The potential use of tetracalcium phosphate in vertebral augmentation: A study in a sheep model

Abstract

BACKGROUND

Percutaneous vertebroplasty (PVP) and percutaneous kyphoplasty (PKP) are widely used to treat vertebral fractures. However, the standard filler material, polymethylmethacrylate (PMMA), presents significant drawbacks, including thermal damage, allergic reactions, and poor biocompatibility. Tetracalcium phosphate (TTCP), a calcium phosphate cement (CPC), has emerged as a promising alternative due to its superior biocompatibility, osteoconductivity, and ability to integrate with natural bone. This study aimed to evaluate the feasibility of TTCP for vertebral augmentation in a preclinical sheep model, focusing on biomechanical stability, biocompatibility, and osteogenic potential.

METHODS

Five Akkaraman sheep underwent PKP with TTCP at three lumbar vertebral levels (L2–L4). Under general anesthesia, TTCP cement was injected into cavities prepared according to the standard PKP procedure. Postoperative care included analgesia and antibiotics. Four animals were followed for 12–14 weeks, and one for 25 weeks. At the end of the study period, the animals were euthanized and vertebrae were harvested for biomechanical testing using a Shimadzu AG-IS 100 kN machine. Histological evaluation was performed to assess ossification stages according to Shapiro’s classification. Statistical analysis was conducted using paired t-tests (p<0.05).

RESULTS

One animal was euthanized prematurely due to infection, while four completed the study without complications. Biomechanical analysis demonstrated no significant difference in compressive strength between treated and untreated vertebrae (p>0.05). Histological examination revealed osteoblastic activity, progressive mineralization, and successful bone integration.

CONCLUSION

TTCP demonstrated promising biomechanical and biophysiological properties for vertebral augmentation. However, its use in infected sites and in the presence of metabolic bone disorders may be limited. Further clinical studies are required to validate its long-term efficacy.

INTRODUCTION

Percutaneous vertebroplasty (PVP) and percutaneous kyphoplasty (PKP) are minimally invasive procedures designed to reinforce weakened vertebrae caused by osteoporosis, tumors, or trauma.^[1] These interventions involve the percutaneous injection of polymethylmethacrylate (PMMA) into the affected vertebrae to restore structural integrity.^[1] First introduced in 1987 by Galibert and Deramond, PVP was initially performed to stabilize a collapsed C2 vertebra affected by a hemangioma through the percutaneous administration of PMMA.^[1]

Over the years, PVP and PKP techniques have been refined to achieve near-optimal clinical outcomes; however, the search for more advanced biomaterials remains ongoing.^[2] Numerous studies, including our own,^[2] have aimed to develop bone replacement materials that are not only biophysiologically compatible but also capable of overcoming the limitations of existing materials.^[3-5] Despite its widespread use, PMMA continues to present several challenges, including the risk of allergic reactions, excessive heat generation during polymerization, biomechanical properties that differ from those of spinal bone, and difficulties in processing.^[6] Most notably, the lack of intrinsic biocompatibility of PMMA has prompted continued exploration of alternative materials.^[6]

Among the candidate materials under investigation, calcium phosphate cements (CPCs) have emerged as a promising class of biomaterials due to their exceptional biocompatibility and osteoinductive properties.^[7-11] These attributes make CPCs particularly suitable for bone repair and replacement applications.^[7-11] One of the most extensively studied types of CPC is tetracalcium phosphate (TTCP), which combines a basic TTCP component with an acidic counterpart, such as brushite or monetite.^[12] This formulation exhibits a self-setting mechanism in which an acid-base reaction, triggered by the addition of a hardening liquid, results in the formation of calcium-deficient hydroxyapatite.^[12] CPCs possess several advantageous properties, including high bioactivity, non-cytotoxicity, and osteoconductivity.^[13] Furthermore, their biodegradability allows gradual replacement by newly formed bone tissue following implantation.^[13] These characteristics have led to their widespread application in dental implantology, where they have been used to reinforce implant sites and stabilize implants in cases of insufficient bone volume.^[13-15]

Given the favorable properties of TTCP and its potential role in bone repair, the present study was designed to evaluate its applicability for vertebral augmentation in a preclinical sheep model. The objective was to determine whether TTCP could serve as a viable alternative to currently used materials for vertebral stabilization, leveraging its superior biocompatibility and osteogenic potential.

