Histological Evaluation of the Effects of Intra-Articular Injection of Caffeic Acid on Cartilage Repair in a Rat Knee Microfracture Model

Introduction

Cartilage is a specialized connective tissue with a unique structure consisting primarily of chondrocytes embedded within an extracellularmatrix (ECM) composed of collagen, proteoglycans, and glycosaminoglycans (GAGs) [1]. It plays a crucial role in maintaining the integrity and function of joints by providing a smooth, low-friction surface essential for movement [2]. Among the different types of cartilage – hyaline, elastic, and fibrous – hyaline cartilage is the most prevalent, found in articular joints and known for its high collagen content, predominantly type II collagen, and its capacity to resist compressive forces [3].

The ECM of articular cartilage is a complex and highly organized structure that allows for the transmission of mechanical loads while maintaining tissue homeostasis [4]. The layers of articular cartilage, from the superficial to the calcified zones, display distinct variations in the arrangement and composition of collagen fibers and proteoglycans, which provide resistance to shearing forces and contribute to cartilage’s resilience under compressive stress [5]. Damage to articular cartilage is particularly challenging due to its avascular nature, which impedes the natural healing process and often leads to degenerative changes such as osteoarthritis. Acute cartilage defects, whether caused by trauma or degeneration, trigger inflammatory processes within the joint, with pro-inflammatory cytokines like IL-1β and TNF-α playing a pivotal role in the breakdown of cartilage matrix components. These molecular events often lead to compromised joint function and long-term clinical problems, necessitating innovative treatments to restore cartilage integrity [6].

Cartilage injury resulting from joint damage aims to restore functionality through the stages of inflammation, cell proliferation, and the resynthesis and remodeling of the extracellular matrix. At the cellular level, the repair cartilage is more densely populated with type 1 collagen but is irregular. Additionally, there is a condition of zonal organization along with neovascularization [7]. Microfracture surgery is one such treatment aimed at stimulating cartilage repair by promoting the infiltration of mesenchymal stem cells into the damaged area, thus facilitating fibrocartilage formation [8]. Given the limitations of articular cartilage’s intrinsic healing capacity, novel approaches are needed to enhance cartilage regeneration following injury. Current methods like microfracture surgery rely on fibrocartilage formation, which lacks the durability and biomechanical properties of native hyaline cartilage [8]. Recent research has focused on the use of biologically active substances to support cartilage repair by modulating the inflammatory environment and promoting the formation of more durable cartilage. In these studies, the International Cartilage Repair Society (ICRS) scoring system is generally used to evaluate cartilage healing both macroscopically and microscopically [9]. In the first stage (ICRS-I), the macroscopic structure of the cartilage surface is assessed, while in the second stage (ICRS-II) the quality of the healing tissue that emerges based on the fundamental differences between fibrocartilage and hyaline cartilage is determined microscopically by scoring its morphological structure, architecture, cell morphology, and matrix staining.

Caffeic acid phenethyl ester (CAPE), a polyphenolic compound with known anti-inflammatory and antioxidant properties, has demonstrated potential in reducing oxidative stres and modulating inflammatory pathways, which are crucial in the early phases of cartilage injury and repair [10]. CAPE has shown the ability to attenuate these effects by modulating inflammatory pathways, as highlighted in various studies on its interactions with molecular targets, including the NF-κB and MAPK pathways [11]. In this context, exploring the intra-articular administration of CAPE as an adjunctive treatment in cartilage repair models may offer new insights into its effects on cartilage regeneration [12]. Therefore, this study was designed to evaluate the effect of intra-articular administration of CAPE on histopathological outcomes of cartilage healing in a rat microfracture model. We hypothosized that intra-articular administration of CAPE significantly improves the histopathological quality of cartilage repair in a rats by reducing inflammation and promoting the formation of durable cartilage compared to control treatments.

Material and Methods

ETHICS STATEMENT:

Ethics approval for the study was obtained from the local ethics committee.

STUDY DESIGN:

This study aimed to evaluate the histopathological effects of CAPE on cartilage healing in a rat model of acute cartilage defects. Due to incomplete knowledge of the study population, power analysis and sample size determination were conducted based on the study by Ulku et al (2017), which utilized 36 knees. According to this study, it was determined that a sample size of 32 subjects would provide a 90% power to represent the study (with values of 0.70 and above considered valid, and 0.80 deemed highly sufficient). The effect size was calculated to be 0.41, indicating a considerable effect size (0.10 small, 0.25 medium, and 0.40 large). The study was designed to include 4 groups (A–B–C–D), with an equalal location planned, resulting in an intra-group sample size of 11. To account for potential subject loss, 12 subjects per group were planned, leading to a total of 48 knees, conducted with 24 rats due to the bilateral design of the study [13].

