Symphytum officinale enhances fracture healing in Wistar albino rats by promoting osteogenesis in fracture callus

Abstract

Background

Symphytum officinale (SO) is a medicinal plant. This study investigated if homoeopathic doses of SO could enhance fracture healing in adult male Wistar albino rats after induction of fracture.

Methods

Adult male Wistar albino rats underwent open osteotomy (femur), were divided into various treatment groups and received homoeopathic doses of oral SO (mother tincture (MT) and 6C), vehicle control and standard care (control). X-ray imaging and serum analysis evaluated fracture healing. The rats were sacrificed at two time points: at 2 weeks (radiological appearance of early-soft callus) and at 5 weeks (late-hard callus). The fracture callus was evaluated with H&E staining, quantitative bone morphometry, and immunohistochemistry. Bone-specific Alkaline phosphatase (B-ALP), and C-terminal telopeptide of type I collagen (CTX) were evaluated in serum using ELISA.

Results

X-rays showed faster formation of callus (both soft and hard callus) in groups treated with MT and 6C doses of oral SO. Quantitative bone morphometry indicated enhanced osteogenesis in both soft (early) and hard (late) callus in animals treated with MT and 6C doses of SO. Immunohistochemical evaluation of soft (early) and hard (late) callus for Col1A1 and Osteocalcin revealed supporting osteogenic mechanisms. Serum levels of B- ALP and CTX indicated osteoanabolic activity in rats treated with the MT dose of SO.

Conclusions

Oral administration of homoeopathic doses of Symphytum officinale (especially mother tincture dose), has the potential to enhance fracture healing in adult male Wistar albino rats.

1. Introduction

Symphytum species belong to the Boraginaceae family and includes Symphytum officinale L. Symphytum officinale (SO), commonly known as comfrey, is a plant with medicinal properties [1]. Homoeopathy is an acknowledged and effective alternative system of medicine and homoeopathic medicines derived from SO have been used to treat various painful muscle and joint conditions [2]. SO by virtue of its bone healing properties, has been used to treat bone fractures, dislocated joints, and damaged muscles and tendons [3,4]. A study that identifies allantoin present in comfrey roots as responsible for triggering cell division and wound healing, thereby promoting bone and cartilage growth, is one of the few studies that have attempted to identify the active ingredients of SO, to help define its osteoanabolic properties [5]. SO root extract also contains phenolic acids, and displays remarkable antioxidant effects, in addition to inducing proliferation and differentiation of osteoblasts [6,7].

Fracture healing is a complex biological process involving the orchestrated interplay of various cells and molecular signaling pathways. Mesenchymal stem cells (MSCs) assist in the regeneration and repair of bones due to their ability to migrate to the site of bone injury and differentiate into chondroblasts [8,9]. MSCs also differentiate into osteoblasts under the influence of growth factors such as Bone Morphogenetic Proteins (BMPs) and Transforming Growth Factor-β (TGF-β), and insulin-like growth factor-1 (IGF-1), replacing the soft callus with the formation of the hard callus [10]. Osteoblasts interact with osteoclasts, which are involved in bone resorption, to maintain balanced bone remodeling [11]. Other ortho-biological agents, such as growth factors like BMP-2 and 7, recombinant parathyroid hormone (PTH), Platelet-Rich Plasma (PRP), and Platelet-Rich Fibrin (PRF), also assist in treating complicated fractures [12,13]. However, there is always a need to develop effective and less expensive osteoanabolic drugs. Despite homoeopathic preparations of SO being clinically used for bone healing, there is a lack of animal studies and data evaluating its efficacy in fracture healing.

This study examines the potential of orally administered SO (MT and 6C potencies) to affect fracture healing in adult male Wistar albino rats following the induction of an open osteotomy in the femoral shaft.

2. Methods

2.1. Study design

This was a randomized, controlled, parallel-group in vivo study designed to evaluate the effect of orally administered homoeopathic preparations of Symphytum officinale (mother tincture [MT] and 6C potency) on fracture healing in adult male Wistar albino rats following standardized open osteotomy of the right femur. In vitro findings from our previous study advocated investigating these doses of Symphytum officinale [6]. Animals were allocated into four experimental groups: control, vehicle control (alcohol), 6C-treated, and MT-treated. Each group was further sub-divided into two pre-defined time-point cohorts: 2 weeks (appearance of early/soft callus) and 5 weeks (appearance of late/hard callus) after osteotomy. The experimental unit was each individual animal. The primary outcome measure was quantitative bone formation in fracture callus, assessed by histomorphometry. Secondary outcome measures included radiographic assessment of fracture healing, histological and immunohistochemical evaluation of callus tissue, and serum biomarkers of bone formation and resorption (B-ALP and CTX).

2.2. Experimental animals

Adult male Wistar albino rats (6–8 weeks old; 150–200 g) were obtained from the Central Animal Facility (CAF), AIIMS, New Delhi. Only healthy animals without any visible signs of disease were included. Female rats were excluded to avoid hormonal variability affecting bone healing. Animals were housed individually under controlled conditions, keeping temperature at 23 ± 2 °C, and relative humidity at 50% ± 5%, and exposed to a 12h dark/light cycle. They were maintained on a semi-synthetic standard dry pellet diet (20 g/day) supplemented with 3 g of chickpeas with ad libitum access to food and water. A preoperative fasting period of 6 h (nil per oral) was followed prior to surgery.

