Novel use of bisphosphonates to improve surgical outcomes in experimental bone tuberculosis

Abstract

BACKGROUND

Tuberculous osteitis is a chronic, granulomatous bone infection that frequently results in impaired bone healing following surgery. Despite surgical intervention and prolonged anti-tuberculous therapy, complete bone regeneration often remains unachieved, contributing to subsequent orthopedic complications.

AIM

To investigate the efficacy and safety of pamidronate in promoting bone regeneration following surgical treatment of experimental animal tuberculous osteitis.

METHODS

A controlled randomized basic study of rabbit femoral tuberculosis induced by Mycobacterium tuberculosis strain H37Rv included surgical removal of infected tissue and implantation of osteoinductive bone grafts with the following animal allocation to one of three groups: (1) Bisphosphonates alone; (2) Bisphosphonates combined with anti-tuberculous therapy; and (3) Anti-tuberculous therapy alone. The control group consisted of animals that received no surgical or medical treatment. Clinical evaluations, biochemical markers, micro-computed tomography imaging, and histomorphometry analyses were conducted at 3 months and 6 months postoperatively.

RESULTS

Pamidronate treatment significantly reduced early implant resorption, increased osteoblastic activity, improved trabecular bone regeneration, and maintained graft integrity compared to the anti-tuberculous therapy-only group. Histologically, pamidronate led to enhanced vascular remodeling and increased bone matrix formation. Crucially, bisphosphonate therapy demonstrated safety, compatibility with anti-tuberculous medications, and did not exacerbate tuberculous inflammation. Furthermore, micro-computed tomography analysis revealed a significant increase in trabecular thickness and density in pamidronate-treated groups, underscoring the anabolic effects of bisphosphonates. Morphometric evaluation confirmed a marked reduction in osteoclast number and activity at graft interfaces. These combined radiological, histological, and biochemical data collectively demonstrate the efficacy of pamidronate as an adjunctive agent in enhancing bone repair outcomes following surgical intervention for tuberculous osteitis.

CONCLUSION

A single intravenous dose of pamidronate significantly enhances bone regeneration and prevents implant resorption following surgical treatment of tuberculous osteitis. The following prospective studies are needed.

Key Words: Tuberculosis; Bone and joint tuberculosis; Pamidronate; Bisphosphonates; Bone regeneration; Bone grafting; Bone resorption

Core Tip: A single intravenous dose of pamidronate significantly enhances bone regeneration after surgical treatment of experimental tuberculous osteitis. Pamidronate effectively inhibits early implant resorption, increases osteoblastic activity, and promotes prolonged osteogenesis, showing compatibility and safety when combined with antituberculosis therapy. This finding suggests that bisphosphonates may be a promising adjunctive therapeutic strategy for enhancing clinical outcomes in patients undergoing skeletal tuberculosis surgery.

INTRODUCTION

Tuberculous (TB) osteitis is a chronic, granulomatous bone infection caused by the Mycobacterium tuberculosis (M. tuberculosis) complex, leading to progressive osteolysis and subsequent skeletal destruction. Despite surgical debridement and long-term anti- TB therapy (ATT), complete bone regeneration remains a challenge due to persistent inflammation and impaired osteogenesis, often resulting in orthopedic sequelae such as deformities, instability, and chronic pain[1-3]. The current surgical osteoregenerative strategies include bone grafts and biologically active substitutes. However, these materials are often subject to early resorption in inflammatory environments, which limits their long-term structural integration[4,5]. Alternative strategies for enhancing bone regeneration include implant materials with antimicrobial properties or the combination of osteoconductive materials with bone morphogenetic proteins (BMPs) or autologous bone marrow[6,7]. Unfortunately, these approaches tend to be costly and involve additional procedures, such as harvesting autologous bone, which increases the surgery time and patient risk.

