicated changes, I am available for a second round of peer review.

Thanks for the opportunity to review this manuscript.

Author Response

1.What is the main question addressed by the research?

To evaluate the long-term clinical outcome of using algae-derived plant hydroxyapatite as a bone grafting material for maxillary sinus floor augmentation vs. demineralized anorganic bovine bone.

2.Is it relevant and interesting?

The article is relevant and interesting.

Thank you so much for your observation.

3.How original is the topic?

The topic is current.

Thank you so much for your observation.

4.What does it add to the subject area compared with other published material?

The authors have collected and analyzed a great deal of original data.

Thank you so much for your observation.

5.Is the paper well written?

Yes, the article is well written.

Thank you so much for your observation.

6.Is the text clear and easy to read?

Yes, but minor English editing is required.

Thank you so much for your observation. The English has been checked and corrected.

7. Are the conclusions consistent with the evidence and arguments presented?

Yes, the conclusions consistent with the evidence and arguments presented but further studies are needed to confirm Authors’ hypothesis.

Thank you so much for your observation. This consideration has been added, as follows: “Further studies are needed to confirm the hypothesis”.

8.Do they address the main question posed?

Yes, the Authors addressed the main question posed.

Thank you so much for your observation.

Other comments:

• English language: Minor spell check required

Thank you so much for your observation. The English has been checked and corrected.

• Summary of abbreviations required.

Following the observations of the all reviewers, the text has been corrected and the abbreviations have been deleted.

• Introduction: This section needs few improvements. For example, Authors may include a brief sentence on osseointegration and factors that can affect it based on the following reference: <<Both macroscopic and microscopic characteristics of dental implants could influence osseointegration and the success of implant-prosthetic procedures [PMID: 32475099]>>. The Authors may improve this section on the theme of alternative instruments for crestal sinus augmentation, implant site preparation, and placement. Allow me to suggest a relevant references to include: “PMID: 35055423”.
• The references have been added after the correction of the introduction section. The introduction has been improved, as follows: “The sinus augmentation to enhance the bone volume and allow for proper implant placement in the atrophic posterior maxilla, remains a challenge in regenerative surgery [1-3]. The primary goal of bone reconstructive surgery is to achieve new functional bone regeneration via the synergistic combination of placing bone grafting materials, cells, and growth factors [2,4-7]. Bone grafting remains the most commonly used surgical procedure to replace bone defects. It supports the repairing process by promoting osteoinduction, osteoconduction and osteogenesis [5,7-9]. Architectural and microstructural properties, and biocompatibility are critical to promote favorable tissue responses [8]. The advantages of the autologous bone grafting technique include the presence the patient’s own osteogenic cells and osteoinductive proteins, such as bone morphogenetic protein 2 (BMP2), and platelet-derived growth factor (PDGF) which provide optimal aforementioned properties without any risk [6, 10-12]. To date, several biomaterials to modulate in situ tissue regeneration have been designed [13, 14]. Bone regeneration consists of transitional steps in which the formation of soft, fibrous connective tissues, cartilage, and woven bone provide the mechanical stability that ultimately leads to bone formation, supporting the scaffold for cell and tissue differentiation [15, 16]. The rationale for any bone biomaterial as a scaffold or graft is not only to replace but to restore function to the physiologically and anatomically replaced part while ensuring complete biocompatibility without causing rejection and collaterals [17-21]. In addition, the optimal bone graft material must assume the role of a bone substitute and respond to various bio-mechanical stresses. Much of the current research has focused on the identification of biomaterials; specifically compounds for sinus floor augmentation [22-26]. The porous fluorohydroxyapatitic (FHA) biomaterial as shown promise as candidate in suitable biomaterial for sinus grafting in severely atrophic maxillae [17-21, 27]. Algipore® FRIOS® is a biomaterial, vegetable-based hydroxyapatite. It is highly analogous to the hydroxyapatite of natural bone and is manufactured from lime-impregnated red marine algae (Corallina officinalis) [28]. The biomaterial is processed through phases involving the pyrolytic segmentation of the native algae and by hydrothermal transformation of calcium carbonate [CaCO3] into FHA [Ca5(PO4)3OHxF1 x]. The structural features of 3D porous particles have shown a hierarchical pore system containing particles with a mean diameter of pores 10 mm periodically septated (mean interval 30 mm) and interconnected by microperforations of 1-4µm. Every pore is limited by one layer of small FHA crystallites with a size of 25–35 nm. he average pore volume decreased from 1.05 cm3/g to 0.93 while the surface area averages 50 m2 /g. Bio-Oss is consists of natural inorganic bovine bone which have shown faster healing process compared to other more conventional procedures applied in soft and hard tissue regeneration [29]. The addition of bovine bone mineral or nonresorbable porous hydroxyapatite to autogenous bone augments bone formation and bone-to-implant contact in augmented sinuses. The morphology of existing bone defect is determinant in the outcome of reconstructive bone surgery. On the other hand, the graft should allow for osteoinduction, osteoconduction, and endogenous osteogenesis of the recipient with adequate vascularity at the implant-bone interface [30-35]. Adequate mechanical stability of the graft or scaffold is also an important point for bone healing and repair. However, both macroscopic and microscopic characteristics of dental implants could influence osseointegration and the success of implant-prosthetic procedures [36-37]. The use of platelet concentrate is at the center of a recent academic debate [38]. Several results have demonstrated the functionality and support of platelet concentrates such as platelet rich plasma (PRP), and platelet rich fibrin (PRF), as key components in combination with bio-grafts to promote wound healing during and following surgery [34, 38]. Autologous PRP have shown to be a solid therapeutic method to improve and induce tissue repair and growth. Platelets contains several molecules like the alpha-granules that are rich in GFs such as the transforming growth factor-β (TGF-β), vascular endothelial growth factor (VEGF) and Platelet-derived growth factors (PDGFs) [39]. These factors play an important role in all phases of healing of the tissue by recruiting mesenchymal stem cells and also during the synthesis of the extracellular matrix. The combined procedure is safe with no relevant secondary reactions [40]. These derivatives alone or in combination with bone grafts and different biomaterials have been shown to promote bone growth and bone homeostasis, vascularization [41]. Our study was aimed to compare the long-term clinical outcome and complications of augmentation technique by using algae-derived plant hydroxyapatite (Algipore) as a bone grafting material for maxillary sinus floor augmentation (MFSA) versus demineralized anorganic bovine bone (Bio-Oss Geistlich Pharma, Wolhusen, Switzerland) with the support of autologous blood-derived PRP.”
• Regarding platelet concentrates Authors may include a brief sentence based on the following reference: "Anyway, the use of platelet concentrate is at the center of a recent academic debate [PMID: 31116189]."
• The reference has been added after the correction of the introduction section.

