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; µm), trabecular separation (Tb.sp; µm). 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.

(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.

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 µm.

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 with and without xenograft in the rhPTH(1-34) and dimeric R25CPTH(1-34)-treated beagle groups. Scale bars, A-C, G-I: 1mm; D-F, J-L: 200 µm. Error bars indicate standard deviation. Data are shown as mean ± s.d. *p<0.05, **p<0.01, n.s., not significant.

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 R25CPTH(1-34), rhPTH(1-34) and control. (A) The study timeline. (B-C) Calcium and phosphorus levels show an upward trend in response to both PTH treatments compared to control, indicating enhanced bone mineralization. (D) P1NP levels, indicative of bone formation, remain relatively stable across time and treatments. (E) 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.

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.