Bio-Compatibility Analysis of Newly Developed Plug and Cuff Electrodes for Future Neuronal Interface Applications

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript reports ISO/ASTM-guided biocompatibility assessments of newly developed plug and cuff electrodes intended for neural interface applications within the NerveRepack program. The topic is clinically relevant, and the authors present a well-organized sequence of cytotoxicity, hemolysis, complement activation, and genotoxicity assays. The regulatory framing of the study is clear, and the manuscript succeeds in documenting early in vitro safety characteristics of the tested devices.

However, the present work remains limited to extract-based biocompatibility testing and provides insufficient engineering, mechanistic, and translational context for readers to assess whether the electrodes are suitable for neural implantation. Critical design details are deferred to a future paper, electrical and mechanical characterization is absent, the biological models only partially reflect the peripheral nerve environment, and complement activation findings for the plug electrode are not adequately analyzed. Furthermore, the manuscript concludes with claims of clinical applicability that are not supported by the data presented. Finally, as submitted to Biomimetics, the work does not articulate a biomimetic or bioinspired design principle, raising concerns about journal–scope alignment.

For these reasons, major revision is required before the manuscript can be considered for publication. Detailed comments follow:

 

Comment 1| The manuscript does not sufficiently differentiate the proposed electrodes from established cuff and penetrating designs. To allow readers to evaluate novelty, please provide a concise comparative analysis of existing neural electrodes, articulate the design rationale behind material stack choices, geometries, and mechanical objectives, and state explicitly what limitations of prior devices the new design resolves.

 

Comment 2| Extract-based assays alone do not replicate the physiologic implant environment, which includes direct tissue contact, micromotion, protein adsorption, material degradation, and chronic exposure. Direct-contact assays, extended exposure paradigms, or, if new experiments are not possible, a clear Statement of Limitations explaining why extract-only data have restricted predictive value for neural interfaces is required.

 

Comment 3| Chronic neural implants commonly elicit macrophage activation, cytokine release, and perineural fibrosis. The manuscript should incorporate an inflammatory assay (e.g., macrophage response, cytokine panel, fibrosis markers) or provide a systematic discussion describing expected immunological risks, material–immune interactions, and plans for follow-up validation.

 

Comment 4| Neural interfaces require baseline stability in impedance, charge-storage capacity, and charge-injection limits, as well as mechanical durability under strain, bending, and cyclic fatigue. Please include electrochemical impedance spectroscopy (EIS), CSC/CIL metrics, delamination resistance, bending-strain profiles, or long-term soak data. Without these measurements, readers cannot evaluate functional readiness.

 

Comment 5| The selected cell lines (SH-SY5Y, NHDF, HaCaT, CHO) do not sufficiently represent the biology of peripheral nerves. Please incorporate Schwann cell or dorsal-root-ganglion (DRG) neuron models, or provide a structured discussion explaining how these physiologically relevant systems will be included in subsequent studies and why their absence limits interpretation.

 

Comment 6| The first extract from the plug electrode demonstrates measurable cytotoxicity, and complement activation occurs at levels comparable to the positive control. Please investigate potential causes (residual solvents, incomplete rinsing, surface chemistry, Ti/Au adhesion interactions) and specify corrective actions (cleaning protocol, fabrication modification, surface passivation) before asserting safety.

 

Comment 7| Given the absence of in vivo data, chronic implantation analysis, electrophysiological function tests, and mechanical durability studies, the concluding statement that the electrodes “can be used in clinical applications” is premature. Please revise to state that the findings represent an initial in vitro safety screen and outline the required preclinical steps (in vivo biocompatibility, fibrosis assessment, nerve conduction/stimulation validation).

 

Comment 8| The Methods section includes extensive procedural details for well-established assays while providing minimal information on the electrode design. Please condense routine assay descriptions and expand the device characterization section (schematics, dimensions, surface morphology, material interfaces, fabrication parameters).

 

Comment 9| The Introduction offers general context but does not clearly define the scientific gap the present work addresses. Similarly, the Discussion should explicitly acknowledge limitations—extract-only testing, limited cell models, absence of function—and offer a coherent roadmap for future work.

 

Comment 10| Although neural interfaces can fall within bio-inspired engineering, the manuscript does not present a biomimetic or bioinspired design mechanism (e.g., structural mimicry, stress-distribution strategies, biologically inspired architectures). Instead, the work reads primarily as an ISO-compliant biocompatibility report without a biomimetic conceptual foundation. To strengthen suitability for the journal, please explicitly define the bioinspired principle underlying the electrode, describe how the design leverages biological architecture or function, or reframe the narrative to clarify the biomimetic contribution.