2.3. Ethical approval and animal welfare

The study was approved by the Institutional Animal Ethics Committee (IAEC), AIIMS (Approval No. 286/IAEC-1/2021), and all procedures followed CPCSEA (Committee for the Purpose of Control and Supervision of Experiments on Animals) guidelines under the Prevention of Cruelty to Animals (PCA) Act, 1960 (India). Animal welfare was prioritized throughout the study. Perioperative and post-operative analgesia and antibiotics were provided to minimize pain, infection and distress. Animals were monitored daily for general health, surgical wound integrity, infection, loss of body weight, gait abnormalities and other behavioral changes.

2.4. Sample size determination

A total of 48 rats were used in this study (n = 12 per group). Sample size was determined based on prior published femoral osteotomy studies in rats using comparable outcome measures and group sizes, as well as feasibility and ethical considerations aimed at minimizing animal use while allowing biologically meaningful differences to be detected.

2.5. Randomization and allocation concealment

The experimental animals were randomly assigned to experimental groups using a computer-generated random number sequence. Allocation was performed by a laboratory member not involved in surgery, treatment administration, or outcome assessment, to ensure allocation concealment.

2.6. Inclusion and exclusion criteria

Inclusion criteria consisted of healthy adult male rats with successful induction of a standardized transverse femoral fracture and stable intramedullary fixation.

Exclusion criteria included perioperative mortality, failure of fracture fixation, or implant displacement identified on radiographic evaluation.

2.7. Blinding

Blinding was followed at multiple stages of the study. Radiographic assessments were performed by a Radiologist blinded to group allocation. Histological and morphometric analyses were conducted by investigators blinded to various treatment groups. Statistical analyses were done using coded datasets to ensure blinding during data interpretation.

2.8. Surgical procedure for open femoral osteotomy

Preoperatively, each rat received intraperitoneal (IP) injections of an antibiotic (cefuroxime-100 mg/kg body weight) and an analgesic (Tramadol-25 mg/kg body weight), along with a combination of Ketamine (75 mg/kg body weight) and Xylazine (10 mg/kg body weight) 15 min before starting the surgical procedure. Before the surgical procedure, hair of the rats was shaved from the right lower limb, from the flank region up to the knee joint, and an open osteotomy was done to obtain a complete fracture. A complete fracture was induced in the middle third of the shaft of the right femur using a chisel and hammer. The fracture was internally fixed with a sterile K-wire, which served as an intramedullary splint to maintain the fracture in a reduced position (Fig. 1). This fixation allowed minimal movement of the fracture ends, facilitating callus formation and healing by secondary intention allowing assessment of bone remodeling [14]. As part of postoperative care, rats were given antibiotics (injection cefuroxime 100 mg/kg) and analgesics (injection tramadol 25 mg/kg/day) intraperitoneally for 5 days after the procedure. Portions of the surgical procedure description are adapted from our previously published protocol [14]. Blood (0.5 ml) was collected from the tail vein of each rat at three time points: before surgery (baseline), two weeks post-osteotomy (T1), and five weeks post-osteotomy (T2). After surgery, rats received two drops of commercially procured homoeopathic medicine Symphytum officinale (MT or 6C) or alcohol (vehicle control) dissolved in 0.5 ml of water, administered orally once a day by gavage (only water was given to control animals). The medication was provided from the day after surgery until the day the animals were sacrificed.

Fig. 1.

Representative images of the open osteotomy model. A & B: Schematic representation of open osteotomy of femoral shaft with internal fixation in Wistar albino rats. C: A complete fracture induced in the middle third of the shaft of the right femur (a), internally fixed with a sterile K-wire (b). D: Sutured wound after surgery.

2.9. Homoeopathic preparations of Symphytum officinale and administration of drug

Homoeopathic preparations of Symphytum officinale of different potencies (mother tincture and 6C) were commercially procured from HAPCO, Kolkata, India. These preparations were extracted from the root of Symphytum officinale, following proper identification, as per the method mentioned in the Homoeopathic Pharmacopoeia of India (HPI). The powdered root was extracted with 50% ethanol by maceration (in the ratio of 1 part of powdered drug by weight to 9 parts 50% alcohol by volume). The liquid was strained and the complete extract was filtered out. This filtered extract constitutes the mother tincture (MT) potency of Symphytum officinale. Subsequently, Symphytum officinale 2x was prepared from Symphytum officinale MT in the decimal scale i.e. 1 part of MT was diluted with 9 parts of 91% ethyl alcohol (HPI), followed by ten strong succussions to the mixture. The potency of 6C was in turn prepared from 2x dilution, following the method of potentization, in the centesimal scale by serial dilution in the ratio of 1:99 with 91% ethyl alcohol (HPI), followed by ten strong succussions after each step of dilution, as mentioned in HPI.