MATERIALS AND METHODS

Ethical Approval and Funding

All experimental procedures were approved by the Animal Research Ethics Committee of our university (Decision No: 2023/12) and were conducted in strict accordance with the European Communities Council Directive (86/609/EEC) on animal welfare. Funding was provided by the Scientific Research Projects Fund of Niğde Ömer Halisdemir University (Project No: 2024/4). The study was conducted in compliance with the principles of the Declaration of Helsinki.

Animal Selection and Surgical Preparation

The study included five Akkaraman sheep (2 years of age; 60–70 kg) obtained from the Ayhan Şahenk Agricultural Research and Application Center of Niğde Ömer Halisdemir University (Fig. 1). The animals were fasted for 24 hours prior to surgery. Rectal temperature was monitored to maintain a stable body temperature of 37°C throughout the procedure. All experiments were conducted in a controlled environment under veterinary supervision (H.K.) and surgical supervision (İ.K.) (Fig. 1). General anesthesia was induced intravenously with xylazine hydrochloride (Rompun^®, Bayer, İstanbul, Türkiye; 0.1 mg/kg) and ketamine hydrochloride (Ketalar^®, Bayer, İstanbul, Türkiye; 2.2 mg/kg). The animals were positioned on their right side to minimize the risk of hypoxia (Fig. 1). Surgical readiness was confirmed by the absence of palpebral and pinch reflexes. Standard aseptic and antiseptic protocols were followed. The surgical field was disinfected with povidone-iodine (Dermosept^®, Algilaç, İstanbul, Türkiye) and sterile draping was applied (Fig. 1).

Figure 1.

Study phases.

PKP Procedure

For the PKP procedure, the L2–L4 vertebral landmarks (transverse and spinous processes) were identified by palpation. A sterile Jamshidi cannula needle was inserted percutaneously at a 45° angle, 3 cm lateral to the midline, advancing into the cancellous bone of the vertebral body. A drilling device was used to create a 1 cm-deep cavity, which was verified with a probe. Subsequently, 3 mL of TTCP compound, comprising alpha-tricalcium phosphate, dicalcium phosphate, tetracalcium phosphate, and calcium silicate powder mixed with a sodium citrate solution (Grandus^® B-One, Permed Health Products, Çanakkale, Türkiye), was injected into the cavity. Each animal underwent PKP at vertebral levels L2–L4 (a total of 15 treated vertebrae). The entry points were closed using sharp-needle 0/0 polypropylene sutures.

Postoperative Care and Follow-up Period

Postoperative pain and inflammation were managed with meloxicam (Maxicam®, Sanovel, İstanbul, Türkiye; 0.5 mg/kg/day for three days). Infection control included oxytetracycline hydrochloride spray (NEO CAF^®; Merck Sharp & Dohme [MSD], İstanbul, Türkiye) and antibiotic injections (Reptopen S^®, Ceva, İstanbul, Türkiye; 3 mL/day for five days). Animals recovered under close observation and were housed individually for five days before being returned to the flock. They were maintained at the Ayhan Şahenk Agricultural Research and Application Center of Niğde Ömer Halisdemir University with unrestricted access to food and water. One animal was euthanized on postoperative day 10 due to systemic infection secondary to wound contamination, likely resulting from the inability to protect the surgical site from contact with surrounding structures during movement. This animal was excluded from the final analysis. The remaining four animals were followed longitudinally: three were observed for 12–14 weeks, and one for 25 weeks. Sutures were removed two weeks postoperatively. No additional complications or adverse effects were observed, and all remaining animals resumed normal flock activity. At the end of the study period, euthanasia was performed using an overdose of xylazine hydrochloride (Rompun^®; Bayer, İstanbul, Türkiye) and ketamine hydrochloride (Ketalar^®; Bayer, İstanbul, Türkiye). Vertebrae L1–L5 were dissected for analysis (Fig. 2).

Figure 2.

Appearance of the isolated vertebra and the treated vertebra, along with the biomechanical force-impact testing procedure performed using the Shimadzu Autograph AG-IS 100 machine.