In contrast, bisphosphonates offer a more cost-effective and efficient solution for improving bone healing, making them an attractive alternative for enhancing post-surgical outcomes, especially in inflammatory conditions such as TB osteitis. Bisphosphonates are agents that could modulate bone turnover, and they have emerged as promising candidates for preserving graft integrity by inhibiting osteoclastic activity and prolonging the osteoconductive phase of healing[8]. Recent studies have also suggested that bisphosphonates may possess anti-inflammatory and angiogenic properties, potentially aiding in the integration of bone implants even in the presence of infection or immune dysregulation[9,10]. Despite the widespread use of bisphosphonates in osteometabolic and oncologic bone diseases, their application in skeletal infections, particularly in bone TB, has been limited. While bisphosphonates have been used to manage osteoporosis and to stabilize metal implants in orthopedic surgeries for patients with skeletal TB, the intravenous administration of bisphosphonates specifically to improve implant integration post-surgery remains unexplored[11]. Studies have indicated the use of bisphosphonates in combination with other treatments for TB, such as soaking allografts in bisphosphonate solutions to enhance their integration in joint prosthesis surgeries[12]. Moreover, bisphosphonates are effective in treating chronic refractory osteomyelitis, where surgical treatment was challenging due to the extent of the infection[13]. However, the targeted use of intravenous bisphosphonates in postoperative settings to enhance bone regeneration and preserve grafts has not been extensively researched, despite its potential advantages over other methods, such as soaking implants[14]. The intravenous infusion ensures controlled systemic concentrations of pamidronate, thereby avoiding concerns regarding implant adhesion and potential drug loss through wound exudate. In light of these considerations, our study aimed to evaluate the efficacy of pamidronate in enhancing bone regeneration following surgical treatment of experimental TB osteitis, and to assess its compatibility with ATT. We hypothesized that pamidronate would inhibit early resorption of bone grafts, reduce osteolytic activity, enhance trabecular bone formation, and preserve graft integrity.

The purpose of this experimental study was to evaluate the efficacy and safety of bisphosphonates, specifically pamidronate, as adjunctive therapy in the surgical treatment of TB osteitis, focusing on effects on implant preservation, bone regeneration, and compatibility with ATT.

MATERIALS AND METHODS

Ethical approval

The study was approved by the Independent Ethical Committee of the Saint Petersburg Research Institute of Phthisiopulmonology, Ministry of Health, Russian Federation (Approval No. 74). All animal handling procedures were strictly followed the principles of the European Commission[15], as well as Russian national standards for laboratory animal care[16,17].

Animal model and study design

The experimental study was conducted on 21 male rabbits (“Soviet chinchilla” breed), with a body weight of 3456 ± 321 g, provided by the certified animal breeding facility (Rappolovo Laboratory Animal Nursery, National Research Center “Kurchatov Institute”, Russia). Animals were housed individually under standardized, controlled conditions: A temperature of 23 °C - 25 °C, a humidity of 50%-70%, a 12-hour light/dark cycle, and ad libitum access to water and food. They were maintained in accordance with local ethical guidelines and regulations.

Experimental TB osteitis model

TB osteitis was induced in the medial condyle of the right femur, according to the previously described and validated method[18]. Under general anesthesia (Zoletil^®, Virbac, France, 25 mg/kg intramuscular injection; Xyla^®, Interchemie werken De Adelaar BV, Netherlands, 2% solution, 1.0-1.5 mL intramuscular injection), the right hind limb was prepared, and a surgical incision (5 cm length) was made on the anteromedial surface of the distal femur. A small bone tunnel (0.5-0.8 cm deep, diameter 0.8 mm) was created using a trocar. A suspension containing 1 × 10⁶ CFU of M. tuberculosis strain H37Rv (drug-sensitive strain tuberculosis #1/47, Institute of Hygiene and Epidemiology, Prague, 1976) was embedded in a sterile collagen sponge (2 mm × 2 mm) and placed inside the bone tunnel. The site of injury was sealed with a bone post from the trocar, and the wound was sutured layer by layer.

Experimental groups

Four weeks post-infection, tuberculin skin tests (Diaskintest^®, Generium, Russia) confirmed successful infection in all animals. Twenty-one rabbits were then randomly allocated into four groups.

Control group (n = 3): No further treatment (infection only, no surgery, no medications).

ATT-only group (n = 4): Surgical debridement, Osteoset^®2DBM pellets (Wright Medical Technology, Inc., TN, United States) implantation, followed by ATT regimen (isoniazid 10 mg/kg, ethambutol 20 mg/kg, pyrazinamide 20 mg/kg orally, 5 days/week).

ATT + pamidronate group (n = 7): Surgical treatment as in group 2, followed by combined ATT and single intravenous pamidronate infusion (Pamidronate Medac, Germany, 1 mg/kg IV at 10 days post-surgery).

Pamidronate-only group (n = 7): Surgical treatment equal to group 2, ATT discontinued post-surgery, single pamidronate infusion (Pamidronate Medac, Germany, 1 mg/kg IV at 10 days post-surgery).

Surgical technique

Six weeks post-infection, rabbits underwent surgical debridement of the lesion under the same anesthesia described above. Necrotic tissue and sequestrations were thoroughly removed through the same incision. Bone defects were filled with osteoinductive Osteoset^®2DBM pellets (Wright Medical Technology, Inc., TN, United States), which contain BMPs (BMP-2, BMP-4), insulin-like growth factor-1, and transforming growth factor-beta1. The wound was closed in layers. Postoperative antibiotic prophylaxis was provided with Cefazolin (KRKA, d.d., Slovenija; 10 mg/kg intramuscular injection, 5 days).

