Author response:
The following is the authors’ response to the original reviews.
Response to Reviewer 1
(Cys25)PTH(1-84) does not show efficacy surpassing that of the previously used rhPTH(1-34). This needs to be discussed biologically and clinically.
Thank you very much for your valuable comments for enhancing the manuscript. We appreciate your input and have noted that this aspect was not addressed in the discussion. The authors have included the following paragraph in discussion section.
“This biological difference is thought to be due to dimeric R25CPTH(1-34) exhibiting a more preferential binding affinity for the RG versus R0 PTH1R conformation, despite having a diminished affinity for either conformation. Additionally, the potency of cAMP production in cells was lower for dimeric R25CPTH compared to monomeric R25CPTH, consistent with its lower PTH1R-binding affinity. (Noh et al., 2024) One of the potential clinical advantages of dimeric R25CPTH(1-34) is its partial agonistic effect in pharmacodynamics. This property may allow for a more fine-tuned regulation of bone metabolism, potentially reducing the risk of adverse effects associated with full agonism, such as hypercalcemia and bone resorption by osteolcast activity. Moreover, the dimeric form may offer a more sustained anabolic response, which could be beneficial in the context of long-term treatment strategies. (Noh et al., 2024) Also, the effects of dimer were prominent, as we mentioned better bone formation than the control group.” (2nd paragraph, Discussion section)
The terms (Cys25)PTH(1-84) and Dimeric R25CPTH(1-34) are being used interchangeably and incorrectly. A unification of these terms is necessary.
We totally agree with the reviewer’s notion. R25CPTH(1-84) represents mutated human PTH, rhPTH(1-34) and dimeric R25CPTH(1-34) are synthesized PTH analogs. To clarified the terminology, we thus have changeed the terminology in the manuscript appear in red.
The figure legend is incorrect. Not all figures are described, and even though there are figures from A to I, only up to E is explained, or the content is different.
We apologize for our negligence. As suggested by a reviewer, we've fixed the figure legends throughout before the list of references in the manuscript as follows.
“Figure legends
Figure 1. Micro-CT analysis (A-D) Experimental design for the controlled delivery of rhPTH(1-34) and dimeric R25CPTH(1-34) in ovariectomized beagle model. Representative images for injection and placement of titanium implant. (E) Micro-CT analysis. bone mineral density (BMD), bone volume (TV; mm3), trabecular number (Tb.N; 1/mm), trabecular thickness (Tb. Th; um), trabecular separation (Tb.sp; ㎛). Error bars indicate standard deviation. Data are shown as mean ± s.d. *p<0.05, **p<0.01, ***p<0.001, n.s., not significant. P, posterior. R, right
Figure 2. (A-I) Histological analysis of the different groups stained in Goldner’s trichrome. The presence of bone is marked by the green color and soft tissue in red. Red arrows indicate the position with soft tissues without bone around the implant threads. The area of bone formed was the widest in the rhPTH(1-34)-treated group. In the dimeric R25CPTH(1-34)treated group, there is a greater amount of bone than vehicle-treated group. Green arrows represent the bone formed over the implant. blue dotted line, margin of bone and soft tissue; Scale bars: 1mm
Figure 3. Histological analysis using Masson trichrome staining results in the rhPTH(1-34) and dimeric R25CPTH(1-34)-treated group (A-L) Masson trichrome-stained sections of cancellous bone in the mandibular bone. The formed bone is marked by the color red. Collagen is stained blue. Black dotted box magnification region of trabecular bone in the mandible. Scale bars, A-C, G-I: 1mm; D-F, J-L: 200 ㎛
Figure 4. Immunohistochemical analysis using TRAP staining for bone remodeling activity (A-L) TRAP staining is used to evaluate bone remodeling by staining osteoclasts. Osteoclasts is presented by the purple color. Black dotted box magnification region of trabecular bone in the mandible. (M, N) The number of TRAP-positive cells in the mandible of the rhPTH(1-34) and dimeric R25CPTH(1-34)-treated beagles. Scale bars, A-C, G-I: 1mm; D-F, J-L: 200 ㎛. Error bars indicate standard deviation. Data are shown as mean ± s.d. *p<0.05, **p<0.01, n.s., not significant
Figure 5. Measurement of biochemical Marker Dynamics in serum. The serum levels of calcium, phosphorus, P1NP, and CTX across three time points (T0, T1, T2) following treatment with dimeric dimeric R25CPTH(1-34), rhPTH(1-34), or control. (A-B) Calcium and phosphorus levels exhibit an upward trend in response to both PTH treatments compared to control, suggesting enhanced bone mineralization. (C) P1NP levels, indicative of bone formation, remain relatively unchanged across time and treatments. (D) CTX levels, associated with bone resorption, show no significant differences between groups. Data points for the dimeric R25CPTH(1-34), rhPTH(1-34), and control are marked by squares, circles, and triangles, respectively, with error bars representing confidence intervals.