Biomechanical Testing

Biomechanical compressive testing of vertebrae L1, L2, L4, and L5 was performed using a Shimadzu Autograph AG-IS 100 kN testing machine (Shimadzu, Kyoto, Japan). Testing was conducted with a compressive tool at a crosshead displacement rate of 1 mm/min to generate force-stroke curve data in the Hydraulic and Pneumatic Laboratory, Faculty of Engineering, Department of Mechanical Engineering, Niğde Ömer Halisdemir University (Fig. 2). Force-stroke data for each specimen were recorded using the Trapezium software integrated with the Shimadzu testing system. Data analysis was conducted on a computer equipped with an Intel Xeon 2.2 GHz processor, 28 GB RAM, and a 16 GB NVIDIA Tesla P100 GPU. All tests were conducted in air at 20°C under an atmospheric pressure of 656 mmHg. Further biomechanical evaluation of TTCP was considered infeasible due to ethical constraints, limited animal availability, financial limitations, and fundamental anatomical and postural differences inherent to the species.

Histological Processing and Evaluation

The L3 vertebrae were fixed immediately in 10% formalin following dissection of L1-5 for histological analysis. After completion of biomechanical testing, L2 and L4 vertebrae were also fixed in formalin and processed histologically together with L3 at the Department of Pathology, Faculty of Medicine, Niğde Ömer Halisdemir University. Bone specimens underwent decalcification in 3% nitric acid, followed by paraffin embedding, sectioning at 4 µm thickness, and hematoxylin-eosin (H&E) staining. All histological evaluations were performed in a blinded manner by a professional histopathologist (C.Ö.). Microscopic findings related to bone formation were assessed according to the “Classification of Stages of Woven Bone Formation” described by Shapiro et al.^[16] (Table 1).

Table 1.

"Classification of Stages of Woven Bone Formation" by Shapiro et al.^[16]

Stage 1	Highly cellular, densely packed accumulations of differentiating pre-osteoblasts present de novo at sites with no evidence of pre-existing bone tissue. These pre-osteoblasts are derived from the undifferentiated mesenchymal cell pool.
Stage 2	Mesenchymal osteoblasts surround themselves in a 360° arc with randomly oriented matrix fibers: a. The cell area is greater than the matrix area. b. The cell area is equal to the matrix area. c. The matrix area is greater than the cell area.
Stage 3	The matrix is sufficient to act as a scaffold upon which surface osteoblasts begin to synthesize bone in a lamellar configuration.
Stage 4	There is progressive reduction of woven bone within the woven bone/lamellar bone complex. This reduction is relative due to increased synthesis of lamellar bone and absolute due to osteoclastic resorption: a. Woven bone exceeds lamellar bone. b. Woven bone equals lamellar bone. c. Lamellar bone exceeds woven bone.
Stage 5	When the entire bone tissue developmental sequence from undifferentiated mesenchymal cells to exclusively lamellar bone is considered, stage 0 is defined as mesenchymal cell development from undifferentiated elongated cells to progressively differentiating oval cells, whereas Stage V represents the stage at which all bone observed in a region is lamellar.

Statistical Analysis

Statistical analyses were performed using IBM SPSS Statistics version 23 (IBM Corp., Armonk, NY, USA) and Microsoft Excel (version 17). Paired-sample t-tests and percentage analyses were conducted. Statistical significance was defined as p<0.05.

RESULTS

Macroscopically, no abnormalities were observed in the vertebrae. The entry points were located within 1–4 mm of the intended target regions (Fig. 2), and a penetration depth of at least 1 cm was achieved in all treated vertebrae.

Regarding mechanical resistance, no statistically significant differences were detected between treated and untreated vertebrae (p>0.05) (Table 2, Fig. 3). Despite the anatomical proximity of the vertebrae assigned to the treatment (L2, L4) and control (L1, L5) groups, as well as the inclusion of craniocaudally mixed levels, the comparable force–impact curve profiles observed in both groups suggest that TTCP effectively replicates the load-bearing characteristics of a healthy spine (Table 2, Fig. 3).

Table 2.

Key parameters of the force-impact curve data obtained using a Shimadzu Autograph AG-IS 100 kN testing machine (Shimadzu, Kyoto, Japan) at a constant test speed of 1 mm/min

Study Groups	Treated Vertebrae (L2, L4)	Control Vertebrae (L1, L5)
Minimum load	10,726.9 N*	10,342.5 N
Maximum load	9,920 N	10,713.8 N
Average maximum load	10,210.4 N	10,528.2 N
Standard deviation	448.436 N	262.549 N

^*Newton

Figure 3.

