Efficacy of intralesional propolis on tendon healing: A study of macroscopic and histological parameters in partial Achilles tendon rupture model in Wistar rats

INTRODUCTION

Tendon injuries are increasingly recognized as a common challenge in contemporary orthopedic practice worldwide. Among them, the Achilles tendon – despite its structural strength – is the most frequently affected. The incidence of Achilles tendon injuries has risen markedly in recent decades, from approximately 11 to 37 cases/100,000 population.^[1] Surgical repair or reconstruction remains the standard of care for restoring optimal function, preserving quality of life, and minimizing complications.^[2] In recent years, various adjuvant therapies have been investigated to enhance tendon healing, including mesenchymal stem cells (MSCs),^[3] platelet-rich plasma,^[4] 2-octyl cyanoacrylate,^[3-5] and intermittent pneumatic compression.^[4]

Propolis, a natural resinous compound produced by honey bees by combining plant exudates (such as sap, resin, and bark or leaf secretions) with bee-derived enzymes, has attracted scientific interest due to its diverse biological effects.^[5] Its principal constituents – polyphenols and flavonoids – are known to exhibit antioxidant, antibacterial, antifungal, antihyperglycemic, and even cytotoxic activities against cancer cells.^[6,7] In the field of orthopedics, propolis has been reported to promote the proliferation of bone marrow-derived MSCs, support chondrogenic and adipogenic differentiation, enhance cell migration,^[8] and demonstrate potent antibacterial activity against Staphylococcus aureus and Staphylococcus epidermidis, two common pathogens in orthopedic infections.^[9] Moreover, the addition of propolis to bone grafts is safe and increases MSC concentration at the graft site.^[10]

However, studies evaluating propolis as an adjuvant therapy in Achilles tendon repair have not been widely conducted. Desteli et al. reported no significant improvement in tendon healing with oral propolis supplementation.^[11] No studies have used propolis locally or intralesionally on tendons. However, Meimandi-Parizi et al. found that local administration of propolis demonstrated that local propolis application enhanced bone formation in a rat radius defect model.^[12]

Therefore, our research question was: how is the effect of intralesional propolis administration on tendon healing, using macroscopic and histological assessments, in a partial Achilles tendon rupture model in Wistar rats?

MATERIALS AND METHODS

We conducted an actual experimental study with a post-test only control group design in the Pharmacology Laboratory, Universitas Sumatera Utara, Medan, North Sumatera, Indonesia. We calculated the sample size using the Federer formula [(n-1) (t-1) ≥15], where n=sample size for each intervention and P=number of interventions. In our study, there were three interventions, so the minimum sample size per group was 8 subjects. We added a subject for each group to anticipate any dropouts. Therefore, we included 27 healthy adult male Wistar rats (2–3 months old) weighing 200– 250 g. All samples underwent surgical partial tenotomy of the Achilles tendon. We randomly assigned the rats to three equal groups: A test group (given an intralesional injection of 50% propolis solution), a placebo group (given an intralesional injection of normal saline), and a control group (no additional treatment). All rat groups were euthanized after 35 days of observation. Then, Achilles tendon tissue was collected for macroscopic and histological examination.

Achilles partial tenotomy and adjuvant therapy administration

All experimental animals were acclimatized in the laboratory for 2 weeks before surgery. Anesthesia was induced with an intraperitoneal injection of ketamine (40 mg/kg) combined with midazolam (2 mg/kg), administered 5 min before the surgical procedure. Analgesics and antibiotic prophylaxis were provided with subcutaneous injections of tramadol (5 mg/kg) and gentamicin (4 mg/kg), respectively. Postoperatively, gentamicin administration was continued once daily for 5 consecutive days to prevent infection.

The distal leg was disinfected with chlorhexidine and shaved. Then, the rat was placed on an operating table and covered with a sterile surgical drape. The skin was incised longitudinally in the midline approximately 5 mm proximal to the insertion of the Achilles tendon at the calcaneus to expose the Achilles tendon. Partial transverse tenotomy of the Achilles tendon was performed at the midpoint of the tendon, approximately 50% of the total width of the tendon. Samples in the test group then received a 0.1 cc local intralesional injection of 50% propolis solution, whereas those in the placebo group received a 0.1 cc injection of normal saline [Figure 1]. Samples in the control group did not receive additional administration. The skin was closed using 5.0 monofilament non-absorbable suture and covered with a sterile dressing. Postoperatively, analgesics are administered (paracetamol 7.5 mg) orally divided into three doses for 3 days. The rats were not given any casting immobilization. The general condition of the rats and the surgical wound were evaluated daily.