Comments for author File: Comments.pdf

Author Response

Reviewer 1

First, we would like to thank the Reviewer for the time and effort to review our manuscript and provide us with constructive comments.

 

Comments:

This manuscript reports ISO/ASTM-guided biocompatibility assessments of newly developed plug and cuff electrodes intended for neural interface applications within the NerveRepack program. The topic is clinically relevant, and the authors present a well-organized sequence of cytotoxicity, hemolysis, complement activation, and genotoxicity assays. The regulatory framing of the study is clear, and the manuscript succeeds in documenting early in vitro safety characteristics of the tested devices.

However, the present work remains limited to extract-based biocompatibility testing and provides insufficient engineering, mechanistic, and translational context for readers to assess whether the electrodes are suitable for neural implantation. Critical design details are deferred to a future paper, electrical and mechanical characterization is absent, the biological models only partially reflect the peripheral nerve environment, and complement activation findings for the plug electrode are not adequately analyzed. Furthermore, the manuscript concludes with claims of clinical applicability that are not supported by the data presented. Finally, as submitted to Biomimetics, the work does not articulate a biomimetic or bioinspired design principle, raising concerns about journal–scope alignment.

For these reasons, major revision is required before the manuscript can be considered for publication. Detailed comments follow:

 

Comment 1| The manuscript does not sufficiently differentiate the proposed electrodes from established cuff and penetrating designs. To allow readers to evaluate novelty, please provide a concise comparative analysis of existing neural electrodes, articulate the design rationale behind material stack choices, geometries, and mechanical objectives, and state explicitly what limitations of prior devices the new design resolves.

 

Answer:

We have now added detailed information on the background of plug and cuff electrodes and their rational for construction. In Table 1 and 2 the key differences between today’s gold standard cuff electrodes (manufactured by subtractive laser processing of platinum sheet or wire materials with silicon molding) and the new NerveRepack cuff electrode (manufactured by additive micro system technology processes based on a thermoplastic polyurethane carrier with gold electrodes and Nitinol as support material) are shown and outlined. Applying additive micro system technology with hybrid Nitinol support allows a very versatile design of significant thinner and smaller electrodes from soft thermoplastic polymers with other materials (e.g. gold electrodes) in micrometer precision. Further on, using gold as electrode material in the new cuff design will also allow future designs with significantly improved electrical characteristics (e.g. by applying high charge injection coatings such as modified PEDOT:PSS).

 

Comment 2| Extract-based assays alone do not replicate the physiologic implant environment, which includes direct tissue contact, micromotion, protein adsorption, material degradation, and chronic exposure. Direct-contact assays, extended exposure paradigms, or, if new experiments are not possible, a clear Statement of Limitations explaining why extract-only data have restricted predictive value for neural interfaces is required.

 

Answer:

We agree with the Reviewer that there are many limitations of this study. Nevertheless, we have used incubation times and conditions as described in the protocols from ISO and ASTM. We have also prepared a new section to address all the limitations of our study (Section 4 “Limitations of the study”).

 

Comment 3| Chronic neural implants commonly elicit macrophage activation, cytokine release, and perineural fibrosis. The manuscript should incorporate an inflammatory assay (e.g., macrophage response, cytokine panel, fibrosis markers) or provide a systematic discussion describing expected immunological risks, material–immune interactions, and plans for follow-up validation.

 

Answer:

The limitations have been described in a new section (Section 4). Further, citation to a recent study Ionescu et al. 2024 Biosensor (new Ref 30) that describes the use of two plug electrodes in pigs has been given. No signs of inflammation or fibrosis were found after 10 days of implantation.

 

Comment 4| Neural interfaces require baseline stability in impedance, charge-storage capacity, and charge-injection limits, as well as mechanical durability under strain, bending, and cyclic fatigue. Please include electrochemical impedance spectroscopy (EIS), CSC/CIL metrics, delamination resistance, bending-strain profiles, or long-term soak data. Without these measurements, readers cannot evaluate functional readiness.

 

Answer:

We have conducted electrical and mechanical performance testing along with physical and functional testing and design verification. All electrodes (plug and cuff) passed the necessary requirements for longterm implant (LTI) based materials as they have been built and processed with proven medical grade processes and controlled LTI materials. Besides that, it is new to apply additive micro system technology processes similar materials are applied in cardiac rhythm mangement electrodes. Therefore, existing representative scientific literature can be used to justify our neural plug and cuff electrode technologies to demonstrate functional readiness for our concepts. Futher on, it is not the key intention of the current paper to focus on a complete biocompatibility evaluation of the overall implant device design at this early stage. Only that our biomedical tests demonstrate that our selected electrode implant platforms, materials and processes are justified. In order to align with this comment, however, the paper title and conclusion was slightly adopted, too.