Force-impact test results.

Microscopic examination demonstrated osteoblastic activity and mineralization within the bone specimens. The margins of the treated regions were interwoven with native bone tissue, rendering the distinction between treated and untreated areas difficult. Additionally, the material used for ossification appeared as a crystal-like foreign body in certain regions (Figs. 4, 5). Early ossification foci were also observed (Fig. 6). According to Shapiro et al.’s [16] "Classification of Stages of Woven Bone Formation" (Table 1), ossification at three months was classified as Stage 2c, whereas ossification at six months corresponded to Stage 4a (Figs. 4-6). These findings indicate that TTCP undergoes progressive replacement by newly formed bone over time.

Figure 4.

Representative images of microscopic ossification from the 14-week sample (A-B: Hematoxylin and eosin staining, ×100; C-D: Hematoxylin and eosin staining, ×200).

Figure 6.

Representative image of an early ossification focus (Hematoxylin and eosin staining, ×200).

Figure 5.

Representative images of woven bone formation with tetracalcium phosphate (TTCP) from the 25-week sample (A: Hematoxylin and eosin staining, ×100; B: Hematoxylin and eosin staining, ×200).

DISCUSSION

Since its introduction in 1987 by Galibert and Deramond,^[1] PVP and PKP techniques have undergone substantial refinement.^[2] Despite numerous advancements, ranging from improvements in Jamshidi cannulas to the development of stent-assisted systems, progress in filler materials has remained largely confined to experimental trials, with PMMA continuing to be the only universally accepted material in clinical practice to date.^[2-5] Considering its widespread use, PMMA has several notable limitations, including thermal injury, allergic reactions, difficulties in revision procedures when used in conjunction with implants, incompatibility with subsequent implant placement at treated sites, and, most critically, its lack of biocompatibility.^[6,17]

In this context, TTCP, a member of the CPC family, has emerged as a promising alternative for PVP and PKP applications. Its unique properties make it particularly suitable for vertebral augmentation. TTCP hardens through an exothermic reaction at temperatures below 30°C, a process reported to be non-cytotoxic.^[18] It achieves 50% of its ultimate compressive strength within one hour and 80% within four hours, thereby providing early mechanical stability and adhesive support in bone defects.^[18] A key characteristic of TTCP is its higher calcium-to-phosphorus (Ca/P) ratio compared to hydroxyapatite, the principal mineral component of bone. Specifically, TTCP has a Ca/P ratio of 2.0, whereas hydroxyapatite has a ratio of 1.67.^[19] This composition enables TTCP to form hydroxyapatite during the setting process.^{[19, 20]} The resulting structure is approximately 50–60% porous and negatively charged, creating an environment conducive to protein binding.^[19,20] This, in turn, facilitates the attachment of circulating endogenous growth factors, thereby supporting bone healing and remodeling processes.^[13,21] Recognized for its osteoconductive, bioactive, and biodegradable characteristics, TTCP promotes bone regeneration by facilitating the migration of osteogenic cells to the treatment site.^[13,22] Following implantation, TTCP cements undergo gradual resorption and are progressively replaced by host bone through cell-mediated remodeling.^[18,22] Importantly, while TTCP is resorbed, mechanical strength is maintained through its progressive replacement by newly formed bone tissue.^[22]

Based on these properties, TTCP offers several potential advantages and clinical applications. Unlike PMMA,^[6] TTCP does not hinder implant placement or removal, as it gradually converts into native bone tissue over time. Additionally, TTCP demonstrates adhesive strength to both metal and bone that is comparable to that of native bone, as evidenced by long-term applications.^[21,23] A significant advantage of TTCP cements is their potential to reduce the need for bone grafting, thereby eliminating donor-site morbidity associated with traditional graft materials.^[22] Another noteworthy feature of TTCP is the absence of bone-inductive growth factors or osteogenesis-stimulating hormonal components in its formulation, which may render it suitable for use in tumorous pathologies.^[24] Furthermore, TTCP is compatible with the instruments and devices currently used for PVP and PKP procedures with PMMA.^[21,25] In our study, TTCP was successfully applied using these techniques in a sheep model characterized predominantly by cancellous vertebral bone, demonstrating its effectiveness. Tomita et al.^[26] reported that, in cadaveric vertebral models subjected to PVP and PKP, no statistically significant differences were observed between preliminary tricalcium phosphate cement and PMMA under biomechanical testing conditions identical to those employed in our study, even in the absence of osteoregeneration. These findings suggest that, from a purely mechanical standpoint, advanced TTCP formulations may provide immediate load-bearing capacity comparable to that of PMMA following augmentation procedures.^[26] Similarly, our short- and long-term microscopic and biomechanical analyses demonstrated findings comparable to those of normal bone, consistent with previous reports (Table 3).^{[17,19,21,22,25,27-33]}

