In Vivo Engraftment and Functional Efficacy of a 3D-Bioprinted Human Parathyroid Equivalent

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

1. The amount of data presented is extremely limited. Even the data corresponding to the results described in the Results section is insufficient. For example, there is a lack of qualitative and quantitative data on changes over time at 1 week, 2 weeks, 3 weeks, and 3 months after transplantation, as well as changes in dead cells, live cells, and PTH-positive cells. Furthermore, functional evaluation data on PTH secretion capacity should be presented to prove functional efficacy.
2. The numbering and labeling of figures do not match the main text or figure legends. There are several places in the main text where it is unclear which figures are being referenced, preventing readers from accurately understanding the results.
3. The majority of the discussion is limited to a review of general parathyroid surgical treatment, which is not directly related to this study. There is insufficient discussion based on the results of this study, and as a result, the novelty and academic significance of this study are not clearly conveyed. The discussion should be restructured to compare the results of this study with existing research and discuss its significance, limitations, and future challenges.

Comments on the Quality of English Language

The entire English text requires significant revisions. There are many grammatical errors and unnatural expressions, as well as noticeable variations in the spelling of abbreviations. Expert proofreading is recommended.

Author Response

Comment 1: The amount of data presented is extremely limited. Even the data corresponding to the results described in the Results section is insufficient. For example, there is a lack of qualitative and quantitative data on changes over time at 1 week, 2 weeks, 3 weeks, and 3 months after transplantation, as well as changes in dead cells, live cells, and PTH-positive cells. Furthermore, functional evaluation data on PTH secretion capacity should be presented to prove functional efficacy.

 

Response 1:

We thank the reviewer for this important and constructive critique. In response, a Limitations subsection has been added to the Discussion to explicitly acknowledge the constraints of the study. The Results section has been revised and reorganized, and previously missing histopathological data have been added to better illustrate time-dependent changes following implantation. In addition, Figure 4 has been completely redesigned to address the previously missing data and to provide a clearer and more comprehensive representation of the histopathological and immunohistochemical findings across the evaluated time points. While quantitative functional assays of PTH secretion were beyond the scope of this proof-of-concept study, this limitation is now clearly stated and discussed.

“Human parathyroid cells were successfully isolated as freshly isolated primary human parathyroid cells and subsequently incorporated into 3D-bioprinted constructs. Within the alginate-based matrix, the cells exhibited a uniform morphological appearance and were evenly distributed throughout the scaffold (Figure 1A–B).

Immunofluorescence staining demonstrated that the bioprinted human parathyroid cells expressed the parathyroid-specific marker calcium-sensing receptor (CaSR) following isolation and bioprinting (Figure 2). CaSR expression was localized to the cell membrane surrounding the nuclei, confirming the preservation of parathyroid-specific phenotypic characteristics and functional identity within the 3D constructs.

Cell-laden 3D-bioprinted scaffolds generated from primary human parathyroid cells were implanted subcutaneously into the dorsal flanks of nude mice under sterile conditions. The implanted constructs were explanted at predefined time points (1, 2, and 3 weeks post-implantation). With increasing implantation duration, a gradual increase in necrotic tissue was observed; however, the proportion of PTH-positive cells remained relatively stable during the first three weeks (Figure 4).

Histological examination at later stages revealed that the scaffold pores were progressively filled with calcified deposits consistent with dystrophic calcification. The original scaffold architecture was largely replaced by dense fibrotic tissue. Within this fibrotic stroma, histiocytes and occasional foreign body–type multinucleated giant cells were observed adjacent to calcified areas, indicating a chronic foreign body reaction and loss of long-term graft viability.

No marked difference in PTH immunostaining intensity was observed among the first, second, and third weeks post-implantation, indicating preserved functional activity during the early period. In contrast, at the three-month time point, prominent calcification and fibrotic tissue replacement were evident within the scaffold, accompanied by loss of PTH expression. These findings demonstrate maintained short-term functionality but limited long-term graft viability.”

 

Comment 2: The numbering and labeling of figures do not match the main text or figure legends. There are several places in the main text where it is unclear which figures are being referenced, preventing readers from accurately understanding the results.

Response 2:

We thank the reviewer for pointing this out. All figure numbering, labeling, and in-text references have been carefully reviewed and corrected. In particular, Figure 4 has been completely redesigned and its legend revised to clearly reflect the corresponding histopathological and immunohistochemical findings at each time point, ensuring full consistency between the main text and the figures.