 

Comment 5| The selected cell lines (SH-SY5Y, NHDF, HaCaT, CHO) do not sufficiently represent the biology of peripheral nerves. Please incorporate Schwann cell or dorsal-root-ganglion (DRG) neuron models, or provide a structured discussion explaining how these physiologically relevant systems will be included in subsequent studies and why their absence limits interpretation.

 

Answer:

We have now fully addressed this comment and demonstrate the limitations in lines 404-421.

 

Comment 6| The first extract from the plug electrode demonstrates measurable cytotoxicity, and complement activation occurs at levels comparable to the positive control. Please investigate potential causes (residual solvents, incomplete rinsing, surface chemistry, Ti/Au adhesion interactions) and specify corrective actions (cleaning protocol, fabrication modification, surface passivation) before asserting safety.

 

Answer:

We have now expanded this section (lines 486-495) to answer this comment as requested. A thoroughly cleaning protocol must be applied to remove potential residual solvents that are responsible for the observed cytotoxicity.

 

Comment 7| Given the absence of in vivo data, chronic implantation analysis, electrophysiological function tests, and mechanical durability studies, the concluding statement that the electrodes “can be used in clinical applications” is premature. Please revise to state that the findings represent an initial in vitro safety screen and outline the required preclinical steps (in vivo biocompatibility, fibrosis assessment, nerve conduction/stimulation validation).

 

Answer:

The paper outlined a first biocompatibility evaluation of representative key components within the plug and cuff electrodes. Not all biocompatibility testing for the overall implant systems have been defined and conducted in such an early stage (only physical and functional testing). This will be published at a later moment.

For plug electrodes as mentioned there are limited data on preclinical model (2 electrodes implanted in pigs) that shows promising results as no reduction in signal, no fibrosis and no inflammation were found.

Nevertheless, this statement (“can be used in clinical applications”) has been removed.

 

Comment 8| The Methods section includes extensive procedural details for well-established assays while providing minimal information on the electrode design. Please condense routine assay descriptions and expand the device characterization section (schematics, dimensions, surface morphology, material interfaces, fabrication parameters).

 

Answer:

This comment is highly related to Comment 1 and has been addressed.

 

Comment 9| The Introduction offers general context but does not clearly define the scientific gap the present work addresses. Similarly, the Discussion should explicitly acknowledge limitations—extract-only testing, limited cell models, absence of function—and offer a coherent roadmap for future work.

 

Answer:

The introduction has been expanded to include the scientific gaps that this work addresses for plug and cuff electrodes. The scientific gap is explained by the limits of current electrode technology (Table 1 and comment 1).

A new section (Section 4) that discusses the limitations of this study has been added.

 

Comment 10| Although neural interfaces can fall within bio-inspired engineering, the manuscript does not present a biomimetic or bioinspired design mechanism (e.g., structural mimicry, stress-distribution strategies, biologically inspired architectures). Instead, the work reads primarily as an ISO-compliant biocompatibility report without a biomimetic conceptual foundation. To strengthen suitability for the journal, please explicitly define the bioinspired principle underlying the electrode, describe how the design leverages biological architecture or function, or reframe the narrative to clarify the biomimetic contribution.

 

Answer:

We have decided to submit our paper in the journal Biomimetics due to the connection of the electrodes with the exoskeletons and/or exoprostheses that are biomimetic devices. Further, this paper nicely fits within the scope of the Special Issue entitled “Advances in Bio-Integrated Neural Interfaces: Materials, Devices, and Translational Applications”.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript presents the biocompatibility evaluation of two types of electrodes (plug and cuff). This work addresses a critical step in the translational pathway for neuroprosthetic devices. The Cuff electrode appears to be a promising candidate based on the results. However, the Plug electrode presents significant safety flags (initial cytotoxicity and high complement activation). Here are some suggestions for this research work.