Table 3.

Comparative biophysical properties of grafts^[26-28]

Graft Type	TTCP (Tetracalcium Phosphate) Used in the Study	Autograft	Allograft
Hardening time	10 minutes	6 months	6 months
Time to full load-bearing capacity	4 hours (hardens in 10 minutes)	6 months	6 months
Graft resorption time	4-8 months	4-8 months	4-8 months
Volume loss	2%	10%	10%

Despite its advantages, certain limitations of TTCP must be acknowledged. Unlike PMMA, TTCP does not generate high temperatures during polymerization, thereby preventing thermal damage to vascular lesions. Also, in such regions, the presence of blood may alter the liquid-to-powder ratio of the mixture, interfering with its activation.^[18-20] Similarly, the use of TTCP is not recommended in infected sites.^[34] This limitation is related to its organic composition, which may compromise infection control when implanted into contaminated tissues.^[34] Additionally, alterations in the activator ratio may also occur in infected environments, further complicating the clinical use of TTCP under such conditions.^[18-20] In these cases, alternative materials may be more appropriate, and research into antibiotic-containing materials is ongoing.^[34] Another limitation of TTCP is its inability to achieve bone-level resistance to shear forces until complete replacement by native bone has occurred.[^30] Although this limitation is not critical for PVP or PKP procedures, it presents challenges in long-bone reconstruction, where prolonged immobilization may be necessary.^[30] Furthermore, TTCP is unsuitable in cases of severe degenerative bone disease (Z-scores below -3), as its primary mechanism relies on remodeling into functional bone tissue.^[6] Similarly, conditions that impair osteoblastic activity, such as uncontrolled diabetes, pregnancy, substance or alcohol abuse, and renal failure, may compromise treatment outcomes with TTCP.^[35] Conversely, in patients with hypercalcemia, the osteoinductive properties of TTCP may promote excessive deposition, making it unsuitable for use in such conditions.^[35] Lastly, TTCP requires a bony scaffold for effective application, rendering it inappropriate for environments involving purely soft-tissue environments.^[36]

CONCLUSION

Tetracalcium phosphate emerges as a highly suitable material for PVP and PKP procedures in modern medical practice due to its strong biomechanical and microscopic resemblance to natural bone, along with its faster ossification compared to autografts. However, TTCP has certain limitations, particularly its unsuitability for use in infected tissues or areas containing bodily fluids. Additionally, its effectiveness may be reduced in cases of severe degenerative bone disease, uncontrolled diabetes, hypercalcemia, or other conditions that impair normal bone metabolism. Further large-scale clinical studies are needed to validate its long-term efficacy and safety.

Ethics Committee Approval

This study was approved by the Niğde Ömer Halisdemir University Animal Research Ethics Committee (Date: 16.10.2023, Decision No: 2023/12).

Peer-review

Externally peer-reviewed.

Authorship Contributions

Concept: İ.K., H.Y., H.K.; Design: İ.K.; Supervision: İ.K., H.Y., H.K., C.Ö.; Resource: İ.K., H.Y., H.K., C.Ö.; Materials: İ.K., H.Y., H.K., C.Ö.; Data collection and/or processing: İ.K., H.Y., H.K.; Analysis and/or interpretation: İ.K., H.Y., H.K., C.Ö.; Literature review: İ.K.; Writing: İ.K.; Critical review: İ.K.

Conflict of Interest

None declared.

Financial Disclosure

The authors declare that this study was funded by the Scientific Research Projects Fund of Niğde Ömer Halisdemir University (Project No: 2024/4).