Wistar albino rats (n = 48) were randomly divided into four experimental groups (n = 12 per group) to evaluate the effect of homoeopathic drug Symphytum officinale on fracture healing. The groups were as follows:

• •Control group: Received no experimental drug. Only standard post-operative care (analgesics and antibiotics) was provided after femoral osteotomy.
• •Vehicle control (Alcohol) group: Received two drops of 100% ethanol (used as vehicle control), diluted in 0.5 ml double-distilled water, once daily by oral gavage until they were sacrificed.
• •6C group: Received two drops of Symphytum officinale in 6C potency (homoeopathic dilution), mixed in 0.5 ml double-distilled water and administered once daily by oral gavage until they were sacrificed.
• •Mother Tincture (MT) group: Received two drops of Symphytum officinale mother tincture mixed in 0.5 ml double-distilled water, administered once daily by oral gavage until they were sacrificed.

All the administered formulations were freshly prepared from their respective potencies, just prior to administration.

Each group (n = 12) was further subdivided into two time-point cohorts:

• •T1 (n = 6): Sacrificed at 2 weeks post-osteotomy to assess the appearance of soft (early) callus.
• •T2 (n = 6): Sacrificed at 5 weeks post-osteotomy to evaluate hard (late) callus formation.

Weekly radiographs of the fractured femoral region were taken. Additionally, 0.5 ml of blood was collected from the tail vein on Day 1, Day 14, and Day 35 to monitor serum markers.

2.10. Radiographical evaluation of fracture site

An X-ray of the fractured area was taken a day after surgery and then weekly thereafter in the different treatment groups to evaluate the appearance of soft callus (the beginning of fracture healing) and hard bridging callus (the first time point when the fracture gap was healed), as assessed by the radiologist. Immediately before the x-ray, rats were anesthetized with an intraperitoneal injection of Ketamine (50 mg/kg body weight). An x-ray of the fractured limb was taken with the hip joint flexed and abducted while the knee joint was semi-flexed, using the following exposure settings: Ref. kVp ≈62; Ref. mAS = 6.4; and automatic exposure settings (Ref. mA = 160).

2.11. Animal euthanasia and callus tissue retrieval

Animals were euthanized at predetermined time points (after 2 or 5 weeks of osteotomy) using an overdose of carbon dioxide in accordance with CPCSEA guidelines. Death was confirmed prior to callus tissue harvesting. The K-wire was removed, and the fracture site in the femur was carefully dissected, ensuring not to disturb the callus. It was cleaned with saline and preserved in 10% neutral buffered formalin for 3 days, followed by bone decalcification.

2.12. Decalcification of bone and callus tissue and Hematoxylin and Eosin (H&E) staining

Callus tissue was taken from buffered formalin and kept in a 20% EDTA solution (pH 7) for approximately three weeks for decalcification, with the EDTA solution being changed every two days. After complete decalcification, the bone was cut into longitudinal sections, photographed using a D5600 digital camera (Nikon, Tokyo, Japan), and preserved in paraffin blocks. Five micro-meter thick sections were deparaffinized using xylene, followed by rehydration with graded ethanol. They were stained with hematoxylin for 10 min and then rinsed in running tap water for 3 to 5 min. The sections were then destained with 0.3% acid alcohol (1% HCl in 70% ethanol) for 1 to 2 s to remove excess nuclear stain, rinsed again, and counterstained with eosin for 30 s, followed by another rinse under tap water. Thereafter, the slides were dehydrated by passing through graded ethanol and mounted using DPX mounting medium. Images of H&E stained sections were captured using a Nikon Eclipse Ti–S fluorescence microscope (Nikon, Tokyo, Japan).

2.13. Quantitative morphometry of callus tissue

Quantitative analysis of characteristic areas corresponding to bone, fibrous tissue, and cartilage was conducted using ImageJ software (1.54j, NIH, USA). After selecting each region of interest (ROI), its corresponding percentage was calculated by dividing it by the total calculated area. Five images of the specific callus areas were analyzed for each group (biological replicates, n = 5).

2.14. Immunohistochemistry

Immunohistochemistry (IHC) was employed to detect specific antigens associated with the extracellular matrix and osteogenic markers in the tissue sections. After deparaffinizing the tissue sections with heat and xylene, they were passed through an alcohol gradient and rinsed in running tap water. Antigen retrieval involved boiling the tissue sections in citrate buffer for 20 min, followed by rinsing them under tap water for another 10 min. The sections were incubated in a permeabilization and peroxide blocking solution for 30 min (non-specific blocking was prevented using SuperBlock), then washed twice with TBST. Primary antibodies, diluted in citrate buffer, were added to the sections and incubated overnight in a humidified chamber at 4 °C. The following primary antibodies were used: anti-rat Col1A1 antibody (1:100, Cat. No. STJ111079), St John's Laboratory Ltd., (London, UK); anti-rat Osteocalcin/BGLAP (1:100, Cat. no. STJ502017), St John's Laboratory Ltd., (London, UK). After washing the sections three times with TBST, a polyvalent secondary antibody (HRP-tagged anti-rabbit secondary antibody (1:2000, Cat. no. 7074S), Cell Signaling Technology (MA, USA) was added and incubated for 30 min at room temperature. For antigen detection, 50 μl of DAB solution was added, and color development was observed. The sections were washed with TBST and counterstained with hematoxylin, followed by a rinse in tap water. Finally, the sections were cleared in xylene for 5 min, mounted with DPX, and covered with a coverslip. Images were captured using a Nikon Eclipse Ti–S fluorescence microscope (Tokyo, Japan) and analyzed with NIS-Elements BR software.