Pamidronate infusion

Pamidronate was administered intravenously as a single infusion at a dose of 1 mg/kg body weight, diluted in 20 mL of sterile saline, delivered slowly over 60 minutes under careful veterinary monitoring. The infusion was performed 10 days following surgical intervention, ensuring optimal conditions for osteogenesis and implant stabilization. Animals were continuously monitored during and after the infusion to detect any adverse reactions, such as changes in vital signs, signs of distress, or injection site reactions. The dose and administration protocol were based on previous pharmacokinetic studies and aimed at achieving effective osteoclast inhibition while maintaining safety and compatibility. Throughout the study period, no significant adverse events or side effects related to pamidronate administration were observed.

Clinical and laboratory assessments

Animal health status, activity, food/water intake, body weight, and surgical wound healing were monitored regularly. Blood samples were collected at baseline, 6 weeks post-infection, and at 10 days, 3 months, and 6 months post-surgery. Serum biomarkers related to bone remodeling (total alkaline phosphatase, albumin, osteocalcin, beta-crosslaps, receptor activator of nuclear factor kappa-B ligand, and sclerostin) were analyzed using commercial enzyme-linked immunosorbent assay kits (Eilton LLC, Russia; MyBioSource, Canada; and Rabbit-specific ELISA kits, China).

Radiological examination

Micro-computed tomography (SkyScan 1172, Bruker microCT, Belgium) scans were performed at euthanasia (3 months and 6 months). Scanning parameters were as follows: Pixel size, 13-27 μm; voltage, 100 kV; current, 100 μA; and rotation step, 0.4°. Images were reconstructed (NRecon, Micro Photonics Inc., Poland) and analyzed (RadiAnt DICOM Viewer, Medixant, Poland; CTAn, CTAN Team, Germany). Qualitative (bone trabeculation, implant integrity) and semi-quantitative scoring (0-2 scale: Absence, mild, severe) were performed to evaluate osteoregeneration and osteolysis.

Histological and morphometric analysis

Following euthanasia, the distal epiphysis of the femur was dissected, fixed in neutral buffered formalin (10%), decalcified, embedded in paraffin, and sectioned (3-5 μm thick). Sections were stained with hematoxylin-eosin and Ziehl-Neelsen. Digital slide scanning (Pannoramic, 3DHISTECH Ltd., Hungary) enabled comprehensive morphometric analysis using Orbit Image Analysis software (Actelion Pharmaceuticals Ltd., Switzerland). Histopathological parameters assessed included the area of specific inflammation, fibrosis, necrosis, implant integrity, osteoblastic and osteoclastic activities, bone trabecular thickness, and vascularity.

Euthanasia and disposal of rabbits

Rabbits were withdrawn from the experiment in accordance with the principles outlined by the European Commission[9]. Euthanasia was performed through intravenous administration into the marginal ear vein of a 10% lidocaine solution, at a dose five times higher than the maximum allowable therapeutic level, ensuring rapid, painless, and stress-free cessation of vital functions. Animal welfare procedures were strictly adhered to in compliance with international ethical standards. Following euthanasia, the animal remains and biological waste were safely disposed of by certified personnel using methods compliant with institutional biosafety protocols, ensuring both ethical and environmental safety. The detailed study flow chart is in Figure 1.

Figure 1 The flow chart of the study.

Statistical analysis

Statistical analysis was performed by a biomedical statistician using Statistica version 12.0 (Dell Inc., TX, United States). Data distribution was tested using the Shapiro-Wilk test. Non-parametric tests (Kruskal-Wallis and Mann-Whitney U tests) were applied, and results are presented as median (interquartile ranges). A P-value ≤ 0.05 was considered statistically significant.

RESULTS

Clinical observations

All animals, except one rabbit from the untreated control group, maintained a satisfactory health status, exhibited good physical activity, and had a stable intake of food and water throughout the study. One rabbit from the untreated control group developed generalized tuberculosis infection by 4.5 months post-infection, manifesting poor physical condition and reduced limb function.

A postoperative complication (subcutaneous abscess) occurred in one rabbit (ATT + pamidronate group), successfully treated with local antiseptics without further intervention. Pamidronate infusion was well tolerated, with no observed adverse effects or interference with ongoing ATT.

Body weight dynamics

All groups showed a steady body weight gain throughout the study period (4.8%-15.3%). Notably, pamidronate-treated groups exhibited slightly better weight gain dynamics, although differences were not statistically significant (P > 0.05).

