Assessment of the Osseointegration of Pure-Phase β-Tricalcium Phosphate (β-TCP) Ceramic Cylinder Implants in Critical Segmental Radial Bone Defects in Rabbits

Abstract

Autografts, allografts, and synthetic bone substitutes are essential in reconstructive orthopedic surgery. Although autografts and allografts provide excellent skeletal integration, their use is limited by host morbidity and graft acquisition challenges. Synthetic materials like β-tricalcium phosphate (β-TCP) offer promising osseoconductive properties as a potential substitute. This study evaluated the osseointegration of β-TCP ceramic cylinder implants in bone defects in rabbits. Eighteen New Zealand rabbits underwent radial diaphysis ostectomy to create a critical segmental defect and were divided into three groups: Group A received β-TCP blocks, Group B received allogenous cortical bone grafts, and Group C underwent ostectomy without defect filling. Postoperative assessments included clinical evaluations, radiographs, micro-computed tomography, and histology at various time points to assess osseointegration and implant resorption. At the 120th postoperative day, Group B showed successful bone integration without infection. In contrast, Group A showed no osseointegration or resorption of the β-TCP implants, and Group C exhibited bone non-union. While β-TCP demonstrated biocompatibility, it lacked osseoconductivity, likely due to low porosity. β-TCP implants did not promote bone consolidation, suggesting that further research on porosity and shape is needed to improve their suitability for veterinary orthopedic reconstructive surgery.

Keywords: biomaterial; bone critical defect; bone implant; bone segmental defect.

Figure 1

Photographic images of the limbs of the rabbits a few days after the immediate postoperative period and later at 120 days after the surgical procedure. Note the alignment of operated limbs and absence of edema, swelling, redness, signs of inflammation/infection, or foreign body reaction (A,B) at five days PO. In (C,D), note the good support of the thoracic limbs and load placement on the left thoracic limb at 120 days PO (red and purple arrows in (C) and (D), respectively).

Figure 2

Postoperative radiographic images of the left forelimbs of animals in Groups A, B, and C, respectively, at moments M0 to M4. Craniocaudal images at immediate PO (M0) (A,G,M) and mid-lateral images at immediate PO (M0), and 30 (M1), 60 (M2), 90 (M3), and 120 days (M4) PO (B–F, H–L, N–R, respectively) of animals from Group A (β-TCP), Group B (Bone Graft), and Group C (no defect filling). In both groups with defect filling, note the ceramic implant and bone graft inserted at the ostectomy site (yellow arrow on (B,H)), the 1.5 mm titanium plate (blue arrow in (B,H)), and the 1.5 mm titanium screws (red arrow in (B,H)). In (F), at M5, the integration of the implant and bone cannot be confirmed (solid orange arrow) because of the radiotransparent lines between the bone and the implant, despite its perfect positioning. In (L), at M4, it is possible to notice the integration between the bone and the graft, with the beginning of bone remodeling at the bone critical defect site (solid purple arrow). In (M,N), at M0, we can see the ostectomy site, which is 7 mm in length (yellow arrows). In (O), at M1, there is initial growth of bone inside the defect (blue arrow) that increases until (P) at M2 (red arrow). From that moment, it seems that the bone growing process stops, since the image inside the defect is the same for the next 60 days (P–R) from M2 to M4.

Figure 3

Photographic images of the left forelimb of an animal from Group A (β-TCP; (A–C)) and another from Group B (allogenic cortical bone graft; (D–F)) shortly after euthanasia, with exposure of the operated site for removal of bone fragments for µCT and histological studies. Photographic images showing good limb alignment and no signs of inflammation, infection, or foreign body reactions (A–F). After removing the plate and the screw, it was possible to notice the good alignment of the β-TCP implant (C) and allogenous cortical bone graft (F) in relation to the bone and the possible osseointegration of the implant with the surrounding tissues inside the bone defect and the bone edges. Macroscopically, it seems that there was no implant resorption. The yellow arrows in (C,F) show the positions of the second and third screws (left to right direction), corresponding to the proximal–distal direction of the forelimb. The red and green squares indicate the positions where the β-TCP implant and allogenous graft were inserted in the bone defect. In Group B, osteointegration seems to be more effective and macroscopically visible than Group A. For both groups, it is visible that there was not any kind of foreign bone reaction or infection in the bone or other tissue surfaces below the metallic implants, as seen after plate and screw removal.