2.15. Estimation of bone-specific alkaline phosphatase (B-ALP) in rat serum using ELISA

Measurement of serum B-ALP was performed using ELISA kits (Mybiosource, Cat. No MBS040800; San Diego, USA). These kits utilize a sandwich ELISA format, in which the antigen present in the serum sample is adsorbed onto pre-coated capture antibodies on the wells of the microtitre plate. Briefly, 0.5 ml of blood was collected from the tail vein of both treated and control rats at various time points. The serum was aliquoted and stored at −80 °C. After adding 100 μl of HRP-conjugated anti-ALP antibody reagent to 50 μl of the sample or standard and incubating for 60 min at 37 °C, the plate was washed. Color development was achieved by adding 50 μl each of chromogen solutions A and B. The plate was protected from light, mixed gently, and incubated for 15 min at 37 °C. The reaction was stopped by adding 50 μl of stop solution, and absorbance was measured after 5 min at 450 nm using a multimode microplate reader (Tecan Spark, Tecan Lifesciences, Switzerland). A standard curve was plotted and used to calculate the concentration of bone-specific ALP in rat serum.

2.16. Estimation of C-terminal telopeptide of type I collagen (CTX) in rat serum using ELISA

CTX levels in rat serum were measured using sandwich ELISA according to the manufacturer's protocol (Mybiosource, MBS2602646; San Diego, USA). Briefly, 100 μl of serum samples or standards were added to wells precoated with a monoclonal antibody against CTX and incubated for 90 min at 37 °C. The wells were washed twice, and then 100 μl of biotinylated antibody against CTX was introduced to each well and incubated at 37 °C for 60 min. After three washes, streptavidin-horseradish peroxidase conjugate was added to each well, and the plate was incubated at 37 °C for 30 min. The wells were washed five times, and 100 μl of the TMB substrate was added to all wells. The plate was shielded from light and incubated for 30 min at 37 °C. One hundred microliters of stop solution was added to each well and mixed. Absorbance was measured at 450 nm within 10 min using a multimode microplate reader (Tecan Spark, Tecan Lifesciences, Switzerland). A standard curve was plotted and used to calculate the concentration of CTX in rat serum.

2.17. Statistical analysis

Statistical analyses were performed using GraphPad Prism version 8.0. Data were expressed as mean ± standard deviation. The normality of data distribution was assessed prior to analysis. The quantitative morphometric scoring of cartilage, fibrous tissue, and bone in the T1 and T2 groups in H&E was analyzed using two-way ANOVA with Tukey's multiple comparisons test, with p < 0.05 considered significant. The radiological appearance of early and late callus, along with statistical evaluation, was performed using Fisher's exact test, with p < 0.05 regarded as significant. ELISA data were analyzed using two-way ANOVA with Tukey's multiple comparisons test, with p < 0.05 considered significant.

3. Results

3.1. Surgical fracture induction by open osteotomy and administration of Symphytum officinale

The open osteotomy was performed by an orthopedic surgeon, following standard surgical procedures and precautions (Fig. 1).

3.2. Radiological evaluation of appearance of early and late callus at the fracture site

The comparative time of appearance (in weeks) of early and late callus was analyzed by a radiologist (Fig. 2 and Table 1A, Table 1B). The radiological appearance of soft and hard callus indicated that rats treated with MT exhibited earlier callus formation (both soft and hard); however, a statistically significant result was not obtained using Fisher's exact test (p = 0.1 for soft callus; p = 0.5 for hard callus) (Table 1A, Table 1B).

Fig. 2.

A representative panel of X-ray radiographs from rats in different groups (A-C: Control group; D-F: treated with vehicle control; G-I: 6C treated; J-L: MT treated) displaying the fracture site in the femur at 1 day, 2 weeks (T1), and 5 weeks (T2) after osteotomy, in Wistar rats, illustrating fracture healing through callus formation. Arrows indicate the fracture site and callus formation.

Table 1A.

Radiographic appearance (in weeks) of soft (early) callus following femoral osteotomy in different treatment groups.

	Control	Alcohol	6C	MT
Week 1	0	0	0	2
Week 2	6	4	6	4
Week 3	0	0	0	2
Total	6	4∗	6	8#

∗Two rats in this group experienced K-wire displacement. # Two additional rats were included in this group.

Table 1B.

Radiographic appearance (in weeks) of hard (late) callus following femoral osteotomy in different treatment groups. ∗Two rats in this group experienced displacement of the K-wire. #Two additional rats were included in this group.