• Materials and methods: This section has been properly prepared, anyway Authors should follow CONSORT or STROBE guidelines to improve this section.

The study followed the STROBE guidelines to improve the section, and the following text has been reported: “The study adhered to the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) checklist guidelines.”

• Results: This section has been properly prepared, anyway Authors should follow CONSORT or STROBE guidelines to improve this section.

• The study followed the STROBE guidelines to improve the section, and the following text has been reported: “The study adhered to the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) checklist guidelines.”

• Discussion: What is the main theme that emerges from the authors' analysis? Is the study design a limitation? Please improve.
• The study design represents a limitation. It has been reported in the discussion section, as follows: “Our study showed overlapped results over the techniques. However, our findings are not conclusive. The study design and the sample size represent the major limitations.”
• Conclusion: This section has been properly prepared.

Thank you for your observation.

• Figures and Tables: Please improve figures and tables quality if possible.

Figure and tables have been improved, as required.

After making the indicated changes, I am available for a second round of peer review.

Thanks for the opportunity to review this manuscript.

Reviewer 2 Report

Please indicate exclusively in the title, abstract, and the materials and methods that this is a retrospective study.

The outcomes were supposed to be: Radiographic analysis and the outcomes from clinical implant survival. Where is that discussed in the results section? 

Please re-write the introduction and the discussion to be relevant to the topic. They are almost exclusively discussing the role of growth factors and not much related the sinus surgery or the used biomaterials. The results are not discussed in the discussion or compared with other studies.

Author Response

Apr 16^th, 2022

# REVIEWER 2:

Please indicate exclusively in the title, abstract, and the materials and methods that this is a retrospective study.

Thank you for your correction. It has been applied.

The outcomes were supposed to be: Radiographic analysis and the outcomes from clinical implant survival. Where is that discussed in the results section? 

Some of the results were wrongly presented in the materials and methods section. The correction has been applied, as follows:

At 4 months after the implant placement, radiographic images showed complete osseointegration of the implant (Fig. 11-12, 13-14). We recorded perforation of the sinus membrane during sinus lift during two surgeries, without compromising the surgery and subsequent implantation. The lack of osseointegration distinguished by implant mobility and radiological radiolucency were referred to a failing implant. The orthopantomography checkup show radiographic integration and increased peri-implant bone density maintained at seven years (Fig. 17-18- 19-20).

Fig. 17-18 Follow-up at 7 years implants placement (Test Group, left); (Control Group, right). 

Fig. 19-20 Rx at 7 years implants placement (Test Group, left); (Control Group, right). 

Please re-write the introduction and the discussion to be relevant to the topic. They are almost exclusively discussing the role of growth factors and not much related the sinus surgery or the used biomaterials. The results are not discussed in the discussion or compared with other studies.

The correction has been applied as follows:

The introduction section has been corrected as follows: “The sinus augmentation to enhance the bone volume and allow for proper implant placement in the atrophic posterior maxilla, remains a challenge in regenerative surgery [1-3]. The primary goal of bone reconstructive surgery is to achieve new functional bone regeneration via the synergistic combination of placing bone grafting materials, cells, and growth factors [2,4-7]. Bone grafting remains the most commonly used surgical procedure to replace bone defects. It supports the repairing process by promoting osteoinduction, osteoconduction and osteogenesis [5,7-9]. Architectural and microstructural properties, and biocompatibility are critical to promote favorable tissue responses [8]. The advantages of the autologous bone grafting technique include the presence the patient’s own osteogenic cells and osteoinductive proteins, such as bone morphogenetic protein 2 (BMP2), and platelet-derived growth factor (PDGF) which provide optimal aforementioned properties without any risk [6, 10-12]. To date, several biomaterials to modulate in situ tissue regeneration have been designed [13, 14]. Bone regeneration consists of transitional steps in which the formation of soft, fibrous connective tissues, cartilage, and woven bone provide the mechanical stability that ultimately leads to bone formation, supporting the scaffold for cell and tissue differentiation [15, 16]. The rationale for any bone biomaterial as a scaffold or graft is not only to replace but to restore function to the physiologically and anatomically replaced part while ensuring complete biocompatibility without causing rejection and collaterals [17-21]. In addition, the optimal bone graft material must assume the role of a bone substitute and respond to various bio-mechanical stresses. Much of the current research has focused on the identification of biomaterials; specifically compounds for sinus floor augmentation [22-26]. The porous fluorohydroxyapatitic (FHA) biomaterial as shown promise as candidate in suitable biomaterial for sinus grafting in severely atrophic maxillae [17-21, 27]. Algipore® FRIOS® is a biomaterial, vegetable-based hydroxyapatite. It is highly analogous to the hydroxyapatite of natural bone and is manufactured from lime-impregnated red marine algae (Corallina officinalis) [28]. The biomaterial is processed through phases involving the pyrolytic segmentation of the native algae and by hydrothermal transformation of calcium carbonate [CaCO3] into FHA [Ca5(PO4)3OHxF1 x]. The structural features of 3D porous particles have shown a hierarchical pore system containing particles with a mean diameter of pores 10 mm periodically septated (mean interval 30 mm) and interconnected by microperforations of 1-4µm. Every pore is limited by one layer of small FHA crystallites with a size of 25–35 nm. he average pore volume decreased from 1.05 cm3/g to 0.93 while the surface area averages 50 m2 /g. Bio-Oss is consists of natural inorganic bovine bone which have shown faster healing process compared to other more conventional procedures applied in soft and hard tissue regeneration [29]. The addition of bovine bone mineral or nonresorbable porous hydroxyapatite to autogenous bone augments bone formation and bone-to-implant contact in augmented sinuses. The morphology of existing bone defect is determinant in the outcome of reconstructive bone surgery. On the other hand, the graft should allow for osteoinduction, osteoconduction, and endogenous osteogenesis of the recipient with adequate vascularity at the implant-bone interface [30-35]. Adequate mechanical stability of the graft or scaffold is also an important point for bone healing and repair. However, both macroscopic and microscopic characteristics of dental implants could influence osseointegration and the success of implant-prosthetic procedures [36-37]. The use of platelet concentrate is at the center of a recent academic debate [38]. Several results have demonstrated the functionality and support of platelet concentrates such as platelet rich plasma (PRP), and platelet rich fibrin (PRF), as key components in combination with bio-grafts to promote wound healing during and following surgery [34, 38]. Autologous PRP have shown to be a solid therapeutic method to improve and induce tissue repair and growth. Platelets contains several molecules like the alpha-granules that are rich in GFs such as the transforming growth factor-β (TGF-β), vascular endothelial growth factor (VEGF) and Platelet-derived growth factors (PDGFs) [39]. These factors play an important role in all phases of healing of the tissue by recruiting mesenchymal stem cells and also during the synthesis of the extracellular matrix. The combined procedure is safe with no relevant secondary reactions [40]. These derivatives alone or in combination with bone grafts and different biomaterials have been shown to promote bone growth and bone homeostasis, vascularization [41]. Our study was aimed to compare the long-term clinical outcome and complications of augmentation technique by using algae-derived plant hydroxyapatite (Algipore) as a bone grafting material for maxillary sinus floor augmentation (MFSA) versus demineralized anorganic bovine bone (Bio-Oss Geistlich Pharma, Wolhusen, Switzerland) with the support of autologous blood-derived PRP.”

The discussion has been corrected as follows: Porous phycogenic hydroxyapatite (PHA) derived from red algae (Algipore®), is largely employed as scaffolds in bone regeneration, because of its chemical similarity to bone and interconnected porosity [42-43]. Bone ingrowth is affected by several mechanical properties of the scaffold, involving the internal porous structure [44]. The pour size influences the permeability and the inadequate dimensions may result in altered bone ingrowth. There are sevral studies conducted on the fluorohydroxyapatite (FHA) FRIOSs Algipores as a proper biomaterial for the reconstruction of severely atrophic maxillae [45-47]. Schopper et al. investigated the histomorphological and histomorphometrical examination of 69 trephine specimens who were submitted to maxillary sinus grafting with FRIOSs Algipore [48]. The authors demonstrated that the scaffold elicited generation of new bone in the grafted sinuses, about 23.0% over an observation time of 7 months. The findings accord with other authors who found a comparable bone formation after 6-7 months, combining porous hydroxyapatite and autogenous bone for sinus grafting. Deproteinized bovine bone Bio-Oss is biocompatible and osteoconductive, while is missing of osteoinductive property [49]. Sartori et al. [50] observed a slow but continuous resorption of the Bio-Oss scaffold. These results were contrasting with Schlegel et al. [51] who reported a low resorption capacity of deproteinized bovine bone. The present investigation compared the FHA biomaterial FRIOSs Algipore and deproteinized bovine bone in triggering the formation of new bone in the grafted sinuses of severely atrophic maxillae. We noticed a significant increase in the new bone formation in the areas augmented with both biomaterials. Our findings are in agreement with other authors using a combination of porous hydroxyapatite or deproteinized bovine bone and autogenous bone for sinus grafting [52-58]. 

The maxillary bone in edentulous upper premolars and molars undergoes atrophy and pneumatization of the maxillary sinus, which decreases the amount of bone in horizontal and vertical dimensions, making it insufficient for implant placement. Sinus elevation via a lateral approach was applied. It is classified as a technique-sensitive procedure due to the high risk of Schneiderian membrane perforation that can occur quite frequently up to 35%, in this procedure piezosurgery offers a positive long-term prognosis and a higher survival rate than the placement of ungrafted maxillary, and in particular, rough-surface implants [59-61].