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Figure 1:

(a and b) Achilles partial tenotomy and (c) intralesional propolis solution administration.

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Histological examination

The entire length of each Achilles tendon specimen was processed using standard histological techniques. Tissue sections were prepared and stained with hematoxylin and eosin for examination. Histological evaluation was performed using a light microscope (Olympus^® CX23, Tokyo, Japan) at magnifications of ×40, ×100, and ×400, depending on the parameter being assessed. Microscopic assessment was conducted according to the six parameters of the Movin score: Fiber structure, fiber arrangement, nuclear rounding, inflammation, angiogenesis, and cell density [Table 2, Figure 2].^[14] We blinded the microscopic assessment to the two observers (SU and MS).

Table 2: Movin score.

Parameters	Tendon repair assessment score
	0	1	2	3
Fiber structure	Continue, long fiber	Slightly fragmented	Moderately fragmented	Severely fragmented
Fiber arrangement	Compacted and parallel	Slightly loose and wavy	Moderately loose, wavy, and cross to each other	No identifiable pattern
Nuclear rounding	Long spindle-shaped cells	Slightly rounding	Moderately rounding	Severely rounding
Inflammation (%)	<10	10–20	20–30	>30
Angiogenesis (%)	<10	10–20	20–30	>30
Cell density	Normal pattern	Slightly increase	Moderately increase	Severely increase

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Figure 2:

Histological appearance in Movin score.

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RESULTS

Macroscopic evaluation

A total of 27 samples were included in this study, equally divided into three groups. In the test group, all subjects achieved complete tendon healing; by contrast, 33.3% and 55.6% of samples in the placebo and control groups, respectively, failed to achieve complete tendon healing (P = 0.034) [Table 3, Figure 3]. No adhesion occurred in 88.9% of samples in the test group, which was significantly higher than in other groups (P = 0.037) [Table 3, Figure 4].

Table 3: Results of macroscopic evaluation.

Parameters	Test group n (%)	Placebo group n (%)	Control group n (%)	P-value
Tendon healing
Complete	9 (100)	6 (66.7)	4 (44.4)	0.034*
Incomplete	0 (0)	3 (33.3)	5 (55.6)
Adhesion status
No adhesions	8 (88.9)	3 (33.3)	2 (22.2)	0.037*
Mild adhesions	1 (11.1)	3 (33.3)	5 (55.6)
Moderate adhesions	0 (0)	3 (33.3)	2 (22.2)
Severe adhesions	0 (0)	0 (0)	0 (0)

*significant P-value (P<0.05)

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Figure 3:

Macroscopic appearance of tendon healing. (a) Complete tendon healing (red arrow) and (b) incomplete tendon healing (red arrow).

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Figure 4:

Macroscopic appearance of adhesion status. (a) No adhesion; (b) mild adhesion; and (c) moderate adhesion.

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Histological evaluation

There were significant differences based on the parameters of fiber structure (P = 0.004), nuclear rounding (P = 0.027), angiogenesis (P = 0.003), and cell density (P = 0.022). Contrary to our expectations, we did not find significant differences in fiber arrangement (P = 0.056) or inflammation (P = 0.127) [Table 4, Figures 5-10]. The interobserver reliability test between the two observers showed almost perfect agreement for the fiber structure parameter (κ = 0.947) and the angiogenesis parameter (κ = 0.95), and perfect agreement (κ = 1) for the other parameters (P= 0.0001).

Table 4: Results of histological evaluation.