 

Comment 3: The majority of the discussion is limited to a review of general parathyroid surgical treatment, which is not directly related to this study. There is insufficient discussion based on the results of this study, and as a result, the novelty and academic significance of this study are not clearly conveyed. The discussion should be restructured to compare the results of this study with existing research and discuss its significance, limitations, and future challenges.

Response 3:

We thank the reviewer for this important and constructive comment. In response, the Discussion section has been revised and reorganized to reduce the emphasis on general parathyroid surgical approaches and to focus more directly on the results of the present study. A new discussion segment has been added to compare our findings with the existing literature on 3D bioprinting and endocrine tissue engineering, highlighting that previous studies were largely limited to in vitro models and that comparable in vivo data on 3D-bioprinted human parathyroid constructs are scarce.

 

The revised Discussion now explicitly addresses the short-term preservation of PTH expression, the time-dependent loss of graft functionality due to calcification and fibrosis, and the translational implications of these findings. In addition, a dedicated Limitations subsection has been added at the end of the Discussion to clearly outline methodological constraints and future challenges related to scaffold design, long-term graft viability, and host–graft interactions.

 

 

Comments on the Quality of English Language

The entire English text requires significant revisions. There are many grammatical errors and unnatural expressions, as well as noticeable variations in the spelling of abbreviations. Expert proofreading is recommended.

 

Response:

We thank the reviewer for this comment. The manuscript has undergone comprehensive language editing, and we have arranged for professional English proofreading through the MDPI English Editing Service to address grammatical issues, improve clarity, and ensure consistency in terminology and abbreviation usage throughout the text.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript, titled "In Vivo Engraftment and Functional Efficacy of a 3D-Bioprinted Human Parathyroid Equivalent," presents a compelling study on the development of a bioengineered solution for post-surgical hypoparathyroidism. The authors address a significant clinical challenge by utilizing advanced 3D bioprinting technology to create functional human parathyroid tissue substitutes.

Overall, the work is scientifically sound and well-structured. The methodology for isolating primary human parathyroid cells and integrating them into an alginate-based bioink is clearly described and follows established protocols in tissue engineering. 

The conclusions are realistic and well-grounded in the observed data.

Minor Points for Revision:

1) Animal Sample Size: In the Materials and Methods section, the authors describe the implantation of constructs into CD1 athymic mice; however, the specific number of mice used for the study is not mentioned.

Please clarify:

The total number of CD1 athymic mice used (n)

The number of mice euthanized at each specific interval (n per time point)

Whether each mouse received a single scaffold or multiple constructs.

2) The authors state in the Results (lines 237-238) that human parathyroid cells were "successfully isolated, expanded in culture, and subsequently incorporated into 3D bioprinted constructs." However, the timeline provided in the Materials and Methods (lines 122–131) explicitly states that the entire process took only 9 hours. A 9-hour window allows for cell isolation and bioprinting but precludes any biological cell expansion.

The authors must correct the statement in the Results. The cells should be described as "freshly isolated primary cells" rather than "expanded" cells. This distinction is vital, as the behavior of primary cells versus expanded (passaged) cells can differ significantly in tissue engineering applications.

3) The specific cross-linking agent and its concentration (0.5 M CaCl2​) should be clearly stated in the Materials and Methods section rather than appearing for the first time in the Discussion.

Author Response

Comment 1: This manuscript, titled "In Vivo Engraftment and Functional Efficacy of a 3D-Bioprinted Human Parathyroid Equivalent," presents a compelling study on the development of a bioengineered solution for post-surgical hypoparathyroidism. The authors address a significant clinical challenge by utilizing advanced 3D bioprinting technology to create functional human parathyroid tissue substitutes.

Overall, the work is scientifically sound and well-structured. The methodology for isolating primary human parathyroid cells and integrating them into an alginate-based bioink is clearly described and follows established protocols in tissue engineering. 

The conclusions are realistic and well-grounded in the observed data.

Reponse 1: We thank the reviewer for the careful evaluation of our manuscript and for the positive assessment of the scientific rationale, methodology, and conclusions of the study. We appreciate the constructive suggestions, which have helped us improve the clarity and methodological transparency of the manuscript. All comments have been addressed as detailed below.