The split crest procedure was only performed with very fine chisels as the use of burs could compromise bone preservation. According to Misch and Judy [44] the use of this procedure, with type C bone defect should respect a crestal width between 1.5 and 2.5 mm with a height ranging between 8 and 12 mm. Implant placement in the ideal prosthetic position may be compromised by bone resorption due to the presence of an increased interarch distance or an unfavorable horizontal and sagittal intermaxillary relationship [62-64]. It was necessary to subject resorbed ridges to regeneration treatment before or concurrently with implant placement in order to increase the amount of hard and soft tissues [65-68]. This allowed us to reduce the crown-to-implant ratio, place axial implants, and achieve good occlusion and a quality aesthetic appearance. The alveolar ridge split is a predictable and reliable procedure, characterized by its low invasiveness. This procedure allowed us to achieve significant bone augmentation in the horizontal plane. In the case were we registered vertical bone lost,before to proceed to implantation, we performed bone augmentation according to Khoury’s concept. The bone was collected from retromolar area to reconstructed the vertical defects. Platelet-rich fibrin (PRF) is an autologous platelet concentrate obtained by centrifugation from the patient's own blood without the use of heparin or anti-coagulants. Like PRP, the PRF procedure is easy, safe, and biocompatible. The results suggested the potential role of PRF in periodontal regeneration and tissue bioengineering as a viable material for bio-graft construction. PRF is a material composed mainly of fibrin membranes enriched with platelets and growth factors optimal for promoting the healing process of hard and soft tissues. Thus, PRF is able to regulate inflammation and stimulate the chemotaxis mechanism. In addition, its gelatinous consistency increases the stability of the clot and graft material. However, being a biomaterial formed directly from the patient's blood, the amounts that can be obtained may sometimes be very modest. PRF has the distinguishing trait of polymerizing naturally and slowly during centrifugation. The concentrations of active thrombin and fibrinogen contained in PRF are almost within normal physiologic ranges because the material does not require any addition of bovine or humanized thrombin. Fibrin tends to acquire a three-dimensional structure equivalent to the site where it is inserted supporting the healing process [47-55]. Aggregation of fibrin monomers leads to the formation of a three-dimensional scaffold, forming a thin mesh of soft, porous graft that allows rapid cell colonization of the wound and surrounding tissues [56-61]. This type of bio-scaffold induces a faster physiological healing process and in combination with bone grafting accelerates the formation of new bone tissue [62-65]. Derivatives from different species, usually ovines, undergo a series of tests and processes of demineralization, sterilization, freeze-drying. Although widely used xenografts perform similar osteoconductive activity and are relatively cheaper. In addition, their use reduces the need for a second surgery for bone harvesting [13, 66-69]. However, xenografts have demonstrated a low ability to induce adequate height and width in large defects, especially those of bovine origin. Few results from histomorphometric analysis showed low resorption rate of transplants after several years revealed residual bovine graft up to 40%, data confirmed by several histo-analyses that reported the same amount of graft detected at 3 years to that at 6 months [51, 70-71]. Our study has some limitations: firstly, the study design with intrinsic restriction; secondly, histological examination are needed to reveal a physiologic framework of bone around the biomaterial particles; finally, the inclusion criteria were stringent and eliminated interferences due to systemic disease and other factors that may alter recovery. It follows that while the adoption of mimicry approaches has finally yielded positive results, one must consider the interference of multiple variables such as physical, biochemical, metabolic, immunological, and hormonal conditioning. There are huge differences between an inserted bone graft and a mature healthy tissue microenvironment, but even more so there are crucial changes between an inserted graft and the current health status of the recipient [47,55,72-74]. Consequently, a different intervention should be planned by adapting not only the design of subsequent implants, but emphasizing the treatment plan that fully reflects these differences. Normal healthy adult development occurs in a variety of immunologic, inflammatory, hormonal, and metabolic contexts; the complexity of these factors must necessarily be addressed if the processes are to be united for complete and successful integration of bone grafts and implants [7,47]. Endocrine signaling gradients that function on a scale of healthy conditions can be subverted in a highly deteriorated situation. Modular implants, including those with smaller units including GFs and cells, can be subjected to the unfavorable internal cellular and molecular microenvironment and eventually can be altered leading to infection, necrosis, and ultimately rejection. The immune-endocrine-metabolic environment that modulates the entire process of regeneration, growth, and remodeling and regulates the influx of cells, molecules, and GFs into growing young and adult bone has yet to be fully elucidated. This is probably a crucial time if we are fully committed to unraveling the potential of evolving bioengineering and regenerative medicine, as immune-endocrine-metabolic factors are significant mediators of bone healing and regrowth or, conversely, can cause a delay in healing if they are suppressed and neglected [57,60,73-75]. This last observation serves to highlight the differences between the developmental processes that occur during normal osteogenesis and those involved in the induction of post-traumatic grafting. Indeed, while inflammation, endocrine imbalances, and metabolic dysfunction may be part of the main drivers of bone decay and graft failure, they are fully functional during normal bone development. The significance of interleukins, cytokines, and hormones in the revascularization, mineralization, and bone/cartilage remodeling activities of hPB-SCs has been profoundly elucidated, and their important role is fully appreciated as external supporters in bone graft therapy [7,9, 47]. Our study showed overlapped results over the techniques. However, our findings are not conclusive. The study design and the sample size represent the major limitations. 

Please do not hesitate to contact me if any questions or concerns remain.

We thank you for your consideration.

With kind regards,

Dr. Biagio Rapone, DDS, PG Oral.Surg., MSc, PhD

Interdisciplinary Department of Medicine, University of Bari “Aldo Moro”, Bari, Italy

Piazza Giulio Cesare 11, 70124 Bari, Italy

E-mail: [email protected]

Reviewer 3 Report

1. The introduction is long and should be shortened avoiding the in-depth basic knowledge and should give a hint about the rationale of the study. In addition, there are a lot of grammatical errors and typos that should be corrected. Moreover, using the abbreviation is mistakenly handled in the text.
2. I'm a little bit confusing for the aim of the study. Is it a comparative study between Porous Fluorohydroxyapatite and Anorganic Bovine Bone in combination with autologous PRP and if it is why they used 50% particulate in the test group and 50% autogenous bone in the control group?
3. The study design is flow and the images provided are not helpful in clarifying what the authors intended to produce.
4. Where are the methods of evaluation, just radiographs even without analysis?
5. Overall the manuscript should be edited by a native speaker.