EXPERIMENTAL PROCEDURES:

Twenty-four male Sprague-Dawley rats, averaging 3 months of age, were housed in groups of 3 in 8 separate cages. To acclimate to their environment, the animals were maintained for 2 weeks in a controlled environment with a 12-hour light/dark cycle, at a temperature of 23°C, with ad libitum access to food and water. Cages were cleaned twice daily, and the average weight of the rats was 285 grams.

CAPE was obtained from MedChem Express to ensure purity and consistency. We prepared a 5% dimethyl sulfoxide (DMSO) solution to dissolve the CAPE, achieving a concentration of 150 μg/kg for intra-articular injection. This dosage was selected based on prior research indicating its efficacy in similar models and to ensure a therapeutic concentration that would be both effective and safe for cartilage tissue in this experimental setup [14].

Anesthesia was induced with 80 mg/kg ketamine and 10 mg/kg xylazine administered intraperitoneally. A surgical antibiotic prophylaxis of 30 mg/kg cefazolin sodium was also given intraperitoneally. Both knees were shaved, and the surgical area was disinfected with 10% betadine. A midline incision was made on the anterior aspect of the knee. After incising the skin and subcutaneous tissue, a medial parapatellar incision was performed to facilitate arthrotomy. The patella was laterally retracted to access the distal femoral condyles. This animal study was designed in compliance with the ARRIVE guidelines. To fully assess the effect of CAPE on the repair tissue, rats were divided into 4 groups. In Group A, only a cartilage defect was created, allowing observation of natural healing without any external intervention. In Group B, the healing process of the defect was observed following creation of a microfracture. In the other groups, the effect of CAPE on both the defect alone (Group C) and on the defect repaired with microfracture (Group D) was examined.

A 1.6-mm Kirschner wire was employed to create a cartilage defect in the trochlear groove (Figure 1). For Group B and Group D, microfracture was performed using a 1-mm Kirschner wire with a trocar tip, at a depth of 0.5 mm, extending until the bone marrow was completely exposed. The joint was then irrigated with saline, and the joint capsule was repaired using 4.0 Vicryl sutures (Figure 2). Intra-articular administration of CAPE was carried out at a dosage of 150 μg/kg for Groups C and D. For Groups A and B, the same volume of 5% DMSO solution without CAPE was administered intra-articularly (Figure 3). The skin was closed with 4.0 Prolene sutures, and sterile dressings were applied with 4 sutures securing the surgical site.

Following surgery, the rats were housed in groups of 3 under the specified conditions for 28 days. For the first 3 days after surgery, treatment with 30 mg/kg cefazolin sodium was continued, and 3 mg/kg ketoprofen was administered intraperitoneally for analgesia. On the third day, dressings were removed, and the surgical site was evaluated. Cages were cleaned twice a day, with a 12-hour light/dark cycle and ad libitum access to food and water. No complications were observed during the surgical and post-surgical monitoring period. On day 28, high-dose inhaled sevoflurane was administered, and the rats were euthanized. Bilateral distal femurs were excised for tissue sample collection (Figure 4). Excised tissue samples were independently assessed by 2 researchers (1 orthopedics and traumatology surgeon and 1 histologist) using the macroscopic ICRS-I scoring system. At this stage, the visual assessment of the cartilage tissue filling the defect was conducted with consideration of the surface smoothness and its resemblance to the native cartilage color. Hyaline cartilage was clearly distinguished from fibrocartilage due to its oval and regular cell structure, uniform distribution, dense and homogeneous collagen content, intense presence of proteoglycans, and smooth and even surface (ICRS-II).

HISTOLOGICAL ANALYSES:

After the experiment was completed, paraffin blocks were prepared from tissue samples as described in the Materials and Methods section. Tissue samples were fixed in a 10% neutral formaldehyde solution (pH=7.4) for 7 days at +4°C. Following fixation, the samples underwent decalcification in Morse solution (composed of 22.5% formic acid and 10% sodium citrate) to facilitate bone tissue decalcification. The decalcification process continued for 5 weeks, with solutions renewed bi-weekly. After decalcification, the tissues were washed in running water for 8 hours, followed by dehydration using a graded alcohol series from low to high concentrations. Subsequently, the tissues were immersed in xylene and then in liquid paraffin, with samples embedded in paraffin blocks. The femur was oriented with the cranial part facing upwards, and 5-micron-thick sections were taken in the coronal (frontal) plane. Each sample was stained with hematoxylin and eosin. Stained sections were examined and photographed using a Leica DM 6000 microscope and the Leica Application Suite program. The microscopic evaluation utilized the ICRS-II scoring system [9,15].