	Control	Alcohol	6C	MT
Week 3	1	2	1	3
Week 4	5	1	3	3
Week 5	0	1	2	2
Total	6	4∗	6	8#

3.3. Macroscopic appearance of early and late callus using H&E staining

Characteristic changes were observed in both the early and late callus. The bone marrow (medullary cavity) appeared darker than the rest of the bone. The volume of the early callus exceeded that of the late callus. New bone formation was noted in the form of periosteal lifting or splitting (Fig. 3). This periosteal splitting was also noted in the late callus; however, the space between the old and new cortex decreased with the merging and formation of the neo-cortex. The cartilage in the early callus beneath the periosteum was mucoid (glistening in appearance) and more abundant compared to the late callus, where the cartilage was chalky (lustre-less) rather than mucoid (glistening), indicating new endochondral woven bone formation (Fig. 3). Grey-white spots were seen in the medullary cavity and on the sides, indicating new woven bone formation. Compared to the cartilage, the amount of bone formation in the late callus surpassed that in the early callus. In some sections, a tract (made by the K-wire) was observed within the medullary cavity (the K-wire was removed before bone decalcification). The bone marrow cavity, which appeared obliterated in the early callus, is gradually restored with the formation of new bone marrow. Fibrous, shrunken scar tissue appeared in the late callus, whereas cartilage tissue predominated in the early callus.

Fig. 3.

Representative images of the longitudinal section of the decalcified bone show the formation of early (soft) and late (hard) callus in animals of (A-B: Control group; C-D: Vehicle control treated; E-F: 6C treated; G-H: MT treated at week 2 (T1) and week 5 (T2) respectively after osteotomy. The labeled features are a) bone formation; b) periosteal splitting and the formation of neo-cortex; c) reduction in callus volume.

3.4. Microscopic appearance of early and late callus using H & E staining

Characteristic microscopic areas identified as bone, fibrous tissue and cartilage were observed & quantified (using image J software) in the early and late callus of the different treatment groups (Fig. 4). The following differences in microscopic changes between early and late callus of the different treatment groups were observed. Early callus revealed presence of more cartilage and less endochondral ossification. The new bone formed in early callus is predominantly immature woven bone arranged in a haphazard manner. Additionally, more cellularity was observed within the woven bone in early callus. Late callus showed presence of lesser cartilage and more endochondral ossification compared to early callus. Bone formation in late callus indicated more (mature) lamellar bone and less (immature) woven bone with a more organized structure.

Fig. 4.

Representative images of the microscopic view of H & E stained sections of early (week 2 – T1) and late (week 5 – T2) callus tissue in animals from different groups (A-B: Control group; C-D: Vehicle control treated; E-F: 6C treated; G-H: MT treated) after osteotomy. Scale bar: 500μm. Black arrows indicate fracture site; brown arrows indicate bone formation in the callus.

Quantitative bone morphometric analysis was performed in early (T1) and late (T2) callus across different treatment groups to assess the total amount of cartilage, fibrous tissue, and bone (including both lamellar and woven). At T1, the cartilage in the control animals measured 19.54 ± 10.39%, which decreased slightly in the alcohol-treated group to 15.91 ± 10.20% (p = 0.9617), in the 6C-treated group to 16.05 ± 5.62% (p = 0.9656), and in the MT-treated group to 14.73 ± 8.89% (p = 0.9165) (Fig. 5A). Measurement of fibrous tissue at T1 indicated that the control group had a mean of 47.48 ± 15.30%, which increased slightly in the alcohol-treated group to 50.85 ± 9.88% (p = 0.9689). In the 6C-treated group, fibrous tissue was measured at 38.88 ± 15.71% (p = 0.6567), though nonsignificant, whereas fibrous tissue was significantly reduced in MT-treated group compared with both the control group (24.53 ± 11.76%, p = 0.0172) and the alcohol-treated (vehicle control) group (p = 0.0049), indicating a pronounced reduction in fibrous tissue content after MT treatment at T1 (Fig. 5A).

Fig. 5.

Bar chart illustrating the quantitative morphometry of H&E stained sections of callus tissue, showing the tissue-specific percentages of bone, fibrous tissue, and cartilage in animals from the control group, those treated with vehicle control (alcohol), 6C treated, and mother tincture (MT) treated group at week 2 (A) (n = 5 per group) and week 5 (B) (n = 5 per group). Data is shown as mean ± SD. Statistical comparisons between percentage of (∗) bone and (#) fibrous tissue between treatment groups: ∗/#p < 0.05, ∗∗/##p < 0.01, ∗∗∗/###p < 0.001, ∗∗∗∗/####p < 0.0001.

When bone formation at T1 was examined, both control and alcohol-treated groups showed comparable bone content (32.98 ± 13.47% vs. 33.24 ± 11.74%, p > 0.9999), suggesting minimal effects of alcohol alone on early bone formation. Although bone content appeared higher in the 6C-treated group (45.08 ± 10.88%), this increase was not statistically significant compared with control (p = 0.3745). Interestingly, MT-treated group displayed a significant increase in bone content relative to both the control group (60.75 ± 13.61%, p = 0.0027) and the alcohol-treated group (p = 0.0030), highlighting a robust pro-osteogenic effect of MT at the early stage of callus formation (Fig. 5A).