Comments for author File: Comments.pdf

Author Response

REVIEWER 3:

1. The introduction is long and should be shortened avoiding the in-depth basic knowledge and should give a hint about the rationale of the study. In addition, there are a lot of grammatical errors and typos that should be corrected. Moreover, using the abbreviation is mistakenly handled in the text.

Thank you so much for your observations. The introduction has been shortened. The errors have been corrected.

The introduction section has been corrected as follows: “The sinus augmentation to enhance the bone volume and allow for proper implant placement in the atrophic posterior maxilla, remains a challenge in regenerative surgery [1-3]. The primary goal of bone reconstructive surgery is to achieve new functional bone regeneration via the synergistic combination of placing bone grafting materials, cells, and growth factors [2,4-7]. Bone grafting remains the most commonly used surgical procedure to replace bone defects. It supports the repairing process by promoting osteoinduction, osteoconduction and osteogenesis [5,7-9]. Architectural and microstructural properties, and biocompatibility are critical to promote favorable tissue responses [8]. The advantages of the autologous bone grafting technique include the presence the patient’s own osteogenic cells and osteoinductive proteins, such as bone morphogenetic protein 2 (BMP2), and platelet-derived growth factor (PDGF) which provide optimal aforementioned properties without any risk [6, 10-12]. To date, several biomaterials to modulate in situ tissue regeneration have been designed [13, 14]. Bone regeneration consists of transitional steps in which the formation of soft, fibrous connective tissues, cartilage, and woven bone provide the mechanical stability that ultimately leads to bone formation, supporting the scaffold for cell and tissue differentiation [15, 16]. The rationale for any bone biomaterial as a scaffold or graft is not only to replace but to restore function to the physiologically and anatomically replaced part while ensuring complete biocompatibility without causing rejection and collaterals [17-21]. In addition, the optimal bone graft material must assume the role of a bone substitute and respond to various bio-mechanical stresses. Much of the current research has focused on the identification of biomaterials; specifically compounds for sinus floor augmentation [22-26]. The porous fluorohydroxyapatitic (FHA) biomaterial as shown promise as candidate in suitable biomaterial for sinus grafting in severely atrophic maxillae [17-21, 27]. Algipore® FRIOS® is a biomaterial, vegetable-based hydroxyapatite. It is highly analogous to the hydroxyapatite of natural bone and is manufactured from lime-impregnated red marine algae (Corallina officinalis) [28]. The biomaterial is processed through phases involving the pyrolytic segmentation of the native algae and by hydrothermal transformation of calcium carbonate [CaCO3] into FHA [Ca5(PO4)3OHxF1 x]. The structural features of 3D porous particles have shown a hierarchical pore system containing particles with a mean diameter of pores 10 mm periodically septated (mean interval 30 mm) and interconnected by microperforations of 1-4µm. Every pore is limited by one layer of small FHA crystallites with a size of 25–35 nm. he average pore volume decreased from 1.05 cm3/g to 0.93 while the surface area averages 50 m2 /g. Bio-Oss is consists of natural inorganic bovine bone which have shown faster healing process compared to other more conventional procedures applied in soft and hard tissue regeneration [29]. The addition of bovine bone mineral or nonresorbable porous hydroxyapatite to autogenous bone augments bone formation and bone-to-implant contact in augmented sinuses. The morphology of existing bone defect is determinant in the outcome of reconstructive bone surgery. On the other hand, the graft should allow for osteoinduction, osteoconduction, and endogenous osteogenesis of the recipient with adequate vascularity at the implant-bone interface [30-35]. Adequate mechanical stability of the graft or scaffold is also an important point for bone healing and repair. However, both macroscopic and microscopic characteristics of dental implants could influence osseointegration and the success of implant-prosthetic procedures [36-37]. The use of platelet concentrate is at the center of a recent academic debate [38]. Several results have demonstrated the functionality and support of platelet concentrates such as platelet rich plasma (PRP), and platelet rich fibrin (PRF), as key components in combination with bio-grafts to promote wound healing during and following surgery [34, 38]. Autologous PRP have shown to be a solid therapeutic method to improve and induce tissue repair and growth. Platelets contains several molecules like the alpha-granules that are rich in GFs such as the transforming growth factor-β (TGF-β), vascular endothelial growth factor (VEGF) and Platelet-derived growth factors (PDGFs) [39]. These factors play an important role in all phases of healing of the tissue by recruiting mesenchymal stem cells and also during the synthesis of the extracellular matrix. The combined procedure is safe with no relevant secondary reactions [40]. These derivatives alone or in combination with bone grafts and different biomaterials have been shown to promote bone growth and bone homeostasis, vascularization [41]. Our study was aimed to compare the long-term clinical outcome and complications of augmentation technique by using algae-derived plant hydroxyapatite (Algipore) as a bone grafting material for maxillary sinus floor augmentation (MFSA) versus demineralized anorganic bovine bone (Bio-Oss Geistlich Pharma, Wolhusen, Switzerland) with the support of autologous blood-derived PRP.”

1. I'm a little bit confusing for the aim of the study. Is it a comparative study between Porous Fluorohydroxyapatite and Anorganic Bovine Bone in combination with autologous PRP and if it is why they used 50% particulate in the test group and 50% autogenous bone in the control group?

We are sorry. It was just a transcription error from the draft. It has been corrected with “particulate autogenous”.

The references: Proussaefs, P., Lozada, J., & Rohrer, M. D. (2002). A clinical and histologic evaluation of a block onlay graft in conjunction with autogenous particulate and inorganic bovine mineral (Bio-Oss): a case report. International Journal of Periodontics & Restorative Dentistry, 22(6); Marx, R. E., Miller, R. I., Ehler, W. J., Hubbard, G., & Malinin, T. I. (1984). A comparison of particulate allogeneic and particulate autogenous bone grafts into maxillary alveolar clefts in dogs. Journal of Oral and Maxillofacial Surgery, 42(1), 3-9.