Laboratory results

Skin tests (Diaskintest^®, Generium, Russia) confirmed successful M. tuberculosis infection in all animals (4 weeks after infection). Polymerase chain reaction confirmed the presence of M. tuberculosis DNA in all rabbits, validating the infection model.

Alkaline phosphatase levels significantly decreased in the pamidronate groups (ATT + pamidronate and pamidronate-only; P = 0.07), reflecting reduced bone turnover. Serum sclerostin showed statistically significant intergroup differences at 3 months post-surgery (P = 0.03), with the highest values observed in the control and ATT + pamidronate groups. Levels of osteocalcin, receptor activator of nuclear factor kappa-B ligand, and albumin demonstrated no statistically significant intergroup differences (Table 1).

Table 1 Body weight and biochemical parameters of animals, mean ± SD/medians (interquartile ranges).

Initial level	All animals	Group 1	Group 2	Group 3	Group 4
Weight, g	3456.2 ± 321.6	3446.7 ± 272.9	3008.5 ± 274.8	3572.0 ± 248.3	3600.3 ± 216.9
	3518 (3356-3626)	3546 (3138-3656)	2983 (2802-3215)	3618 (3390-3736)	3526 (3490-3626)
ALP, U/L	95.9 ± 30.7	89.3 ± 24.0	78.0 ± 45.8	100.4 ± 25.7	104.4 ± 30.1
	94.0 (72.0-120.0)	99 (62-107)	64 (45-111)	94 (91-109)	120 (72-134)
Osteocalcin, ng/mL	0.1 ± 0.0	0.1 ± 0.0	0.1 ± 0.0	0.1 ± 0.0	0.2 ± 0.0
	0.1 (0.1-0.2)	0.1 (0.1-0.1)	0.1 (0.1-0.1)	0.1 (0.1-0.1)	0.2 (0.1-0.2)
RANKL, pg/mL	61.5 ± 16.2	81.6 ± 0.0	48.7 ± 0.0	53.7 ± 11.6	67.4 ± 18.3
	61.9 (48.7-66.6)	81.6 (81.6-81.6)	48.7 (48.7-48.7)	52.5 (43.9-63.5)	64 (56.2-78.6)
Albumin, g/L	47.6 ± 3.3	49.0 ± 4.6	47.7 ± 4.9	47.4 ± 3.5	47.0 ± 2.3
	47 (45-50)	50 (44-53)	50 (42-51)	46 (45-51)	47 (45-50)
Sclerostin, ng/mL	105.2 ± 79.8	162.0 ± 0.0	71.5 ± 67.2	104.0 ± 147.1	118.0 ± 0.0
	118.5 (24-162)	162 (162-162)	71.5 (24-119)	104 (0-208)	118 (118-118)
1 month after infection
Weight, g	3608.8 ± 325.1	3481.3 ± 185.5	3212.0 ± 113.3	3779.4 ± 315.9	3719.4 ± 270.9
	3554 (3350-3756)	3466 (3304-3674)	3224 (3119-3305)	3756 (3468-3924)	3606 (3520-3994)
ALP, U/L	65.4 ± 25.5	58.7 ± 18.2	56.3 ± 27.7	60.7 ± 22.3	78.1 ± 29.7
	66 (46-79)	53 (44-79)	65 (37-75.5)	66 (45-76)	81 (46-94)
Osteocalcin, ng/mL	0.2 ± 0.0	0.2 ± 0.0	0.2 ± 0.0	0.1 ± 0.0	0.2 ± 0.0
	0.2 (0.1-0.2)	0.2 (0.2-0.2)	0.2 (0.1-0.2)	0.2 (0.1-0.2)	0.2 (0.1-0.2)
RANKL, pg/mL	58.2 ± 14.1	52.9 ± 9.6	67.9 ± 20.9	60.9 ± 10.4	52.2 ± 13.4
	60.2 (49.6-64.7)	56 (42.1-60.6)	63.8 (55-80.8)	62.5 (49.6-68.9)	53.2 (42.2-62.1)
Albumin, g/L	45.1 ± 2.8	47.3 ± 1.2	43.0 ± 3.7	46.3 ± 2.8	44.1 ± 1.5
	45 (43-48)	48 (46-48)	42.5 (40.5-45.5)	47 (44-48)	44 (43-45)
Sclerostin, ng/mL	178.2 ± 102.2	155.1 ± 82.4	159.7 ± 39.3	231.0 ± 130.8	146.1 ± 99.9