Minor Points for Revision:

Comment 1: Animal Sample Size: In the Materials and Methods section, the authors describe the implantation of constructs into CD1 athymic mice; however, the specific number of mice used for the study is not mentioned.

Please clarify:

The total number of CD1 athymic mice used (n)

The number of mice euthanized at each specific interval (n per time point)

Whether each mouse received a single scaffold or multiple constructs.

Response 1: We thank the reviewer for highlighting this important point. The Materials and Methods section has been revised to clearly specify:

• The total number of CD1 athymic mice used in the study (n = 1),
• The number of animals euthanized at each predefined time point (n 1), and
• That each mouse received a single 3D-bioprinted parathyroid construct.

These details have now been explicitly added to improve methodological transparency and reproducibility.

“A total of five 8-week-old male CD1 athymic nude mice (18–20 g) were used in this proof-of-concept in vivo study. Four mice received cell-laden 3D-bioprinted parathyroid scaffolds, with one animal allocated to each predefined post-implantation time point (1 week, 2 weeks, 3 weeks, and 3 months). Each mouse received a single scaffold implanted subcutaneously into the dorsal flank.
In parallel, one mouse received an acellular alginate scaffold and served as the control.
Animals were housed in individually ventilated cage (IVC) systems under controlled environmental conditions (23 ± 2 °C, 50% relative humidity, and filtered air) with free access to food and water. All experimental procedures were approved by the Hacettepe University Animal Experiments Ethical Committee (Approval No: 2022/646) and were performed in accordance with institutional and international guidelines for the care and use of laboratory animals.”

 

Comments 2) The authors state in the Results (lines 237-238) that human parathyroid cells were "successfully isolated, expanded in culture, and subsequently incorporated into 3D bioprinted constructs." However, the timeline provided in the Materials and Methods (lines 122–131) explicitly states that the entire process took only 9 hours. A 9-hour window allows for cell isolation and bioprinting but precludes any biological cell expansion.

The authors must correct the statement in the Results. The cells should be described as "freshly isolated primary cells" rather than "expanded" cells. This distinction is vital, as the behavior of primary cells versus expanded (passaged) cells can differ significantly in tissue engineering applications.

Response 2: We thank the reviewer for this helpful and important observation. We agree that the term “expanded” was not appropriate given the reported 9-hour workflow. Accordingly, the wording in the Results section has been revised to describe the cells as “freshly isolated primary human parathyroid cells” rather than expanded cells.

This correction ensures full consistency between the Results and the timeline described in the Materials and Methods and eliminates any potential misunderstanding regarding cell passaging or in vitro expansion. This revision does not affect the interpretation of the results but improves the accuracy and clarity of the manuscript.

 

Comment 3: The specific cross-linking agent and its concentration (0.5 M CaCl2​) should be clearly stated in the Materials and Methods section rather than appearing for the first time in the Discussion.

Response 3:

We thank the reviewer for this valuable comment. The Materials and Methods section has been revised to explicitly state the cross-linking agent and its concentration. We now clearly indicate that the 3D-bioprinted alginate constructs were cross-linked using 0.5 M calcium chloride (CaCl₂) to ensure structural stability for in vivo applications. This information has been removed from the Discussion and appropriately relocated to Section 2.2.

 

We thank the reviewer once again for the constructive and insightful comments, which have helped improve the clarity and methodological rigor of the manuscript.

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

Thank you for the possibility to review the manuscript titled: “In Vivo Engraftment and Functional Efficacy of a 3D- Bioprinted Human Parathyroid Equivalent”.  This is an interesting, original and timely study. There are no major objections, however I would recommend minor editing:

- Please exclude citations from the conclusions section. Reference #22 should be moved to discussion section.

-Please include limitations of the study at the end of discussion section

-Please acknowledge in the discussion section that hypocalcemia is usually transient but can be a serious complication if all of the parathyroid glands were removed. This can be seen after thyroidectomy rather than hemithyroidectomy.

-Several other methods are also used for parathyroid gland identification, some of them are mentioned in the manuscript by Dolidze et al

Dolidze D, et al. Prophylaxis of postoperative hypoparathyroidism in thyroid surgery. Folia Med (Plovdiv). 2023 Apr 30;65(2):207-214. doi: 10.3897/folmed.65.e75427. PMID: 37144304.