1. Kindly recheck the accuracy of the error bars in the Figures.
2. The results (Figure 10) show that the plug electrode induces significant complement activation, at levels comparable to the positive control (cellulose membrane). The authors cannot conclude the device is biocompatible with this result standing unexplained. They must either: Provide literature justifying why this specific material/geometry triggers false positives in this assay. Modify the surface coating/material to reduce this activation.
3. The materials in this work were sterilized using "exposure to UV for 10 min per side. UV sterilization is generally considered a surface decontamination method and is often insufficient for implantable devices, especially those with complex geometries (like the plug electrode's needles) or porous materials (potential 3D printed parts). It has poor penetration. The author should provide detailed information on the sterilization process and verify its compliance with relevant standards.
4. The methods section mentions HaCaT cells, and the introduction mentions testing against keratinocytes. However, the results only show HaCaT data for the cuff electrode (Figure 8). Did the authors test the Plug electrode on HaCaT? Even if the plug is fully implanted, the surgical insertion involves skin contact. If it was tested and omitted, or not tested at all, this should be clarified.

Author Response

Reviewer 2

First, we would like to thank the Reviewer for the time and effort to review our manuscript and provide us with constructive comments.

 

Comments:

This manuscript presents the biocompatibility evaluation of two types of electrodes (plug and cuff). This work addresses a critical step in the translational pathway for neuroprosthetic devices. The Cuff electrode appears to be a promising candidate based on the results. However, the Plug electrode presents significant safety flags (initial cytotoxicity and high complement activation). Here are some suggestions for this research work.

1. Kindly recheck the accuracy of the error bars in the Figures.

Answer:

We have carefully checked the error bars in all Figures and they appear to be accurate and correct.

 

1. The results (Figure 10) show that the plug electrode induces significant complement activation, at levels comparable to the positive control (cellulose membrane). The authors cannot conclude the device is biocompatible with this result standing unexplained. They must either: Provide literature justifying why this specific material/geometry triggers false positives in this assay. Modify the surface coating/material to reduce this activation.

 

Answer:

We would like to thank the Reviewer for pointing out this. We have now provided relevant discussion and citations.

 

1. The materials in this work were sterilized using "exposure to UV for 10 min per side. UV sterilization is generally considered a surface decontamination method and is often insufficient for implantable devices, especially those with complex geometries (like the plug electrode's needles) or porous materials (potential 3D printed parts). It has poor penetration. The author should provide detailed information on the sterilization process and verify its compliance with relevant standards.

 

Answer:

The UV-based sterilization process was agreed between partners and was prepared in collaboration with doctors that are using this method to sterilize implants. Further, in the revised version we have added relevant citation.

 

1. The methods section mentions HaCaT cells, and the introduction mentions testing against keratinocytes. However, the results only show HaCaT data for the cuff electrode (Figure 8). Did the authors test the Plug electrode on HaCaT? Even if the plug is fully implanted, the surgical insertion involves skin contact. If it was tested and omitted, or not tested at all, this should be clarified.

 

Answer:

We had a limited number of plug electrode prototypes. Since the plug electrode will not remain in skin contact for a long time, testing the biocompatibility against fibroblasts was the most important part as fibroblasts are present everywhere.

 

 

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Authors have done evaluation study for their electrodes developed for neural implants and neural interface applications. They have designed electrodes for plug and cuff mechanism whereas here only biocompatability part were analysed hence title may be revised as bio-compatability analysis of the developed plug and cuff electrodes. As indirect methods was established along with hemocompatability to study the complaiance, authors need to make an compartitive analysis table for ISO and ASTM standards to thier nearby polymeric materials. It is need to add the table in discussion part and more detail required for Figure 11 & 12. 

Author Response

Reviewer 3

First, we would like to thank the Reviewer for the time and effort to review our manuscript and provide us with constructive comments.

 

Comment:

Authors have done evaluation study for their electrodes developed for neural implants and neural interface applications. They have designed electrodes for plug and cuff mechanism whereas here only biocompatability part were analysed hence title may be revised as bio-compatability analysis of the developed plug and cuff electrodes. As indirect methods was established along with hemocompatability to study the complaiance, authors need to make an compartitive analysis table for ISO and ASTM standards to thier nearby polymeric materials. It is need to add the table in discussion part and more detail required for Figure 11 & 12.

 

Answer:

We have modified the title as requested. The new title is “Bio-compatibility analysis of newly developed plug and cuff electrodes for future neuronal interface applications”. Furthermore, the legends of Figure 11 and 12 have been revised accordingly to provide greater detail in the revised version of the manuscript. Finally, a Table with a synopsis on ISO and ASTM standards has been added in the conclusion part as requested.

 

 

 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

With this revision, the major concerns have been adequately addressed and the overall quality of the manuscript has improved substantially. Overall, the revisions are well executed, and I believe the paper is ready to proceed to publication.

Comments on the Quality of English Language

Overall English quality is good and the text is readily understandable. I recommend minor language edits for consistency.