A similar quantitative morphometric analysis was conducted at T2 (5 weeks) to evaluate changes in cartilage, fibrous tissue, and bone content over time. The results at T2 showed that in the control group, cartilage content was 18.15 ± 13.23%, with the alcohol-treated group showing a slight decrease to 15.35 ± 6.68% (p = 0.9848). The 6C-treated group had 13.68 ± 5.87% (p = 0.9427), while the MT-treated group had the lowest cartilage content at 8.82 ± 9.41% (p = 0.6460) (Fig. 5B), indicating an expected reduction in cartilage at this late stage of bone healing.

Fibrous tissue content at T2 was 39.39 ± 17.72% in control animals and was not significantly altered in the alcohol-treated group (50.98 ± 7.18%, p = 0.4695). Additionally, fibrous tissue content in the 6C-treated group (24.26 ± 18.37%) was significantly lower than in the alcohol-treated group (p = 0.0080), although it did not differ significantly from control (p = 0.2396) In contrast, fibrous tissue was significantly reduced in the MT-treated group compared with both the control group (15.70 ± 13.08%, p = 0.0224) and the alcohol-treated group (p = 0.0003) (Fig. 5B).

Analysis of bone formation at T2 showed that bone content in control animals was 42.46 ± 11.52% and remained comparable in the alcohol-treated group (34.01 ± 7.41%, p = 0.7127). Although the 6C-treated group showed an increase in bone content (62.06 ± 17.06%), this increase did not reach statistical significance relative to control (p = 0.0782), despite being significantly greater than the alcohol-treated group (p = 0.0050). However, MT-treated group displayed the highest bone content at T2 (75.48 ± 14.04%), which was significantly higher than both the control group (p = 0.0007) and the alcohol-treated group (p < 0.0001) (Fig. 5B).

3.5. Expression of collagen 1A1 (Co1A1) & osteocalcin in early and late callus

Collagen is present in the extracellular matrix (ECM) of connective tissues such as bone and cartilage. Type I collagen (Col1A1) is commonly found in bone tissue. Collagen secreted in the ECM of woven and lamellar bone during both early and late callus phases was identified using immunohistochemistry. During the early callus, Col1A1 exhibited a haphazard pattern in immature woven bone, which transformed into more organized staining in the ECM of mature lamellar bone during the late callus (Fig. 6). When comparing the staining across different treatment groups in early and late callus, we observed early woven bone formation in the MT and 6C groups during the early callus phase, as well as early lamellar bone formation in the MT and 6C groups during the late callus phase. Even though Col1A1 expression was present in new bone, we could not determine statistically significant differences between the groups due to the challenges associated with quantifying differences in Col1A1 expression.

Fig. 6.

Representative images of immunohistochemistry showing Col1A1 in early (week 2 – T1) and late (week 5 – T2) callus tissue from animals in A-B: Control group; C-D: Vehicle control treated; E-F: 6C treated; G-H: MT treated.

Osteocalcin is specifically expressed by osteoblasts and is considered a marker of osteogenesis. Osteoblasts present in early and late callus expressed osteocalcin (Fig. 7). When comparing the staining in the different treatment groups of early and late callus, we observed increased osteoblastic activity in the MT and 6C treatment groups in both early and late callus; however, we could not identify statistically significant differences between the groups due to the challenges associated with identifying nuclear staining in multiple areas and quantifying the differences.

Fig. 7.

Representative images of immunohistochemistry of Osteocalcin from early (week 2 – T1) and late (week 5 – T2) callus tissue in animals (A-B: Control group; C-D: Vehicle control treated; E-F: 6C treated; G-H: MT treated).

3.6. Bone specific alkaline phosphatase (B-ALP) and C-terminal telopeptide of type I collagen (CTX) in serum

In fracture healing, bone remodeling is regulated by the coordinated activity of osteoblasts and osteoclasts. We evaluated an osteoblastic marker B-ALP, and an osteoclastic marker CTX, using ELISA in the serum of different treatment groups of rats at baseline (before osteotomy), 2 weeks (14 days), and 5 weeks (35 days) (Fig. 8A and B and Table 2A, Table 2BA and 2B).

Fig. 8.

Graphical representation of (A) Bone-specific alkaline phosphatase (B-ALP) and (B) C-telopeptide (CTX) estimated in the serum of different groups of animals (Control, Vehicle control treated (VEH), 6C treated, and MT treated) before osteotomy (day 0), on day 14 (week 2), and on day 35 (week 5) after osteotomy. (n = 4-5 per group). Data is represented as mean ± SD. ∗p < 0.05, ∗∗p < 0.05.

Table 2A.

Bone Alkaline phosphatase (B-ALP) levels in the serum of untreated rats (controls) and different treatment groups before osteotomy (day 0), 14 days and 35 days after osteotomy. ^a

Treatment	Day 0 (ng/mL)	Day 14 (ng/mL)	Day 35 (ng/mL0
Control	0.7266 ± 0.2388	0.9854 ± 0.3362 (p = 0.23)	0.7768 ± 0.1563 (p = 0.86)
Vehicle	0.9194 ± 0.2706	0.7660 ± 0.2129 (p = 0.47)	0.9608 ± 0.2605 (p = 0.61)
Mother tincture	0.7740 ± 0.08658	1.340 ± 0.1133 (p = 0.02)	1.361 ± 0.1286 (p = 0.04)
6C	0.8323 ± 0.2494	0.9368 ± 0.1883 (p = 0.98)	0.8643 ± 0.1865 (p = 0.93)

^aData is presented as mean±SD and p values are calculated on comparison with baseline (day 0) in each group.