Overall, this is an interesting study the evaluates an understudied problem.

Please take into account the recommendations in the spirit of improving the quality of the submission

Author Response

Comments 1: Thank you for the possibility to review the manuscript titled: “In Vivo Engraftment and Functional Efficacy of a 3D- Bioprinted Human Parathyroid Equivalent”.  This is an interesting, original and timely study. There are no major objections, however I would recommend minor editing:

- Please exclude citations from the conclusions section. Reference #22 should be moved to discussion section.

Response 1: We thank the reviewer for this valuable suggestion. Reference #22 has been removed from the Conclusions section and has been appropriately relocated to the Discussion section.

 

-Comments 2: Please include limitations of the study at the end of discussion section

Reponse 2: We thank the reviewer for this valuable suggestion. In response, a “Limitations of the Study” subsection has been added to the end of the Discussion section. This section now explicitly addresses the qualitative nature of the analyses, the lack of quantitative functional PTH measurements, the limitations of the immunodeficient animal model in evaluating long-term host–graft interactions, and the proof-of-concept design of the study. The revised text also outlines directions for future studies to support clinical translation.

“This study has several limitations. Although multiple post-implantation time points were evaluated, the analyses were predominantly qualitative, and quantitative assessments of cell viability, PTH-positive cell proportions, and circulating or secreted PTH levels were not performed. Therefore, functional efficacy was inferred from marker expression rather than confirmed by direct hormone secretion assays.

In addition, the use of immunodeficient athymic mice limits the evaluation of long-term host–graft interactions and immune-mediated rejection. The fibrosis and calcification observed at later time points suggest limited long-term biocompatibility of the scaffold material.

Finally, this study was designed as a proof-of-concept for short-term functional engraftment of 3D-bioprinted human parathyroid constructs rather than to demonstrate clinical efficacy. Further studies incorporating quantitative functional analyses, optimized biomaterials, and immunomodulatory strategies are required for clinical translation.”

 

Comments 3: -Please acknowledge in the discussion section that hypocalcemia is usually transient but can be a serious complication if all of the parathyroid glands were removed. This can be seen after thyroidectomy rather than hemithyroidectomy.

Response 3: The requested statement has been added to the Discussion section to clarify that postoperative hypocalcemia is usually transient but may become severe and persistent when all parathyroid glands are removed or devascularized, particularly after total thyroidectomy.

“Although postoperative hypocalcemia is usually transient, it may become a severe and persistent complication when all parathyroid glands are inadvertently removed or devascularized, a scenario more commonly associated with total thyroidectomy rather than hemithyroidectomy.”

 

Comments 4: -Several other methods are also used for parathyroid gland identification, some of them are mentioned in the manuscript by Dolidze et al

Dolidze D, et al. Prophylaxis of postoperative hypoparathyroidism in thyroid surgery. Folia Med (Plovdiv). 2023 Apr 30;65(2):207-214. doi: 10.3897/folmed.65.e75427. PMID: 37144304.

“

Response 4: In accordance with the reviewer’s suggestion, data from the study by Dolidze et al. have been incorporated into the Discussion section.

Dolidze et al. reported that the use of intraoperative parathyroid identification strategies, including 5-aminolevulinic acid–induced fluorescence and adjunct visual–instrumental techniques, significantly reduced the incidence of transient hypoparathyroidism and effectively prevented permanent hypocalcemia in thyroid surgery (26).

Comment 5: Overall, this is an interesting study the evaluates an understudied problem.

Please take into account the recommendations in the spirit of improving the quality of the submission

Response 5: We thank the reviewer for the positive and encouraging assessment of our work. All recommendations were carefully considered and incorporated where appropriate, with the aim of improving the clarity, scientific rigor, and overall quality of the manuscript.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Although the manuscript has been improved, the figure labeling remains insufficient. Specifically, Figure 4 lacks labels A–D, and Figure 5 lacks labels A and B.

Author Response

Comment 1: Although the manuscript has been improved, the figure labeling remains insufficient. Specifically, Figure 4 lacks labels A–D, and Figure 5 lacks labels A and B.

Response 1: Thank you for your valuable comment. The labeling issue has been corrected. Subpanel labels (A–D) have now been added to Figure 4, and subpanel labels (A and B) have been added to Figure 5. The figures have been revised accordingly to ensure clarity and consistency.

Author Response File: Author Response.docx