	141.4 (115-247.9)	127.3 (90.2-247.9)	165.2 (118-196)	191.9 (119.6-353.3)	133.4 (99.5-227.2)
Before surgery
Weight, g	3659.9 ± 377.6	3821.3 ± 356.1	3153.0 ± 201.7	3872.9 ± 309.9	3667.4 ± 281.5
	3580 (3426-4018)	3682 (3556-4226)	3143 (2996-3310)	3914 (3602-4022)	3514 (3490-4058)
ALP, U/L	55.9 ± 18.5	50.0 ± 24.1	40.5 ± 20.9	52.1 ± 13.2	71.0 ± 10.0
	57 (47-75)	48 (27-75)	40 (23-58)	50 (44-60)	76 (58-80)
Osteocalcin, ng/mL	0.1 ± 0.0	0.1 ± 0.0	0.2 ± 0.0	0.1 ± 0.0	0.1 ± 0.0
	0.1 (0.1-0.2)	0.1 (0.1-0.1)	0.1 (0.1-0.2)	0.1 (0.1-0.2)	0.1 (0.1-0.2)
RANKL, pg/mL	62.9 ± 11.5	66.3 ± 18.1	68.6 ± 5.1	63.3 ± 7.0	57.9 ± 14.5
	65.7 (54.4-69.8)	60.2 (51.9-86.7)	69.7 (65.1-72.1)	66.4 (54.4-68.6)	57.7 (48.5-71)
Albumin, g/L	44.7 ± 2.9	43.7 ± 3.2	47.0 ± 1.0	45.8 ± 3.2	43.3 ± 2.4
	45 (42-47)	45 (40-46)	47 (46-48)	46.5 (42-49)	43 (41-45)
Sclerostin, ng/mL	204.0 ± 104.2	229.6 ± 107.9	145.1 ± 56.2	244.7 ± 149.0	185.8 ± 59.9
	165 (155.6-231.3)	177.6 (157.6-353.7)	137.8 (103.3-186.9)	181.7 (133.5-370.2)	164 (158.5-231.3)
3 months after surgery
Weight, g	3952.9 ± 515.7	3786.7 ± 767.7	3340.0 ± 307.6	4282.9 ± 320.4	4044.3 ± 372.7
	3870 (3680-4390)	3410 (3280-4670)	3270 (3135-3545)	4340 (4150-4440)	3870 (3770-4390)
ALP, U/L	38.3 ± 12.6	41.3 ± 14.0	28.0 ± 9.7	40.7 ± 14.5	40.6 ± 10.9
	37 (28-50)	40 (28-56)	25 (22-34)	39 (28-52)	37 (30-51)
Osteocalcin, ng/mL	0.1 ± 0.0	0.1 ± 0.0	0.1 ± 0.0	0.1 ± 0.0	0.1 ± 0.0
	0.1 (0.1-0.2)	0.1 (0.1-0.1)	0.2 (0.1-0.2)	0.1 (0.1-0.2)	0.1 (0.1-0.2)
RANKL, pg/mL	55.6 ± 13.6	52.7 ± 21.4	54.8 ± 18.6	58.2 ± 10.7	54.8 ± 12.8
	56.3 (47.4-63.5)	48.1 (33.9-75.9)	50.7 (42.4-67.2)	60.9 (57.3-63.5)	56.1 (45-70.5)
Albumin	44.1 ± 3.6	42.3 ± 6.7	42.8 ± 3.3	44.3 ± 2.4	45.4 ± 3.6
	44 (42-46)	39 (38-50)	42.5 (40.5-45)	43 (42-47)	44 (44-46)
6 months after surgery
Weight, g	4033.1 ± 637.9	3720.0 ± 989.9	3400.0 ± 381.8	4498.0 ± 289.4	3925.0 ± 657.5
	4230 (3670-4420)	3720 (3020-4420)	3400 (3130-3670)	4330 (4310-4760)	3885 (3470-4380)
ALP, U/L	23.7 ± 16.8	13.0 ± 0.0	22.5 ± 13.4	19.3 ± 12.2	31.5 ± 24.2
	15 (13-35)	13 (13-13)	22.5 (13-32)	18 (10.5-28)	25.5 (12.5-50.5)
Osteocalcin, ng/mL	0.2 ± 0.1	0.1 ± 0.0	0.1 ± 0.0	0.2 ± 0.2	0.1 ± 0.0
	0.1 (0.1-0.2)	0.1 (0.1-0.1)	0.1 (0.1-0.2)	0.1 (0.1-0.3)	0.1 (0.1-0.2)
RANKL, pg/mL	64.6 ± 12.0	71.0 ± 0.0	55.4 ± 13.1	67.7 ± 14.7	64.3 ± 11.1
	64.7 (52-71)	71 (71-71)	55.4 (46.2-64.7)	66 (57.2-78.3)	64.6 (55.8-72.9)
Albumin, g/L	42.8 ± 2.3	41.0 ± 0.0	45.0 ± 0.0	42.0 ± 1.6	43.5 ± 3.0
	42 (42-44)	41 (41-41)	45 (45-45)	42 (41-43)	42 (42-45)
Sclerostin, ng/mL	216.8 ± 116.6	293.6 ± 0.0	175.5 ± 127.1	272.4 ± 137.8	162.6 ± 99.0
	186.6 (125.7-305.9)	293.6 (293.6-293.6)	175.5 (85.6-265.4)	272.3 (156.9-388)	132.9 (102.3-223)