Table 2B.

CTX levels in the serum of untreated rats (controls) and different treatment groups before osteotomy (day 0), 14 days and 35 days after osteotomy. ^a

Treatment	Day 0	Day 14	Day 35
Control	39.86 ± 16.95	57.93 ± 14.73 (p = 0.65)	49.74 ± 21.09 (p > 0.99)
Vehicle	61.62 ± 18.22	53.54 ± 13.15 (p = 0.96)	47.96 ± 8.599 (p = 0.99)
Mother tincture	42.58 ± 11.66	61.20 ± 10.70 (p = 0.98)	57.27 ± 9.058 (p = 0.86)
6C	55.58 ± 16.08	36.48 ± 8.944 (p = 0.14)	48.74 ± 8.840 (p > 0.9996)

^aData is presented as mean±SD and p values are calculated on comparison with baseline (day 0) in each group.

We observed a significant increase in serum B-ALP levels in rats treated with Symphytum mother tincture at day 14 and day 35 as compared to baseline (day 0). B-ALP levels also increased in MT mice as compared to controls at day 14 (p = 0.04).

Serum CTX levels were also found to higher in Mother tincture treated rats as compared to controls at both day 14 and day 35, however the differences were not found to be statistically significant.

4. Discussion

The human skeletal system has a remarkable ability to regenerate after injury. However, complications in fracture healing are common and contribute to a significant healthcare burden [15]. Bone repair and regeneration following a fracture begin as a local response to tissue damage, and the time required for healing varies based on the type of fracture and several other factors. While simple fractures heal with standard procedures like reduction and immobilization, factors such as age, osteoporosis, and unfavorable location, prolong the healing process [16]. In animal models, delayed healing has been observed in older animals [[16], [17], [18]]. Furthermore, fractures of the skull and vertebral column require extended immobilization periods, making bone healing in these cases particularly challenging [19]. Given these conditions, osteogenic inducers and osteoanabolic agents that accelerate and enhance bone healing can be extremely beneficial [20,21]. One such agent, Symphytum officinale (SO), derived from a medicinal plant, has been found to aid in the healing of bone fractures in homoeopathic literature [7,22,23]. Its ability to augment osteogenesis in vitro has also been established using human bone marrow-derived mesenchymal stem cells [6]. This is the first study to evaluate the role of Symphytum officinale (SO) in accelerating fracture healing in a rat model undergoing open osteotomy, while assessing multiple parameters of bone healing. This rodent model helps to evaluate bone healing following a complete long bone fracture. Specifically it helps to study callus formation using radiological, histological, immunohistochemical, and serological analysis [14].

Radiological evaluation indicated that treatment with MT dose of SO resulted in earlier appearance of soft (early) and hard (late) callus at the fracture site. This finding agrees with previous reports assessing bone density around titanium implants in rats [22,23]. These findings also align with a prior case report of a comminuted bone fracture in the humerus treated with the homeopathic remedy Symphytum officinale, which was found to accelerate bone healing, as confirmed through clinical and radiological assessments [4].

Quantitative morphometry assessment of H&E stained sections from the early callus of the different treatment groups revealed that control and alcohol (vehicle control) behaved similarly. In early callus, all four treatment groups displayed comparable amounts of cartilage tissue, indicating a resemblance in endochondral ossification during early fracture healing. Fibrous tissue was highest in the two controls, slightly lower in 6C, and lowest after MT treatment. Concomitantly, woven bone levels in the early (soft) callus were lowest in the control groups. While the 6C group showed a slight increase, the MT treatment produced the highest amount of woven bone. These novel findings suggest that treatment with MT and 6C doses of SO induces greater bone formation and less scar or fibrous tissue during early bone healing. Although treatment with the 6C dose is better than the controls, MT treatment is significantly superior in terms of the amount of bone formation. Interestingly, quantitative morphometry findings in late callus of the different treatment groups revealed a gradual decline in cartilage tissue in alcohol (vehicle control) and 6C groups, while a significant reduction in cartilage tissue was observed in the MT-treated group, indicating better endochondral ossification after SO treatment. Our novel findings also revealed a significant decrease in fibrous tissue in late callus in 6C and MT treated groups, demonstrating lesser scarring and better bone healing. For the first time, quantitative bone morphometric findings highlighted significantly increased lamellar bone formation in late callus in 6C and MT treated groups. This is the first study to use quantitative morphometry to report the osteoanabolic activity of MT and 6C doses of SO in a fracture model. These results reinforce earlier in vitro findings [6] and studies on rat titanium implants [22,23]. They also align with clinical and radiological evidence of accelerated bone healing found in previous case reports [4].