1. The study design is flow and the images provided are not helpful in clarifying what the authors intended to produce.

We aimed was to compare two bone grafting materials employed to regain lost alveolar structures, and the long-term effect on osteointegration and peri-implant bone density. The images, in our opinion, are sufficient to demonstrate the technique applied and results. Images demonstrated ample bone healing at reentry as early as 4 months.

Images show that radiographic integration and increased peri-implant bone density were maintained at seven years.

1. Where are the methods of evaluation, just radiographs even without analysis?

The long-term stability of implants, as demonstrated by radiographs, represents our analysis.

1. Overall the manuscript should be edited by a native speaker.

Thank you so much. The manuscript has been reviewed.

Reviewer 4 Report

The paper titled “Long-Term Outcomes of Implants Placed in Maxillary Sinus Floor Augmentation with Porous Fluorohydroxyapatite (Algipore® FRIOS®) in Comparison with Anorganic Bovine Bone (Bio-Oss®) and platelet rich plasma (PRP)”, by Biagio Rapone et al. is, in my opinion, not sufficiently sound for publication in the journal. The following suggestions should be considered:

1. The “Introduction”section should be reorganized as it presents very poor readability. And the reason for using porous FHA Algipore® FRIOS® biomaterial as implant should be explained in more detail.
2. Some of the results should be presented in the “ Results”section rather than in the “2. Materials and Methods” section.
3. The advantages of porous biomaterial should be elaborated in detail in the “4. Discussion”
4. The “5. Conclusions”section should be summarized.
5. This is true for the application of HA as implant for bone graft therapy, see e.g. Bioactive Materials, 2021, 6:490-502; Reactive and Functional Polymers, 2021, 160: 104841. This important information should be integrated into the paper.

Author Response

REVIEWER 4:

The paper titled “Long-Term Outcomes of Implants Placed in Maxillary Sinus Floor Augmentation with Porous Fluorohydroxyapatite (Algipore® FRIOS®) in Comparison with Anorganic Bovine Bone (Bio-Oss®) and platelet rich plasma (PRP)”, by Biagio Rapone et al. is, in my opinion, not sufficiently sound for publication in the journal. The following suggestions should be considered:

1. The “Introduction” section should be reorganized as it presents very poor readability. And the reason for using porous FHA Algipore® FRIOS® biomaterial as implant should be explained in more detail.

Thank you so much for your observations. The introduction section has been corrected as follows: “The sinus augmentation to enhance the bone volume and allow for proper implant placement in the atrophic posterior maxilla, remains a challenge in regenerative surgery [1-3]. The primary goal of bone reconstructive surgery is to achieve new functional bone regeneration via the synergistic combination of placing bone grafting materials, cells, and growth factors [2,4-7]. Bone grafting remains the most commonly used surgical procedure to replace bone defects. It supports the repairing process by promoting osteoinduction, osteoconduction and osteogenesis [5,7-9]. Architectural and microstructural properties, and biocompatibility are critical to promote favorable tissue responses [8]. The advantages of the autologous bone grafting technique include the presence the patient’s own osteogenic cells and osteoinductive proteins, such as bone morphogenetic protein 2 (BMP2), and platelet-derived growth factor (PDGF) which provide optimal aforementioned properties without any risk [6, 10-12]. To date, several biomaterials to modulate in situ tissue regeneration have been designed [13, 14]. Bone regeneration consists of transitional steps in which the formation of soft, fibrous connective tissues, cartilage, and woven bone provide the mechanical stability that ultimately leads to bone formation, supporting the scaffold for cell and tissue differentiation [15, 16]. The rationale for any bone biomaterial as a scaffold or graft is not only to replace but to restore function to the physiologically and anatomically replaced part while ensuring complete biocompatibility without causing rejection and collaterals [17-21]. In addition, the optimal bone graft material must assume the role of a bone substitute and respond to various bio-mechanical stresses. Much of the current research has focused on the identification of biomaterials; specifically compounds for sinus floor augmentation [22-26]. The porous fluorohydroxyapatitic (FHA) biomaterial as shown promise as candidate in suitable biomaterial for sinus grafting in severely atrophic maxillae [17-21, 27]. Algipore® FRIOS® is a biomaterial, vegetable-based hydroxyapatite. It is highly analogous to the hydroxyapatite of natural bone and is manufactured from lime-impregnated red marine algae (Corallina officinalis) [28]. The biomaterial is processed through phases involving the pyrolytic segmentation of the native algae and by hydrothermal transformation of calcium carbonate [CaCO3] into FHA [Ca5(PO4)3OHxF1 x]. The structural features of 3D porous particles have shown a hierarchical pore system containing particles with a mean diameter of pores 10 mm periodically septated (mean interval 30 mm) and interconnected by microperforations of 1-4µm. Every pore is limited by one layer of small FHA crystallites with a size of 25–35 nm. he average pore volume decreased from 1.05 cm3/g to 0.93 while the surface area averages 50 m2 /g. Bio-Oss is consists of natural inorganic bovine bone which have shown faster healing process compared to other more conventional procedures applied in soft and hard tissue regeneration [29]. The addition of bovine bone mineral or nonresorbable porous hydroxyapatite to autogenous bone augments bone formation and bone-to-implant contact in augmented sinuses. The morphology of existing bone defect is determinant in the outcome of reconstructive bone surgery. On the other hand, the graft should allow for osteoinduction, osteoconduction, and endogenous osteogenesis of the recipient with adequate vascularity at the implant-bone interface [30-35]. Adequate mechanical stability of the graft or scaffold is also an important point for bone healing and repair. However, both macroscopic and microscopic characteristics of dental implants could influence osseointegration and the success of implant-prosthetic procedures [36-37]. The use of platelet concentrate is at the center of a recent academic debate [38]. Several results have demonstrated the functionality and support of platelet concentrates such as platelet rich plasma (PRP), and platelet rich fibrin (PRF), as key components in combination with bio-grafts to promote wound healing during and following surgery [34, 38]. Autologous PRP have shown to be a solid therapeutic method to improve and induce tissue repair and growth. Platelets contains several molecules like the alpha-granules that are rich in GFs such as the transforming growth factor-β (TGF-β), vascular endothelial growth factor (VEGF) and Platelet-derived growth factors (PDGFs) [39]. These factors play an important role in all phases of healing of the tissue by recruiting mesenchymal stem cells and also during the synthesis of the extracellular matrix. The combined procedure is safe with no relevant secondary reactions [40]. These derivatives alone or in combination with bone grafts and different biomaterials have been shown to promote bone growth and bone homeostasis, vascularization [41]. Our study was aimed to compare the long-term clinical outcome and complications of augmentation technique by using algae-derived plant hydroxyapatite (Algipore) as a bone grafting material for maxillary sinus floor augmentation (MFSA) versus demineralized anorganic bovine bone (Bio-Oss Geistlich Pharma, Wolhusen, Switzerland) with the support of autologous blood-derived PRP.”