ALP: Alkaline phosphatase; RANKL: Receptor activator of nuclear factor kappa-B ligand.

Radiological findings

Micro-computed tomography analysis at 3 months and 6 months showed substantial improvements in bone regeneration parameters within the pamidronate-treated groups. These groups demonstrated greater preservation and integration of the implanted osteoinductive material (Osteoset^®2DBM pellets), as evidenced by robust trabecular formation and significantly reduced osteolytic lesions compared to the ATT-only and control groups.

Specifically, at 6 months, the pamidronate-treated groups showed significantly higher scores for bone trabecular integrity and regeneration (median total score, 7.5 vs 4.0 in ATT-only; P < 0.05), with minimal evidence of osteolysis (median score, 0.5 in pamidronate-treated vs 2.0 in ATT-only; P < 0.05; Figure 2).

Figure 2 Computed tomography images of the distal part of the right femur of a rabbit from group 4 (autopsy at 6 months control point). A: Three-dimensional reconstruction showing the heterogeneous density of the implanted material with visible inclusions of calcium sulfate and bone trabeculae ingrowth; B: Computed tomography cross-sectional slice confirming the implant structure and absence of peri-implant resorption.

Histological and morphometric analysis

Histological examination of untreated controls revealed severe and extensive specific granulomatous inflammation (40%-50% of the bone area), accompanied by abundant caseous necrosis and numerous acid-fast bacilli (Figure 3A and B).

Figure 3 Pathological evolution of bone tuberculosis under different treatment regimens: A comparative histological and computed tomography analysis. A and B: Group 1 (control) - infection was induced; no surgical or medical treatment was performed; autopsy at the 3-month control point. Scanned histologic section stained with hematoxylin and eosin, histological section showing specific inflammation occupying 405% of the specimen area, involving the metaphysis and diaphysis zones, including the articular surface (A); computed tomography (CT) cross-section of the corresponding macroscopic specimen of the epiphysis of the right femur, corresponding computed tomography image of the same distal femur region, the metaphyseal plate is not visualized due to severe inflammation (B); C and D: Group 2 - surgical or medical treatment + anti-tuberculous therapy-only, autopsy at 3 months control point. Scanned histologic section stained with hematoxylin and eosin, the area of specific displacement in the diaphysis contains necrosis with detritus in the center (C); CT cross-section of the corresponding macroscopic specimen of the epiphysis of the right femur (D); E and F: Group 2 - surgical or medical treatment + anti-tuberculous therapy-only, autopsy at 6 months control point. Scanned histologic section stained with hematoxylin and eosin, specific inflammatory infiltrates are determined in an area of no more than 1% of the epiphyseal zone, a single productive epithelioid cell granuloma surrounded by a ridge of lymphocytes is revealed (E); CT cross-section of the corresponding macroscopic specimen of the epiphysis of the right femur (F); G and H: Group 4 - surgical or medical treatment + pamidronate-only, autopsy at 6 months control point. Scanned histologic section stained with hematoxylin and eosin, the histoarchitecture of the epiphyseal zone is altered due to inflammation, the beam structure is partially restored, and the beams form a looped network in the epiphysis (G); CT cross-section of the corresponding macroscopic specimen of the epiphysis of the right femur (H). In the epiphysis zone, closer to the cortical bone, according to CT data, implant granules are visualized. Nonspecific inflammation, primarily characterized by lymphoplasmacytic infiltration, is also visible in this zone on histological preparations. Growing beams with active osteoblasts on the surface, full-blooded vessels, and isolated bone sequestra are found in this zone, a mature connective tissue capsule is present in some areas around the foci. Around the foci of inflammation, there is bone marrow remodeling, characterized by active mesenchyme and granulation tissue of varying maturity, with an increased number of full-blooded vessels. Stromal cords with lymphocytic infiltration extend from the inflammation zone; single multinucleated cells of the “foreign body” type are encountered, and diffuse single lymphocytic infiltration is found in the thickness of the bone marrow tissue.