Figure 4

Tomographic images of selected fragments of animals from Group A (β-TCP; (A–D)) and Group B (cortical allogeneic bone graft; (E–H)). Tomographic images of 2 cm bone with β-TCP implant (Group A) and bone graft (Group B). (A,E) Middle part of the implant and graft as region of interest (ROI) for μCT analysis and acquisition of cross-sectional images. Note the implant and bone graft inserted in the receptor bones (black arrows in (A) and (E), respectively). (B,F) Trans-axial cut. (C,G) Coronal cut. (D,H) Sagital cut. * β-TCP implant. # Allogenous cortical bone graft. Note that there is no implant–bone osteointegration seen in μCT images of Group A (blue arrows in (C,D)), but on the contrary, there is positive osteointegration between the bone graft and bone, as shown in (H) (yellow arrows).

Figure 5

Histological analysis of calcified sections to visualize the positions of the β-TCP implant (Group A; (A–D)) and bone graft (Group B; (E–H)), as well as their interfaces with the receptor bone. Staining was performed using Exakt protocol. In Group A, the β-TCP implant reacted with the resin during the fixation protocol, leading to its dissolution (A–D), except for some remaining intact portions (black arrows in (A,D)). Notably, no newly formed bone was observed within the implant area. The receptor bone edges remained intact, encompassing the implant (red arrows in (A,C)), except in certain areas (yellow arrows in (B,D)) where minor resorption was observed. There were no signs of foreign body reactions, indicating the implant’s biocompatibility. The implant appeared to retain its original shape 120 days post-implantation, showing no evidence of resorption. In Group B (E–H), the allogenous bone graft successfully integrated with the receptor bone, forming direct connections and undergoing remodeling. The integration was evident along the graft edges and surfaces (yellow arrows in (E–H)). In most cases, the graft and receptor bone communicated through a medullary channel (blue arrows). Center of the implant (*); proximal and distal portions of the bone (#); center of the allogenous cortical bone graft (†); ×40 magnification. Histological slides with 40 µm thickness.

Figure 6

Histological analysis of a section of bone fragment/implant from an animal from Group A, 120 days after receiving a β-TCP cylinder implant. Protocol using hematoxylin and eosin in a decalcified bone fragment. (A) Observe the cavitation lined by a delicate fibrous capsule (*) and surrounded by a thin deposition of fibrous connective tissue (ǂ) between the bone lamellae of the host mature bone (Φ) and adipose tissue (¤). (B) At higher magnification (×10), thickening of the capsule (*) is noted, characterized by fibrocytes (yellow arrowhead) and an extracellular matrix (mature fibrous connective tissue/fibrosis) closely associated with the bone matrix (Φ). Additionally, an immature fibrous matrix (red arrowhead) is observed lining the bone lamella (Φ) in contact with extramedullary adipose tissue (¤). (C) Presence of amorphous granular black material (β-TCP implant remnant/◊) interspersed among bone lamellae (Φ) in direct contact with the implant (×5 magnification). (D) At higher magnification (×40), a thin layer of connective tissue is observed in direct contact with the implant (◊), surrounded by extramedullary adipose tissue (¤). No newly formed bone cells are present.

Figure 7

Histological analysis of a section of bone fragment/graft from an animal from Group B, 120 days after receiving an allogenous cortical bone graft. Protocol using hematoxylin and eosin in a decalcified bone fragment. (A) Photomicrography of grafted region at ×2.5 magnification. In the image, we can see a newly formed osteoid matrix (Ω) surrounded by bone lamellae (Φ), adipose tissue (¤), and fibrous connective tissue (ǂ). (B) At higher magnification (×10), a delicate demarcation is observed between the fibrous connective tissue (ǂ) surrounding the bone graft remnants (◊) and newly formed osteoid (Ω). (C) At ×20 magnification, fibrous connective tissue (ǂ) is observed to closely adhere to the newly formed osteoid (Ω), which is rich in osteocytes (green arrowhead). (D) Higher magnification of the allograft region (×20) reveals a moderate presence of amorphous and intensely eosinophilic residues (◊), surrounded by newly formed bone matrix/osteoid (Ω) with prominent osteocyte activity (green arrowhead), as well as fibrous connective tissue (ǂ).