1. Some of the results should be presented in the “Results” section rather than in the “2. Materials and Methods” section.

The correction has been applied as follows:

At 4 months after the implant placement, radiographic images showed complete osseointegration of the implant (Fig. 11-12, 13-14). We recorded perforation of the sinus membrane during sinus lift during two surgeries, without compromising the surgery and subsequent implantation. The lack of osseointegration distinguished by implant mobility and radiological radiolucency were referred to a failing implant. The orthopantomography checkup show radiographic integration and increased peri-implant bone density maintained at seven years (Fig. 17-18- 19-20).

Fig. 17-18 Follow-up at 7 years implants placement (Test Group, left); (Control Group, right).

Fig. 19-20 Rx at 7 years implants placement (Test Group, left); (Control Group, right).

1. The advantages of porous biomaterial should be elaborated in detail in the “4. Discussion”

The advantages of porous biomaterial have been added as follows: Porous phycogenic hydroxyapatite (PHA) derived from red algae (Algipore®), is largely employed as scaffolds in bone regeneration, because of its chemical similarity to bone and interconnected porosity [42-43]. Bone ingrowth is affected by several mechanical properties of the scaffold, involving the internal porous structure [44]. The pour size influences the permeability and the inadequate dimensions may result in altered bone ingrowth. There are sevral studies conducted on the fluorohydroxyapatite (FHA) FRIOSs Algipores as a proper biomaterial for the reconstruction of severely atrophic maxillae [45-47]. Schopper et al. investigated the histomorphological and histomorphometrical examination of 69 trephine specimens who were submitted to maxillary sinus grafting with FRIOSs Algipore [48]. The authors demonstrated that the scaffold elicited generation of new bone in the grafted sinuses, about 23.0% over an observation time of 7 months. The findings accord with other authors who found a comparable bone formation after 6-7 months, combining porous hydroxyapatite and autogenous bone for sinus grafting. Deproteinized bovine bone Bio-Oss is biocompatible and osteoconductive, while is missing of osteoinductive property [49]. Sartori et al.[50] observed a slow but continuous resorption of the Bio-Oss scaffold. These results were contrasting with Schlegel et al. [51] who reported a low resorption capacity of deproteinized bovine bone. The present investigation compared the FHA biomaterial FRIOSs Algipore and deproteinized bovine bone in triggering the formation of new bone in the grafted sinuses of severely atrophic maxillae. We noticed a significant increase in the new bone formation in the areas augmented with both biomaterials. Our findings are in agreement with other authors using a combination of porous hydroxyapatite or deproteinized bovine bone and autogenous bone for sinus grafting [52-58].

The maxillary bone in edentulous upper premolars and molars undergoes atrophy and pneumatization of the maxillary sinus, which decreases the amount of bone in horizontal and vertical dimensions, making it insufficient for implant placement. Sinus elevation via a lateral approach was applied. It is classified as a technique-sensitive procedure due to the high risk of Schneiderian membrane perforation that can occur quite frequently up to 35%, in this procedure piezosurgery offers a positive long-term prognosis and a higher survival rate than the placement of ungrafted maxillary, and in particular, rough-surface implants [59-61].