Supplementary Figure. Three-dimensional reconstructed image of the bone surrounding the implants. Three-dimensional reconstructed images of the peri-implant bone depicting the osseointegration after different therapeutic interventions. (A) Represents the bone response to recombinant human parathyroid hormone fragment (rhPTH 1-34) treatment, showing the most robust degree of bone formation around the implant in the three groups. (B) Shows the bone response to a modified PTH fragment (dimeric R25CPTH(1-34)), indicating a similar level of bone growth and integration as seen with rhPTH(1-34), although to a slightly lesser extent. (C) Serves as the control group, demonstrating the least amount of bone formation and osseointegration. The upper panel provides a top view of the bone-implant interface, while the lower panel offers a cross-sectional view highlighting the extent of bony ingrowth and integration with the implant surface.”
In Figure 5, although the descriptions of T0, T1, T2 are mentioned in the method section, it would be more clear if there was a timeline like in Figure 1.
Based on the reviewer’s advice, we have indicated the timing of T0, T1, and T2 in the materials & methods section describing the serum biochemical assay, and we have shown a timeline in figure 5.
In Figure 5, instead of having calcium, phosphorus, P1NP, CTX graphs all under Figure 5, it would be more convenient for referencing in the text to label them as Figure 5A, Figure 5B, Figure 5C, Figure 5D.
We totally understood the reviewer’s comment. As the reviewer’s suggested, we have corrected the labeling in the text for figure 5 as follows.
“The levels of calcium, phosphorus, CTX, and P1NP were analyzed over time using RM-ANOVA (Figure 5). There were no significant differences between the groups for calcium and phosphorus at time points T0 and T1 (Figure 5A). However, after the PTH analog was administered at T2 (Figure 5A), the levels were highest in the rhPTH(1-34) group, followed by the dimeric R25CPTH(1-34) group, and then, lowest in the control group, which was statistically significant (Figure 5B,C). (P < 0.05) The differences between the groups over time for CTX and P1NP were not statistically significant (Figure 5D, E).”
Significance should be indicated in the figure (no asterisk present).
As the reviewer’s comment, we put the asterisk in the figure 5.
Addition of Figures in Text:
Line 112: change from "figure 2" to "figure 1" / Line 115: mention "figure 1. E"
Line 120: refer to "figure 1. E" / Line 123: change from "figure 3" to "figure 2"
Line 128: refer to "figure 2.A-C" / Line 137: mention "figure 3"
Line 138: refer to "figure 3. A-L" / Line 143: mention "figure 3. A-L"
Line 144: refer to "figure 3. E,F,K,L" / Line 148: mention "figure 4"
Line 150: refer to "figure 4 M,N" / Line 152: mention "figure 4. M,N"
Line 155: refer to "figure 5" / Line 157: mention "figure 5"
Line 159: refer to "figure 5" / Line 171: mention "figure 1 E"
Line 175: refer to "figure 2 M, N"/ Line 194: mention "figure 3"
Above all, thank you for the reviewer’s notion. We corrected detailed figure labeling in text to red color.
Response to Reviewer 2
First, the authors should clarify why they compared the effects of rhPTH(1-34) and of dimeric R25C2 PTH(1-34)? In most of the parameters, rhPTH(1-34) seems to be superior to dimeric R25C2 PTH(1-34). Why did the authors insist that the anabolic effects of dimer were prominent? Even though implication of dimeric R25C2 PTH(1-34) was drawn from genetic mutation studies, the authors should describe more clearly in the discussion the potential clinical benefits of the dimeric R25C2 PTH(1-34) compared to rhPTH(1-34), especially if dimeric R25C2 PTH(1-34) has just partial agonistic effect in pharmacodynamics.