Each parameter was scored on a scale of 0 to 100, with the best and worst scores indicated for each parameter:

STATISTICAL ANALYSIS:

Descriptive statistics for continuous variables were presented as mean ± standard deviation. The normality of data distribution was assessed using the Kolmogorov-Smirnov test. Given the non-normal distribution of the data (P<0.01) and the sample sizes being below 30, non-parametric methods were applied. Comparisons between groups were performed using the Kruskal-Wallis test, while pairwise comparisons employed the Mann-Whitney U test. The consistency of measurements was evaluated using the intraclass correlation coefficient (ICC). Relationships between scores and evaluations were analyzed using the Spearman correlation coefficient. A P value of less than 0.05 was considered significant for decision-making. Data were analyzed using SPSS version 25.0.

Results

BASELINE DATA:

A high level of consistency was observed between the observers (rICC=0.911, P<0.001). This indicates that both experts provided assessments that were highly consistent. Consequently, the equal weighting of evaluations in this study contributed to a reduction in variance, leading to the inclusion of averaged results from both experts.

NUMBERS ANALYZED:

The analysis of ICRS-I scores revealed significant differences among the study groups (P<0.01). Specifically, it was found that the ICRS-I scores in Group A were significantly lower compared to Groups B, C, and D (P<0.01).

OUTCOMES AND ESTIMATION:

There were significant differences in the levels of chondrocyte clustering among the study groups (P<0.01). Groups A and B exhibited higher chondrocyte clustering levels compared to Groups D and C, while Group D also showed higher levels than Group C (P<0.01). Matrix staining levels differed significantly across the groups (P<0.01), with Groups A and B displaying higher levels than Groups D and C, and Group D showing higher levels than Group C (P<0.01) (Table 1).

Cell morphology levels were significantly different among the groups (P<0.01), where Groups A and B had lower levels than Groups D and C, with Group D also exhibiting lower levels than Group C (P<0.01). In Groups C and D, the morphological structures of chondrocytes were preserved, whereas fibrous tissue cells had become spindle-shaped in Groups A and B. The normal histological arrangement of chondrocytes in the articular cartilage at the bone surface in Group C was markedly distinct compared to the other groups, where a significant presence of fibrous connective tissue was noted in Groups A and B. In contrast, Group D exhibited a histology that was close to normal compared to Groups A and B (Table 1; Figure 5A–5D).

Tissue morphology levels also differed significantly across groups (P<0.01), with Groups A and B showing lower levels than Groups D and C, and Group D having lower levels than Group C (P<0.01). Surface architecture levels revealed significant differences (P<0.01), with Group C displaying distinct differences in surface architecture compared to all other groups (P<0.01) (Table 2; Figure 6A–6D).

Tidemark formation levels showed significant differences (P<0.01), where Groups A and B had lower levels than Groups D and C, and Group D showed lower levels than Group C (P<0.01). In Groups A and B, there was fibrous connective tissue at the bone surface and tissue loss due to inflammation, compromising the integrity of the surface structure. In contrast, Groups C and D exhibited a highly intact surface composed of cartilage (Table 2; Figure 6A–6D).

Subchondral bone anomaly levels were significantly different among the groups (P<0.01), with Group A showing lower levels compared to Groups B, D, and C. Additionally, Groups B and D also exhibited lower levels compared to Group C (P<0.01). Basal integration levels were significantly different (P<0.01), with Groups A and B showing lower levels compared to Groups D and C, while Group D also demonstrated lower levels than Group C (P<0.01). Abnormal calcification levels showed significant differences (P<0.01), with Groups A and B exhibiting lower levels compared to Groups D and C, while Group C had higher levels than Group C (P<0.01) (Table 3).

Inflammation within the fibrous connective tissue in Groups A and B was observed to adversely affect both cellular and bone matrix ossification. Conversely, this adverse condition was reversed in Groups C and D. Vascularization levels exhibited significant differences among the groups (P<0.01), with Groups A and B showing lower levels than Groups D and C (P<0.01). Inflammation levels were significantly different (P<0.01), with Group A displaying lower inflammation levels compared to Groups B, D, and C. In contrast, Groups C and D exhibited higher inflammation levels than Group B (P<0.01) (Table 4).