At 3 months post-surgery, ATT-only animals exhibited incomplete trabecular regeneration and persistent small inflammatory foci. Conversely, the pamidronate-treated groups showed substantially improved histological outcomes, characterized by active osteoblast proliferation, increased vascularity, and organized granulation tissue around the implants (Figures 3C-H and 4).

Figure 4 Quantitative histomorphometry analysis of bone remodeling in the femoral epiphysis. A and B: Thickness of bone trabeculae in the epiphysis at 3 months and 6 months control points, measured in micrometers (μm); C and D: Number of osteoblasts per high-power field in the femoral epiphysis at 3 months and 6 months control points (magnification × 1000), median; E: Number of osteoclasts per high-power field in femoral epiphyses (magnification × 1000), median.

By 6 months, pamidronate-treated animals demonstrated remarkable improvements in trabecular thickness and overall bone matrix area, significantly greater compared to other groups (P < 0.001). Osteoclast activity markedly reduced following pamidronate administration (0.1 cells per high-power field vs 0.3 cells in ATT-only; P < 0.05). Specific inflammatory changes were minimal or absent in 75% of pamidronate-treated cases, in contrast to persistent residual lesions in ATT-only animals (Figures 3E-H and 4).

An important histological finding in pamidronate-treated animals was the presence of organized fibrous stromal strands extending from the implant, indicative of a favorable remodeling environment. Small fragments of the implant are visualized; histological preparations in this area reveal specific inflammation with small areas of nonspecific lympho-plasmacytic infiltration.

Correlations and clinical relevance

Statistically significant correlations were established between osteoclast numbers and implant density (r = -1.0 at 3 months and r = -1.0 at 6 months; P < 0.05), confirming the direct role of bisphosphonates in preserving implanted materials by reducing osteoclast-mediated resorption.

A strong correlation (r = 1.0 at 3 months; r = 0.9 at 6 months) was also observed between serum sclerostin levels and osteocyte numbers, suggesting that active bone remodeling dynamics are influenced by bisphosphonate therapy.

DISCUSSION

This experimental, randomized, controlled study demonstrates that pamidronate, a potent bisphosphonate, significantly enhances bone regeneration following surgical treatment of TB osteitis in rabbits. Pamidronate treatment improved the preservation and integration of implanted osteoinductive materials, increased trabecular thickness, and notably suppressed osteoclast activity. These findings highlight the therapeutic potential of bisphosphonates as adjunctive agents for enhancing bone healing and preventing complications after surgical interventions for skeletal TB[2,19-21].

TB osteitis, a form of extrapulmonary TB, poses considerable treatment challenges due to persistent inflammatory osteolysis and poor regenerative responses, even after optimal surgical intervention and extended courses of ATT[22,23]. Prior clinical and experimental data have consistently reported that residual bone defects and incomplete healing are common, leading to significant orthopedic morbidity, deformities, and functional impairment[1,22,24,25]. The search for adjunctive therapies capable of mitigating these complications is therefore of paramount clinical importance.

Despite extensive use in metabolic bone conditions, the application of bisphosphonates in infectious osteitis, particularly bone and joint TB, has been minimally explored[26,27]. Our study addressed this knowledge gap by investigating pamidronate in a validated rabbit model of TB osteitis. We observed significant structural benefits, with enhanced preservation and integration of osteoinductive bone grafts, confirming bisphosphonates’ capacity to protect implants from premature resorption and contribute to bone regeneration[27-29]. One of the key mechanisms underlying the observed improvements in bone regeneration is angiogenesis. Recent studies have shown that bisphosphonates can modulate angiogenic pathways, particularly through the vascular endothelial growth factor (VEGF) signaling pathway. VEGF is a critical mediator of endothelial cell proliferation and new blood vessel formation during tissue repair and regeneration. Bisphosphonates, including pamidronate, have been shown to enhance VEGF-mediated vascular remodeling in bone tissues, promoting improved blood flow to the healing site. The enhanced vascularization in pamidronate-treated animals may contribute to the better integration of the bone grafts and provide an environment conducive to osteogenesis. The increased blood supply ensures the delivery of nutrients, growth factors, and oxygen to the regenerating bone, thus facilitating the repair process and supporting the survival of osteoblasts at the graft site[19,20,30,31].

