Review Reports - Factors That Determine the Adhesive Strength in a Bioinspired Bone Tissue Adhesive

Round 1

Reviewer 1 Report

Thank you for submitting this interesting manuscript. Pujari-Palmer et al evaluate the adhesion strength of phosphoserine modified cements (PMCs) by failure mode, scatter plots and regression analysis of several factors. According to their testing PMCs are relative insensitive to the adherent surface properties and not affected by uneven bond lines. In this study the authors report a strong correlation between failure mode and PMC adhesive strength, which was unexpected since prior studies suggested a poor correlation of this factors.

Comments

The authors didn´t describe the oxygen amount during the experiments. Since bone and cartilage are rather hypoxic environments and assuming the authors used 21% during their experiments, do they think there is an influence of oxygen concentration on curing time and stability of PMCs?

The authors declare “the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.” In the authors contributions they describe the conceptualization, metholody, validation, investigation, data curation visualization etc. was done by one or more of the funders of the company GPBio. Please clarify.

Line 67: “… produce a thin, even bond line (300g)” Please explain the abbreviation.

In table 1 the authors describe the experimental layout. Since all tested curing environments didn´t contain proteins like proteases or even amino acids, the reviewer suggests the use of a protein based buffer. Please discuss why you used the mentioned buffers as surrounding environment. At the end of the discussion (line 582ff) the authors quickly mention this problem, which needs to be expanded.

For shear testing the authors used steel and aluminium cubes. Please explain shortly why metal was used in this test setup. Would bone or a bone like structures (for example Dentin) be more suitable for this test system?

The authors explain the concern that adhesives can impair bone healing (line 536f). How can this concern be tested ex vivo or in vitro? Can the authors cite in vitro data of PMCs tested for example in cell culture (osteoblasts, osteoclasts)?

The authors explain the mismatch between the optimal laboratory test performance and the more unpredictable surgical conditions for bone adhesives. Do the authors think PMCs are suitable for animal model testing? Suitable animal models are available and this data could provide important information about PMC behavior in vivo.

Minor comments

Figure 2F: Scale bar would increase information about surface roughness

Figure 2G: Legend description (specially Aluminium (1.63µm) could be separated from graph for easier identification

X-axis labeling can be improved (for example: Figure 1C Average bond thickness, Figure 3A Cure type, Figure 3B Grip time, Figure 4B Phosphoserine (mole))

Author Response

Reviewer #1

Comments

The authors didn´t describe the oxygen amount during the experiments. Since bone and cartilage are rather hypoxic environments and assuming the authors used 21% during their experiments, do they think there is an influence of oxygen concentration on curing time and stability of PMCs?

Author response:

The authors thank the reviewer for this insightful question- the oxygen content has not been investigated prior. Since the cements are expected to be hand mixed in the clinic by a surgeon, for use on tissues, we expect the cement to encounter an oxygen rich environment up until it comes into contact with the bone or cartilage tissue. Thus far we have seen significant bonding strength between the adhesive and cartilage, and bone tissues, ex vivo (citation #10 in the manuscript), and to cancellous bone in vivo (ongoing work, not published yet). Therefore the evidence suggests that the oxygen content will not influence curing, but we cannot address concern in the present study. The authors elect to address this concern in the revised manuscript, in line 579 of the discussion section, with the following text: “It should ne noted that select tissues have low oxygen content, particularly cartilage. While PMCs have shown good bond strength to such tissues ex vivo [10], additional testing is needed to confirm whether the adhesive effect or setting reactions are affected by hypoxia.”

The authors declare “the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.” In the authors contributions they describe the conceptualization, metholody, validation, investigation, data curation visualization etc. was done by one or more of the funders of the company GPBio. Please clarify.

Author response:

The authors thank the reviewer for this insightful question as well, and clarify in the conflict of interests section with the following revised text: “The funders (SSF and EIT) had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.” The authors received funding only from SSF and EIT, not from the company holding the IP, or from any other sources. The authors that are part owners of GPBio did not contribute, or receive any funding for this study. Therefore, as indicated in the text, the only funding organizations had no role in designing the study. If the reviewer would prefer more information or clarity, the authors would readily add more text as the reviewer suggests.

Line 67: “… produce a thin, even bond line (300g)” Please explain the abbreviation.

Author response:

The authors thank the reviewer for catching this error. The 300g refers the applied force that can be expected from a surgeon, to establish a thin layer of adhesive during surgery. We have revised the text as follows: “with minimal pressure applied by the surgeon (300g or 3N) during fixation/ reconstruction to produce a thin, even bond line [22].”

In table 1 the authors describe the experimental layout. Since all tested curing environments didn´t contain proteins like proteases or even amino acids, the reviewer suggests the use of a protein based buffer. Please discuss why you used the mentioned buffers as surrounding environment. At the end of the discussion (line 582ff) the authors quickly mention this problem, which needs to be expanded.