Parameters	Test group n (%)	Placebo group n (%)	Control group n (%)	P-value
Fiber structure
Continue, long fiber	7 (77.8)	1 (11.1)	0 (0)	0.004*
Slightly fragmented	2 (22.2)	1 (11.1)	4 (44.4)
Moderately fragmented	0 (0)	6 (66.7)	4 (44.4)
Severely fragmented	0 (0)	1 (11.1)	1 (11.1)
Fiber arrangement
Compacted and parallel	6 (66.7)	1 (11.1)	0 (0)	0.056
Slightly loose and wavy	2 (22.2)	5 (55.6)	6 (66.7)
Moderately loose and wavy, cross to each other	1 (11.1)	2 (22.2)	2 (22.2)
No identifiable pattern	0 (0)	1 (11.1)	1 (11.1)
Nuclear rounding
Long spindle cell	6 (66.7)	1 (11.1)	0 (0)	0.027*
Slightly rounding	2 (22.2)	3 (33.3)	6 (66.7)
Moderately rounding	1 (11.1)	3 (33.3)	2 (22.2)
Severely rounding	0 (0)	2 (22.2)	1 (11.1)
Inflammation
<10%	2 (22.2)	1 (11.1)	0 (0)	0.127
10–20%	7 (77.8)	3 (33.3)	6 (66.7)
20–30%	0 (0)	2 (22.2)	2 (22.2)
>30%	0 (0)	3 (33.3)	1 (11.1)
Angiogenesis
<10%	6 (66.7)	1 (11.1)	0 (0)	0.003*
10–20%	3 (33.3)	3 (33.3)	3 (33.3)
20–30%	0 (0)	2 (22.2)	5 (55.6)
>30%	0 (0)	3 (33.3)	1 (11.1)
Cell density
Normal pattern	2 (22.2)	1 (11.1)	0 (0)	0.022*
Slightly increase	6 (66.7)	0 (0)	2 (22.2)
Moderately increase	1 (11.1)	3 (33.3)	3 (33.3)
Severely increase	0	5 (55.6)	4 (44.5)

*significant P-value (P<0.05)

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Figure 5:

Histological findings of fiber structure (yellow arrows) (×100 magnification using haematoxylin-eosin staining) (a) continue, long fiber; (b) slightly fragmented; (c) moderately fragmented; and (d) severely fragmented.

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Figure 6:

Histological findings of fiber arrangement (green arrows) (×100 magnification using haematoxylin-eosin staining) (a) Compacted and parallel; (b) slightly loose and wavy; (c) moderately loose, wavy, and cross to each other; and (d) no identifiable pattern.

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Figure 7:

Histological findings of nuclear rounding (blue arrows) (×400 magnification using haematoxylin-eosin staining) (a) Long spindle cell; (b) slightly rounding; (c) moderately rounding; and (d) severely rounding.

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Figure 8:

Histological findings of inflammation (red arrows) (×40 magnification using haematoxylin-eosin staining) (a) <10%; (b) 10–20%; (c) 20–30%; and (d) >30%.

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Figure 9:

Histological findings of angiogenesis (green arrows) (×40 magnification using haematoxylin-eosin staining) (a) <10%; (b) 10– 20%; (c) 20–30%; and (d) >30%.

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Figure 10:

Histological findings of cell density (blue arrows) (×400 magnification using haematoxylin-eosin staining) (a) normal pattern; (b) slightly increase; (c) moderately increase; and (d) severely increase.

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DISCUSSION

In our study, we found a significant difference in tendon healing between the three groups (P = 0,034), contrary to Desteli et al., who found no significant effect of orally given propolis supplementation on tendon healing.^[11] No studies have examined intralesional administration of propolis for tendon injury. However, Yang et al. concluded that directly given propolis improved wound healing.^[15]

There was a significant difference in adhesion status between the three groups (P = 0.037). Yang et al. reported similar findings, indicating that propolis reduces adhesion and fibrosis during wound healing.^[15] Askari et al. also found that intraperitoneal administration of propolis reduced intraperitoneal adhesion in the postoperative period, attributable to its antioxidant and anti-inflammatory effects.^[16]

We found a significant difference in fiber structure (P = 0.004). Propolis contains high concentrations of flavonoids and other phenolic compounds. It improved the collagen fiber structure through collagen synthesis and deposition, particularly of type I collagen, resulting in thicker, more mature, and more organized fibers than type III collagen. In addition, it reduced the activity of matrix metalloproteinases, which are responsible for degrading collagen.^[17]