Additionally, bisphosphonates are known for their anti-inflammatory properties, which could further explain the improved healing observed in our study. Pamidronate has been shown to reduce the release of pro-inflammatory cytokines, such as tumor necrosis factor-α, interleukin-6, and interleukin-1β, which are typically elevated in inflammatory environments. By modulating the immune response, pamidronate may reduce the inflammatory burden at the surgical site, thereby preventing excessive bone resorption that often accompanies chronic inflammation, including TB osteitis. The reduction in inflammatory cytokines not only limits osteoclast activity but also fosters a more favorable environment for osteoblast activity, which is crucial for bone regeneration. Our data, showing a reduction in inflammation and enhanced osteoblast activity in pamidronate-treated groups, support this hypothesis[20,21,32,33].

Moreover, pamidronate may influence osteocyte signaling, an important aspect of bone remodeling. Osteocytes play a critical role in bone homeostasis by coordinating bone formation and resorption through their mechanosensory function and interaction with osteoblasts and osteoclasts. Bisphosphonates, including pamidronate has been shown to affect osteocyte-mediated signaling by altering the release of factors like sclerostin and sclerostin-like molecules. Sclerostin is a potent inhibitor of osteoblastic activity, and its suppression has been associated with increased bone formation. Our study observed an increase in osteoblast numbers and activity in the pamidronate-treated groups, which can be attributed in part to the modulation of osteocyte signaling pathways. Pamidronate may have a dual effect, inhibit osteoclast activity and promote osteoblast function, which ultimately leads to improved bone formation and graft retention.

The significant clinical implication of our study is that even a single administration of pamidronate can substantially enhance bone regeneration outcomes after surgical treatment of TB osteitis. This approach could represent a feasible and effective strategy to reduce postoperative complications, minimize structural deformities, and potentially shorten rehabilitation times for patients with skeletal tuberculosis[34]. However, clinical trials are warranted to confirm these experimental findings in human subjects[21,23].

The underlying pathology determines standard pamidronate dosing regimens. For example, in osteolytic breast cancer bone metastases, pamidronate is typically given at 90 mg intravenous every 3-4 weeks (according to the instructions for use). In situations where there is no ongoing bone destruction (i.e., no progressive osteolysis), there are currently no established protocols for the repeat administration of bisphosphonates. In our study, we elected to administer a single dose of pamidronate in the early postoperative period, primarily for practical reasons. A one-time infusion administered immediately after surgery is easier to integrate into clinical practice, as it enhances patient adherence and is more cost-effective, as it eliminates the need for repeated hospital visits or prolonged therapy. Moreover, when we designed this study, no evidence-based protocols existed for multiple pamidronate doses to enhance bone regeneration in such scenarios. We therefore opted for the most straightforward and safest strategy initially. From a mechanistic standpoint, administering a single dose is supported by the pharmacodynamics of bisphosphonates. Pamidronate binds strongly to the bone matrix and is then slowly released over periods of weeks to even years, providing a long-lasting antiresorptive effect. Therefore, one dose is sufficient to cover the critical early postoperative period, when bone resorption around the implant is at its peak. Additionally, bisphosphonates possess anti-inflammatory properties in addition to their antiresorptive effects. Pamidronate administered postoperatively may have attenuated the local inflammatory response and consequent bone resorption during the initial phase of implant integration (i.e., the first few months after surgery).

Certain limitations must be acknowledged. Firstly, the relatively small number of animals, inherent to ethical and logistical constraints of experimental research, may limit statistical power. Secondly, this study employed only a single bisphosphonate administration; the effects of multiple or prolonged treatments remain to be studied. Lastly, the extrapolation of animal model findings directly to clinical practice requires cautious interpretation. Nevertheless, the significant positive outcomes strongly justify further clinical investigations.

Our experimental data demonstrate that pamidronate significantly promotes bone regeneration and effectively inhibits osteoresorption in TB osteitis following surgical treatment. Given the excellent tolerability and compatibility with ATT, as well as the promising osteoanabolic potential demonstrated in this model, bisphosphonates should be considered as adjunctive therapy for managing patients with skeletal tuberculosis[19-21].

CONCLUSION

This experimental randomized controlled study demonstrates that a single intravenous dose of pamidronate significantly enhances bone regeneration, inhibits osteoresorption, and improves the integration and stability of bone graft materials following surgical treatment of TB osteitis. Pamidronate therapy was found to be safe and fully compatible with concurrent ATT, effectively mitigating local inflammatory and osteolytic processes. These findings strongly support further clinical evaluation of bisphosphonates as an adjunctive treatment strategy to improve outcomes in patients undergoing surgical intervention for skeletal tuberculosis.

ACKNOWLEDGEMENTS

We sincerely thank the staff of the Saint Petersburg Research Institute of Phthisiopulmonology and the Interregional Laboratory Center for their technical support during laboratory and histological analyses, and special thanks to Natalia M. Blum for histological consultations.