Thank you for your insightful comments and questions regarding our results between rhPTH(1-34) and dimeric R25CPTH(1-34). rhPTH(1-34) is a well-characterized therapy for osteoporosis. In this study, rhPTH(1-34) generally showed superior outcomes in most parameters tested, the dimeric R25CPTH(1-34) exhibited specific anabolic effects that are not as pronounced with rhPTH(1-34). We recognized R25CPTH(1-34) as a anabolic effector. One of the potential advantages of dimeric R25CPTH(1-34) is its partial agonistic effect in pharmacodynamics. This property may allow for a more fine-tuned regulation of bone metabolism, potentially reducing the risk of adverse effects associated with full agonism, such as hypercalcemia and bone resorption by osteolast activity. Moreover, the dimeric form may offer a more sustained anabolic response, which could be beneficial in the context of long-term treatment strategies. Also, based on our results, we notes that the effects of dimer were prominent, as we mentioned better bone formation than the control group. We appreciate your input and have noted that this aspect was not addressed in the discussion. As a result, we have included the following paragraph in discussion section.
“This biological difference is thought to be due to dimeric R25CPTH(1-34) exhibiting a more preferential binding affinity for the RG versus R0 PTH1R conformation, despite having a diminished affinity for either conformation. Additionally, the potency of cAMP production in cells was lower for dimeric R25CPTH compared to monomeric R25CPTH, consistent with its lower PTH1R-binding affinity. (Noh et al., 2024) One of the potential clinical advantages of dimeric R25CPTH(1-34) is its partial agonistic effect in pharmacodynamics. This property may allow for a more fine-tuned regulation of bone metabolism, potentially reducing the risk of adverse effects associated with full agonism, such as hypercalcemia and bone resorption by osteolcast activity. Moreover, the dimeric form may offer a more sustained anabolic response, which could be beneficial in the context of long-term treatment strategies. (Noh et al., 2024) Also, the effects of dimer were prominent, as we mentioned better bone formation than the control group.” (2nd paragraph, Discussion section)
Second, please describe the intermittent and continuous application of PTH analogues. Many of the readers may misunderstand that the authors' daily injection of PTHs were actually to mimic the clinical intermittent application or continuous one. Incorporation of the author's intention for experimental design would be more helpful for readers.
Thank you for your insightful comments regarding the need for clearer differentiation between intermittent and continuous applications of PTH analogs in this study. We appreciate your concern that the readers may not fully grasp whether our daily injection protocol was intended to mimic clinical intermittent or continuous PTH administration. To address this, we have revised the manuscript to explicitly clarify that the daily injections of rhPTH(1-34) and dimeric R25CPTH(1-34) were designed to simulate the intermittent dosing regimen commonly used in clinical practice. This regimen is known to maximize the anabolic effects on bone while minimizing potential catabolic actions associated with more frequent or continuous hormone exposure. We have added detailed explanations in the Introduction, Methods, and Discussion sections to help readers understand our experimental design and its relevance to clinical settings.
Introduction section
“Administration of prathyroid hormone (PTH) analogs can be categorized into two distinct protocols: intermittent and continuous. Intermittent rhPTH(1-34) therapy, typically characterized by daily injections, is clinically used to enhance bone formation and strength. This method leverages the anabolic effects of rhPTH(1-34) without significant bone resorption, which can occur with more frequent or continuous exposure. On the other hand, continuous rhPTH(1-34) exposure, often modeled in research as constant infusion, tends to accelerate bone resorption activities, potentially leading to bone loss (Silva and Bilezikian, 2015; Jilka, 2007). Understanding these differences is crucial for interpreting the therapeutic implications of rhPTH(1-34) in bone health.”
Silva, B. C., & Bilezikian, J. P. (2015). Parathyroid hormone: anabolic and catabolic actions on the skeleton. Current Opinion in Pharmacology, 22, 41-50.
Jilka, R. L. (2007). Molecular and cellular mechanisms of the anabolic effect of intermittent PTH. Bone, 40(6), 1434-1446.
Materials and Methods section
“Each animal received one injection per day, aimed at replicating the intermittent rhPTH(1-34) exposure proven beneficial for bone regeneration and overall skeletal health in clinical settings (Neer et al., 2001; Kendler et al., 2018). This regimen was chosen to investigate the potential anabolic effects of these specific PTH analogs under conditions closely resembling therapeutic use.”
Neer, R. M., Arnaud, C. D., Zanchetta, J. R., Prince, R., Gaich, G. A., Reginster, J. Y., Hodsman, A. B., Eriksen, E. F., Ish-Shalom, S., Genant, H. K., Wang, O., and Mitlak, B. H. (2001). Effect of Parathyroid Hormone (1-34) on Fractures and Bone Mineral Density in Postmenopausal Women with Osteoporosis. The New England Journal of Medicine, 344(19), 1434-1441.