The split crest procedure was only performed with very fine chisels as the use of burs could compromise bone preservation. According to Misch and Judy [44] the use of this procedure, with type C bone defect should respect a crestal width between 1.5 and 2.5 mm with a height ranging between 8 and 12 mm. Implant placement in the ideal prosthetic position may be compromised by bone resorption due to the presence of an increased interarch distance or an unfavorable horizontal and sagittal intermaxillary relationship [62-64]. It was necessary to subject resorbed ridges to regeneration treatment before or concurrently with implant placement in order to increase the amount of hard and soft tissues [65-68]. This allowed us to reduce the crown-to-implant ratio, place axial implants, and achieve good occlusion and a quality aesthetic appearance. The alveolar ridge split is a predictable and reliable procedure, characterized by its low invasiveness. This procedure allowed us to achieve significant bone augmentation in the horizontal plane. In the case were we registered vertical bone lost,before to proceed to implantation, we performed bone augmentation according to Khoury’s concept. The bone was collected from retromolar area to reconstructed the vertical defects. Platelet-rich fibrin (PRF) is an autologous platelet concentrate obtained by centrifugation from the patient's own blood without the use of heparin or anti-coagulants. Like PRP, the PRF procedure is easy, safe, and biocompatible. The results suggested the potential role of PRF in periodontal regeneration and tissue bioengineering as a viable material for bio-graft construction. PRF is a material composed mainly of fibrin membranes enriched with platelets and growth factors optimal for promoting the healing process of hard and soft tissues. Thus, PRF is able to regulate inflammation and stimulate the chemotaxis mechanism. In addition, its gelatinous consistency increases the stability of the clot and graft material. However, being a biomaterial formed directly from the patient's blood, the amounts that can be obtained may sometimes be very modest. PRF has the distinguishing trait of polymerizing naturally and slowly during centrifugation. The concentrations of active thrombin and fibrinogen contained in PRF are almost within normal physiologic ranges because the material does not require any addition of bovine or humanized thrombin. Fibrin tends to acquire a three-dimensional structure equivalent to the site where it is inserted supporting the healing process [47-55]. Aggregation of fibrin monomers leads to the formation of a three-dimensional scaffold, forming a thin mesh of soft, porous graft that allows rapid cell colonization of the wound and surrounding tissues [56-61]. This type of bio-scaffold induces a faster physiological healing process and in combination with bone grafting accelerates the formation of new bone tissue [62-65]. Derivatives from different species, usually ovines, undergo a series of tests and processes of demineralization, sterilization, freeze-drying. Although widely used xenografts perform similar osteoconductive activity and are relatively cheaper. In addition, their use reduces the need for a second surgery for bone harvesting [13, 66-69]. However, xenografts have demonstrated a low ability to induce adequate height and width in large defects, especially those of bovine origin. Few results from histomorphometric analysis showed low resorption rate of transplants after several years revealed residual bovine graft up to 40%, data confirmed by several histo-analyses that reported the same amount of graft detected at 3 years to that at 6 months [51, 70-71]. Our study has some limitations: firstly, the study design with intrinsic restriction; secondly, histological examination are needed to reveal a physiologic framework of bone around the biomaterial particles; finally, the inclusion criteria were stringent and eliminated interferences due to systemic disease and other factors that may alter recovery. It follows that while the adoption of mimicry approaches has finally yielded positive results, one must consider the interference of multiple variables such as physical, biochemical, metabolic, immunological, and hormonal conditioning. There are huge differences between an inserted bone graft and a mature healthy tissue microenvironment, but even more so there are crucial changes between an inserted graft and the current health status of the recipient [47,55,72-74]. Consequently, a different intervention should be planned by adapting not only the design of subsequent implants, but emphasizing the treatment plan that fully reflects these differences. Normal healthy adult development occurs in a variety of immunologic, inflammatory, hormonal, and metabolic contexts; the complexity of these factors must necessarily be addressed if the processes are to be united for complete and successful integration of bone grafts and implants [7,47]. Endocrine signaling gradients that function on a scale of healthy conditions can be subverted in a highly deteriorated situation. Modular implants, including those with smaller units including GFs and cells, can be subjected to the unfavorable internal cellular and molecular microenvironment and eventually can be altered leading to infection, necrosis, and ultimately rejection. The immune-endocrine-metabolic environment that modulates the entire process of regeneration, growth, and remodeling and regulates the influx of cells, molecules, and GFs into growing young and adult bone has yet to be fully elucidated. This is probably a crucial time if we are fully committed to unraveling the potential of evolving bioengineering and regenerative medicine, as immune-endocrine-metabolic factors are significant mediators of bone healing and regrowth or, conversely, can cause a delay in healing if they are suppressed and neglected [57,60,73-75]. This last observation serves to highlight the differences between the developmental processes that occur during normal osteogenesis and those involved in the induction of post-traumatic grafting. Indeed, while inflammation, endocrine imbalances, and metabolic dysfunction may be part of the main drivers of bone decay and graft failure, they are fully functional during normal bone development. The significance of interleukins, cytokines, and hormones in the revascularization, mineralization, and bone/cartilage remodeling activities of hPB-SCs has been profoundly elucidated, and their important role is fully appreciated as external supporters in bone graft therapy [7,9, 47]. Our study showed overlapped results over the techniques. However, our findings are not conclusive. The study design and the sample size represent the major limitations.

1. The “5. Conclusions” section should be summarized.

The conclusion section has been summarized as follows: At 7 years follow up, the combination of the particulate autogenous bone with Algipore® FRIOS® or Bio-Oss® showed predictable results over the time in the presence of a small amount of residual bone. However, further studies are needed to confirm the hypothesis.

1. This is true for the application of HA as implant for bone graft therapy, see e.g. Bioactive Materials, 2021, 6:490-502; Reactive and Functional Polymers, 2021, 160: 104841. This important information should be integrated into the paper.

We are sorry, but we have not identified the references.

Please do not hesitate to contact me if any questions or concerns remain.

We thank you for your consideration.

With kind regards,

Dr. Biagio Rapone, DDS, PG Oral.Surg., MSc, PhD

Interdisciplinary Department of Medicine, University of Bari “Aldo Moro”, Bari, Italy

Piazza Giulio Cesare 11, 70124 Bari, Italy

E-mail: [email protected]

Round 2

Reviewer 1 Report

After the changes made the article may be suitable for publication.

Author Response

After the changes made the article may be suitable for publication.

Thank you so much.

Reviewer 2 Report

Thank you for doing the suggested corrections.

One thing that must be done before the manuscript is accepted though is to be read, edited, and refined by a native English speaker. The number of typos and sentences that don't read well is just too much right now for the article to be accepted.

Thanks

Author Response

Thank you for doing the suggested corrections.

One thing that must be done before the manuscript is accepted though is to be read, edited, and refined by a native English speaker. The number of typos and sentences that don't read well is just too much right now for the article to be accepted.

Thanks

Thank you so much for your evaluation. Our expert in scientific English corrected the manuscript. The manuscript has been resubmitted.

Reviewer 3 Report

No further comments

Author Response

No further comments

Thank you so much.