Surface evaluation levels revealed significant differences among the groups (P<0.01), with Group C achieving higher surface evaluation scores compared to Groups A, B, and D (P<0.01). Deep-layer evaluation levels differed significantly (P<0.01), with Group B exhibiting lower levels than Groups A, C, and D, while Groups A and D were also lower than Group C (P<0.01) (Table 4).

Overall assessment in ICRS-II scores showed significant differences among the groups (P<0.01), with Group A’s ICRS-II scores being lower than those of Groups B, C, and D. Furthermore, Group B was found to have lower scores than Groups C and D, while Group C had higher scores than Group D (P<0.01). When calculating the effect sizes for the visual assessments of chondrocyte clustering, an effect size of −1.19 was found between Group B and Group D. In contrast, for tissue morphology, the effect size between Group D and Group C was 1.51.

ADVERSE EVENTS:

To illustrate the prevalence of inflammation and vascularization within the tissue, various magnifications were utilized, as shown in Figure 7. At lower magnifications, vascularization and accompanying inflammation were widespread and severe in Groups A and B. While vascularization is generally considered beneficial for bone materials, the persistence of inflammation in Groups A and B hindered ossification. In contrast, Groups C and D clearly exhibited the cessation of inflammation, and the vascular structures facilitated the transformation of ossification materials towards the tissue (Figure 7A–7D).

Discussion

In the groups administered CAPE (Groups C and D), the morphological structures of the cells were preserved, whereas in the non-administered groups (Groups A and B), spindle-shaped fibroblast-like cells were predominantly observed. Additionally, in Groups C and D, the arrangement and alignment of chondrocytes appeared to be more aligned with normal histological structure. In contrast, Groups A and B exhibited a predominance of fibroticconnective tissue. Tissue losses, fissures, and disruptions in continuity were observed on the cartilage surface in Groups A and B. Conversely, in Groups C and D, it was evident that the integrity of the cartilage surface was maintained.

The level of inflammation observed in Groups A and B negatively impacted basal integration and subchondral ossification, both at the cellular and extracellular matrix levels. Furthermore, it was noted that increased vascularization associated with inflammation adversely affected subchondral ossification in Groups A and B. In contrast, in Groups C and D, both basal integration and subchondral ossification progressed favorably in terms of tissue repair. Current histopathological evaluations indicate that intra-articular application of CAPE has positive effects on cartilage healing at a microscopic level in a microfracture treatment model for acute cartilage defects.

Isolated chondral and osteochondral defects in the knee present a challenging clinical issue, particularly in young patients for whom alternatives like partial or total knee arthroplasty are rarely recommended. Inadequate healing associated with cartilage injuries typically leads to the production of type I collagen and fibrocartilaginous tissue instead of normal hyaline cartilage. The optimal targeted treatment for focal articular cartilage lesions involves organizing hyaline cartilage, which is a practical and minimally invasive approach, effectively restoring the tissue over an extended period, with minimal morbidity. Numerous surgical techniques have been developed for the treatment of focal cartilage defects. Cartilage treatment strategies can be characterized as palliative (eg, chondroplastyanddebridement), repair (eg, drilling and microfracture), or restoration (eg, autologous chondrocyte implantation [ACI], osteochondral autograft transfer [OAT], and osteochondral allograft transplantation) [16].

The microfracture (MF) procedure used in our study is one of the bone marrow stimulation methods based on perforation from the lesion to the bone marrow. This aims to deliver mesenchymal stemcells and growth factors to the damaged area. Mesenchymal stemcells that reach the damaged area differentiate into fibrocartilaginous cells, resulting in the formation of fibrous cartilage. Compared to hyaline cartilage, the collagen accumulation and arrangement in fibrous cartilage differ, resulting in lower capacity for load-bearing and shear forces [17].

The development and application of the microfracture technique, which facilitates the migration of mesenchymal stemcells to the defect, began in the early 1980s. The surgical goal is to create microfractures in the subchondral bone perpendicular to the surface and to ensure access to all areas of the joints with instruments. The microfracture technique has been shown to be an arthroscopic treatment for full-thickness cartilage lesions and degenerative lesions of joints. This technique is low-cost and not technically complex, with an extremely low associated morbidity rate and leaving options for further treatment. Adherence to rehabilitation, knee congruence, and the depth of the cartilage defect surrounding the lesion are among the factors that can affect outcomes following microfracture [18].