Kendler, D. L., Marin, F., Zerbini, C. A. F., Russo, L. A., Greenspan, S. L., Zikan, V., Bagur, A., Malouf-Sierra, J., Lakatos, P., Fahrleitner-Pammer, A., Lespessailles, E., Minisola, S., Body, J. J., Geusens, P., Moricke, R., & Lopez-Romero, P. (2018). Effects of Teriparatide and Risedronate on New Fractures in Post-Menopausal Women with Severe Osteoporosis (VERO): A Multicenter, Double-Blind, Double-Dummy, Randomized Controlled Trial. The Lancet, 391(10117), 230-240.
Discussion section
“The use of daily injections in this study was intended to simulate intermittent PTH therapy, a well-established clinical approach for managing osteoporosis and enhancing bone regeneration. Intermittent administration of PTH, as opposed to continuous exposure, is critical for maximizing the anabolic response while minimizing the catabolic effects that are associated with higher frequency or continuous hormone levels. Our findings support the notion that even with daily administration, both rhPTH(1-34) and dimeric dimeric R25CPTH(1-34) promote bone formation and osseointegration, consistent with the outcomes expected from intermittent therapy. It’s important for future research to consider the dosage and timing of administration to further optimize the therapeutic benefits of PTH analogs (Dempster et al., 2001; Hodsman et al., 2005).”
Dempster, D. W., Cosman, F., Kurland, E. S., Zhou, H., Nieves, J., Woelfert, L., Shane, E., Plavetic, K., Müller, R., Bilezikian, J., & Lindsay, R. (2001). Effects of Daily Treatment with Parathyroid Hormone on Bone Microarchitecture and Turnover in Patients with Osteoporosis: A Paired Biopsy Study. Journal of Bone and Mineral Research, 16(10), 1846-1853.
Hodsman, A. B., Bauer, D. C., Dempster, D. W., Dian, L., Hanley, D. A., Harris, S. T., Kendler, D. L., McClung, M. R., Miller, P. D., Olszynski, W. P., Orwoll, E., Yuen, C. K. (2005). Parathyroid Hormone and Teriparatide for the Treatment of Osteoporosis: A Review of the Evidence and Suggested Guidelines for Its Use. Endocrine Reviews, 26(5), 688-703.
Third, please unify the nomenclature. Ensure consistency in the nomenclature throughout the article. Unify the naming conventions for PTH analogues, such as rhPTH(1-34) vs teriparatide and (Cys25)PTH(1-84) vs R25CPTH(1-34) vs R25CPTH(1-34) vs (1-84). Choose one nomenclature for each analogue and use it consistently throughout the article.
We totally agree with the reviewer’s notion. R25CPTH(1-84) represents mutated human PTH, rhPTH(1-34) and dimeric R25CPTH(1-34) are synthesized PTH analogs. To clarified the terminology, we thus have changed the terminology in the manuscript appear in red.
Response to Reviewer 3
I would recommend to rewrite the manuscript in a form that is more understandable to the readers. In fact, it appears to me that this work was originally formatted in a way that would need the Materials and Methods to precede the results. As presented (and as requested by the eLife formatting) the Materials and Methods are available only at the end of the reading and, as a consequence, the readers needs to refer to the Materials and Methods to have a general and initial understanding of the study design (i.e. type of treatment for each group, etc are not well specified in the Results section).
Thank you for you constructive comments and suggestions regarding the manuscript. We appreciate your feedback on the organization of the manuscript entirely. As reviewer mentioned, Materials and methods were placed after the discussion section in accordance with the format of the elife journal. For a better and initial understanding, a description of each experimental group has been added to the Results section as follow. Thank you again for your valuable comments.
“To investigate evaluating and comparing the efficacy of rhPTH(1-34) and the dimeric R25CPTH(1-34) in promoting bone regeneration and healing in a clinically relevant animal model. In our study, beagle dogs were selected as the model due to their anatomical similarity to human oral structures, suitable size for surgeries, human-like bone turnover rates, and established oral health profiles, ensuring comparable and ethically sound research outcomes. The normal saline injected-control group, injected with 40ug/day PTH (Forsteo, Eli Lilly) group, and 40ug/day PTH analog-injected group. Animals in each group were injected subcutaneously for 10 weeks.”