There was no significant difference in fiber arrangement (P = 0.056). However, a larger percentage of samples in the test group were categorized as having a normal, compacted, and parallel arrangement compared to the other groups. ElSakhawy et al. found that propolis administration showed a more organized fiber arrangement.^[17] During tendon healing, fiber arrangement gradually changes from irregular to organized, parallel, and axial arrangements, particularly during the proliferation and remodeling phases. These phases were also promoted by mechanical burden and cellular activity to achieve an integrated arrangement with the surrounding tissue.^[18]

We found a significant difference in nuclear rounding (P = 0.027). Nuclear rounding is a morphological change in which the cell nuclei in injured or degenerated tendons transition from an elongated (spindle) shape to a more rounded shape. This change indicates the loss of nuclear confinement within the dense, parallel collagen fibers of healthy tendons. It is associated with altered cell phenotype, high cellularity, matrix disruption, and impaired tissue function. Nuclear rounding is a characteristic feature of tendinopathy and can be caused by excessive mechanical use, injury, and inflammation.^[19] This showed that subjects who received propolis had better cellular morphology than other groups.

There was no significant difference in inflammation (P = 0.127). The inflammatory process during the tendon-healing phase decreased as the healing process progressed, particularly on entering the remodeling phase.^[20] This also occurred in the other group samples. This may be a factor contributing to the statistically insignificant differences found in our study.

We found a significant difference in angiogenesis (P = 0.003). Angiogenesis is crucial for facilitating tendon healing, as it supplies oxygen and nutrients, removes metabolic waste products, and regulates the immune response. Vascular endothelial growth factor is among the most important angiogenic factors regulating blood vessel formation during tendon healing. However, prolonged or abnormal angiogenesis leads to persistent hypervascularity, which can potentially cause prolonged inflammation, pain, and abnormal extracellular matrix formation, as seen in chronic tendinopathy.^[21] Our study demonstrated that propolis exerted a controlled effect on angiogenesis during tendon healing.

We found a significant difference in cell density (P = 0.022). In tendon healing, cell density refers to the number of cells in injured tendon tissue, which increases significantly compared to intact tendons during the proliferation phase.^[22] The remodeling phase is characterized by a decrease in cell density and an increase in type I collagen production, as the tissue transforms to resemble a healthy tendon. During this prolonged phase, the less organized type III collagen is replaced by type I collagen, which is stiffer and more structurally robust, thereby restoring mechanical strength.^[23] This indicates that the subjects given propolis in this study had entered the remodeling phase. In contrast, in other groups, where cell density tends to be higher, they were still in the proliferation phase or transitioning to the remodeling phase.

We performed a reliability test using the Kappa test and showed almost perfect to perfect agreement among the histological parameters. This supported the fact that the Movin score had already been established for its validity and reproducibility.^[24,25]

A limitation of our study is that it cannot definitively elucidate the mechanism underlying the propolis effect on tendon healing, as it is an in vivo study in Wistar rats. However, this study found that intralesional administration of propolis in a partial Achilles tendon rupture model has a beneficial effect. Further studies may also focus on the effect of propolis on chronic tendon injuries and tendinopathy.^[13] Propolis has also been shown to have antimicrobial activity ^[11] and further research could investigate its effects on tendon healing in cases of infectious tendinitis.

Besides, future research should isolate flavonoid and phenolic compounds found in propolis, quantify their contribution, and evaluate whether targeted formulations at higher concentrations improve tendon healing outcomes. In a study conducted by Subramanian et al., a flavonoid-rich fraction was isolated from Dodonaea viscosa and evaluated for its effect on wound healing. They found that the formulated flavonoid-rich fraction promoted wound healing in wound granulation tissue.^[26]

CONCLUSION

Intralesional propolis administration in a partial Achilles tendon rupture model in Wistar rats showed a significant beneficial effect on tendon healing, adhesion status, fiber structure, nuclear rounding, angiogenesis, and cell density. This is the first study to support the current theories on the effect of intralesional propolis on tendon rupture. However, further research should focus on the effect of propolis on chronic tendon injuries associated with infectious tendinitis.