Author response:

The reviewer raises a very good point. The authors considered the use of proteins in the curing buffer, to more closely mimic curing in physiological fluids. However reproducibility is of higher importance to this study than physiological relevance. Metal cubes were washed and cleaned between use, however exposure to protein containing fluids can significantly affect the surface properties, and adhesive bonding behavior. The following text has been added to the discussion section: “Despite rigorous surface cleaning methods, proteins are difficult to remove completely and leftover proteins can affect how the surface of the adhesive orients, nucleates, and bonds to the surface of the metal cube. Protein was avoided, as a possible source of variation, because the correlation model developed in the present study would require duplication of a large number of samples, to account for the effects of protein on the correlation model. It should be noted that protein rich bone cubes were used in the present study, and comparable bond strengths were obtained.” The authors also have an ongoing in vivo study where PMC has demonstrated comparable strength in vivo, on live bone, as in vitro and ex vivo. Based on this information, the authors suggest that the expected effect of proteins on the adhesive strength is minimal in vivo, and have elected to avoid proteins in the present study, but will certainly investigate them in future studies.

For shear testing the authors used steel and aluminium cubes. Please explain shortly why metal was used in this test setup. Would bone or a bone like structures (for example Dentin) be more suitable for this test system?

Author response

As mentioned in line 532, in reference to citation #10, we have previously shown that PMC bonds with comparable strength to metal and bone. The authors have added the following text in the discussion section: “While the present study sought to investigate and predict the behavior of PMC on tissues, large variability in the surface properties, sample preparation (i.e. cutting with band saw, polishing, etc.), and tissue architecture (variation between regions of the bone, within and between different animal tissue sources, etc.) could easily obscure the correlation results. Therefore, in the present study metal samples were used to ensure control of the surface properties, and reproducibility of the testing methods and results.” The authors also included bone samples to demonstrate that the bonding strength was comparable, and the likelihood that the current findings (correlation model, etc.) can be extended from metal to tissue adhesion. In future testing the authors will address these questions on bone tissue.

The authors explain the concern that adhesives can impair bone healing (line 536f). How can this concern be tested ex vivo or in vitro? Can the authors cite in vitro data of PMCs tested for example in cell culture (osteoblasts, osteoclasts)?

Author response

As mentioned in the introduction (line 43, citation 8, 10, 12-14), it has already been demonstrated that PMCs remodel in vivo, when implanted in bone. The authors acknowledge the reviewers’ point- this is a critical property for a proposed bone adhesive. However, the studies cited have in-vivo data showing that this is not a concern for PMCs; they resorb to form healthy, new bone tissue in vivo. Therefore, the authors have acknowledged this concern, but have focused instead on the physical behavior of the adhesive in this study. To address the reviewers’ concerns, the authors have added the following text to the introduction section: “Prior in vivo studies have demonstrated that PMCs are safe (no adverse reactions) and readily remodel into bone [8, 12-14]. Therefore, the focus of the present study was the physical (i.e. bond strength), rather than biological properties of PMCs.”

The authors explain the mismatch between the optimal laboratory test performance and the more unpredictable surgical conditions for bone adhesives. Do the authors think PMCs are suitable for animal model testing? Suitable animal models are available and this data could provide important information about PMC behavior in vivo.

Author response

Please see the answer to the prior reviewer comment. We have ongoing animal studies, two of which demonstrate no toxicity, and 1 of which shows measurable adhesive strength on screw pull-out testing, AND bone healing and replacement of adhesive with new bone (results are still being analyzed, so we cannot yet claim this in a publication until the results are verified and completely analyzed.

Minor comments

Figure 2F: Scale bar would increase information about surface roughness

Author response

Figure 2F has a scale bar on the X and Y axis of the picture. To clarify, the following text has been added to the Figure legend: “(F) Optical image (profilometer) of steel surface, with scale (0-1681 μm) indicated on the X- and Y-axis”

Figure 2G: Legend description (specially Aluminium (1.63µm) could be separated from graph for easier identification

Author response

Figure 2G: The figure has been revised as the review suggests. Thank you for this helpful suggestion.

X-axis labeling can be improved (for example: Figure 1C Average bond thickness, Figure 3A Cure type, Figure 3B Grip time, Figure 4B Phosphoserine (mole))

Author response

The authors have reviewed the suggested figures, but are unsure of what changes the authors is suggesting? Is the font not spaced appropriately? Is it the positioning of X-axis, or scale? Can the reviewer provide additional information. The figures appear easily legible and understandable at present, but we would be happy to revise if more information is provided.

Author Response File: Author Response.docx

Reviewer 2 Report

Well executed research and well written manuscript. Will provide excellent contribution to the literature.

Author Response

No comments provided by reviewer. We thank the reviewer.

Reviewer 3 Report

In this work the authors studied the failure mode of PMC adhesive with varied formulation, application method, adhesion surface profile and demonstrated that the failure mode strongly correlates to adhesive strength. This study is significant for identifying the determining factors of failure mode and fill the gap between lab study and medical practice.

Author Response

No comments provided by reviewer. We thank the reviewer.

Reviewer 4 Report

The article "Factors that determine the adhesive strength in a bioinspired bone tissue adhesive" is well written and experiments are sound. The adhesive properties of the bone tissue adhesive are very well presented and described. I recommend acceptance of the article in the present form for publication.

Author Response

No comments provided by reviewer. We thank the reviewer.

Round 2

Reviewer 1 Report

The authors reviewed the manuscript and answered all questions. This revisions increased the value of the manuscript which makes it suitable for publication in Chemengineering.

Short note to the last question:

"X-axis labeling can be improved (for example: Figure 1C Average bond thickness, Figure 3A Cure type, Figure 3B Grip time, Figure 4B Phosphoserine (mole))"

The x-achsis lable in Figure 1C "Average bond thickness" : The "g" in Average is not completly visible. The other figures are fine in the current version.