Symphytum officinale (especially mother tincture) is known to promote osteogenesis by enhancing the differentiation of MSCs into osteoblasts in vitro. These osteoblasts then synthesize the bone matrix [6]. Thus, bone-specific alkaline phosphatase (B-ALP) activity, a marker of osteoblast function, was assessed in serum samples of the various treatment groups. MT-treated animals exhibited a significant increase in B-ALP activity at time points corresponding to soft (early) and hard (late) callus, indicating greater osteoblastic activity following MT treatment at both these time points. Additionally, it was noted that treatment with 6C did not produce any significant increase in B-ALP activity. While alcohol treatment did not result in an increase in B-ALP activity at any time point, standard controls demonstrated a mild increase in B-ALP activity at two weeks, followed by a decline after five weeks. These findings indicate that while B-ALP activity is initially elevated in controls, it does not remain consistently high over a longer period. In contrast, its activity remains persistently and significantly elevated in the serum of the MT treated group. Multiple reports highlighting the utility of assessing ALP activity as a measure of osteogenic activity have been published [24,25]. Our novel findings suggest that MT treatment of SO enhances osteoblastic activity and bone formation in the animal model.

As serum CTX (C-terminal telopeptide of type I collagen) serves as a marker of osteoclastic activity, its levels were evaluated to gain insight into bone remodeling. Analyzing CTX levels revealed distinct patterns among the treatment groups. The control group showed an increase in CTX levels, followed by a decline, indicating an intermediate rise in osteoclastic activity normally associated with fracture healing. Such a trend was not observed in the vehicle control (alcohol). 6C treatment demonstrated a decline in CTX levels at both time points, suggesting reduced bone remodeling at these time points. Interestingly, treatment with MT dose of SO showed early and consistently increased CTX levels, indicating consistently increased osteoclastic activity and hence greater bone remodeling after MT treatment of SO. During bone remodeling, osteoblasts continue to build new bone, while osteoclasts reshape and refine the bone to restore its original structure and strength [26,27]. Our findings suggest that MT treatment may uniquely influence the balance of bone remodeling by promoting early osteoclastic activity. Measurement of serum CTX is subject to high intrinsic biological variations [28] which could partially account for the variability in serum CTX levels observed in this study.

To the best of our knowledge, this is the first multiparametric animal study demonstrating that the MT dose of SO has osteoanabolic effects on fracture healing. Our findings indicate that Symphytum officinale (especially mother tincture) not only enhances osteoblastic and osteoclastic activity but also accelerates bone healing, emphasizing its potential as an osteoanabolic agent. The limitation of this study is that immunohistochemistry of Col1A1 and Osteocalcin could not identify statistically significant differences among the various treatment groups, as it is challenging to accurately quantify differences in their staining due to multiple factors.

5. Conclusion

The present study provides experimental evidence that Symphytum officinale, particularly its mother tincture, was associated with enhanced osteogenic activity and improved characteristics of fracture callus in a rat femoral osteotomy model. These findings represent empirical observations within the limitations of an animal study. Although several biological mechanisms may be hypothesized to contribute to the observed effects, the current data does not directly establish a definitive mechanistic pathway. In particular, the biological basis of the effects observed with highly diluted preparations remains uncertain, and warrants cautious interpretation. Therefore, Symphytum officinale may merit further investigation as a complementary adjunct in fracture repair. Additional studies incorporating pharmacological characterization, dose-response evaluation, and mechanistic assays will be required before any clinical relevance or therapeutic positioning with approved osteoanabolic therapies such as recombinant BMP-2 or teriparatide can be reliably inferred. Further rigorous preclinical and controlled clinical studies are required to clearly demarcate its therapeutic value, appropriate indications, and possible role as an adjunct in the management of challenging conditions, including non-union, compound fractures, and skull and vertebral fractures, particularly in resource-limited settings.

Author contributions

ST: Writing the original draft, methodology, investigation, validation, formal analysis.

PKP: Methodology, investigation, validation, formal analysis, review.

VS: Methodology, investigation, validation, formal analysis, review and editing.

DD: Methodology, investigation, review.

AA: Methodology, investigation, validation, review.

AB: Methodology, investigation, validation, review.

SR: Investigation, review.

BG: Supervision, resources, review.

PK: Methodology, resources, review.

AG: Conceptualization, supervision, resources, methodology, review and editing.

DN: Methodology, review.

RKS: Methodology, resources, review and editing.

ARM: Conceptualization, supervision, resources, methodology, review and editing.

SS: Funding acquisition, conceptualization, supervision, project administration, resources, review and editing.

Data statement

Data will be made available on request.

Ethics statement

This research was approved by the Institutional Animal Ethics Committee (IAEC), AIIMS (Approval No. 286/IAEC-1/2021), and all procedures followed CPCSEA (Committee for the Purpose of Control and Supervision of Experiments on Animals) guidelines under the Prevention of Cruelty to Animals (PCA) Act, 1960 (India).

Declaration of generative AI in scientific writing

The authors declare that no generative artificial intelligence (AI) tools were used in the writing, editing, data analysis, or preparation of this manuscript.

Sources of funding

The authors are grateful to Central Council for Research in Homoeopathy (CCRH), Ministry of AYUSH, Govt. of India, for financial support – extramural research grant (Grant ID – 17-8/2018-19/CCRH/Tech/Coll/Symphytum-AIIMS).

Conflict of interest

The authors declare that they have no conflicts of interest.