Xu et al conducted a study involving 24 New Zealand rabbits to evaluate the effect of the intra-articular injection of cartogenin on the restoration of full-thickness cartilage defects treated with microfracture. One group underwent microfracture and intra-articular cartogenin application after the creation of a full-thickness cartilage defect in the patellar groove, while the other group only received microfracture. Histopathological examinations at 4 and 6 weeks after treatment revealed no hyaline cartilage tissue formation in the group treated solely with microfracture. They reported that the defects were weakly covered by fibrous cartilage tissue and noted poor subchondral bone formation, weak organization of repair tissue, and the absence of tidemark formation [19].

Gudas et al conducted a randomized prospective study comparing the outcomes of mosaic-type osteochondral autograft transfer (OAT) and microfracture (MF) procedures for the treatment of knee joint cartilage defects. Of the 57 athletes included in the study, 28 underwent OAT and 29 underwent microfracture. Post-treatment evaluations were performed using modified Hospital for Special Surgery (HSS) and International Cartilage Repair Society (ICRS) scores, radiography, magnetic resonance imaging, and clinical assessment. After an average follow-up of 37 months, they reported that OAT was superior to microfracture. Biopsy samples were obtained from 58% of patients, and better results were reported for the groups that underwent OAT according to the ICRS scoring system. Histological examination of patients who underwent microfracture showed the formation of softer fibroelastic tissue with fibrous cartilage, as well as fibrillation and disruption of the surface architecture [20].

Knutsen et al compared autologous chondrocyte implantation and microfracture in a study involving 80 patients with symptomatic cartilage defects who did not exhibit osteoarthritis. Forty patients received autologous chondrocyte implantation, while the other 40 were treated with microfracture. After 2 years of follow-up, clinical evaluations were conducted using the Lysholm and Short Form 36 (SF-36) scores, and histologically using the ICRS scoring system. They reported significant clinical improvement in both groups after 2 years. Additionally, the clinical improvement in the microfracture group was reported to be better than that in the autologous chondrocyte implantation group. No significant differences were observed between the 2 groups in histological evaluation, and they found no significant relationship between histological improvement and clinical improvement measured by Lysholm and SF-36 scores [21].

In the current literature, microfracture treatment for knee joint cartilage defects is regarded as a widespread, easily applicable treatment modality with good clinical outcomes. Furthermore, histopathological studies involving microfracture application indicate that the healing tissue is characterized by fibrocartilage, and the cartilage surface architecture is disrupted. In our study, consistent with the existing literature, fibroelastic healing tissue was observed in the microfracture groups. On the other hand, in the CAPE-treated groups, the cellular morphologies were preserved, and their arrangement was closer to normal histological structure.

Elmalı et al investigated the effects of intra-articular CAPE injection on the progression of disease in an experimental osteoarthritis model in rabbits. Twelve New Zealand rabbits were randomly divided into 2 groups after the establishment of the experimental osteoarthritis model. One group received intra-articular CAPE, while the other was given a solution containing the same amount and composition without CAPE. After 4 weeks, the rabbits were killed for histological evaluation. The assessment revealed that in the group treated with intra-articular CAPE, the destruction of cartilage and loss of matrix proteoglycans significantly decreased. In the control group, fraying and fissuring on the cartilage surface were observed. These evaluations concluded that CAPE application prevens progression of disease in the experimental osteoarthritis model [22].

A study conducted by Huang et al investigated the protective effects of CAPE in chondrocytes and the mechanisms underlying these efects. It was reported that CAPE effectively suppressed the degradation of collagen II and aggrecan inducedby IL-1β and reduced the release of inflammatory mediators, including eNOS, COX2, MMPs, and ADAMTS5. These reported suppressive effects are thought to be mediated through the NF-κB- and MAPK-related JNK pathway [23].

Sun et al assessed the anti-inflammatory effects of CAPE in vitro on IL-1β-stimulated chondrocytes and in vivo in a rat model of experimental osteoarthritis. They reported that CAPE decreased the expression of inducible nitric oxidesynthase and cyclooxygenase-2 in IL-1β-stimulated chondrocytes, as well as the extracellular secretion of nitric oxide and prostaglandin E2 in cell culture supernatants. Additionally, CAPE alleviated the degradation of extracellular matrix by increasing the expression of aggrecan and collagen II and decreasing the expression of metalloproteinases. In vivo, CAPE was shown to protect cartilage from destruction and delay the progression of osteoarthritis in rats [24].

The literature also includes studies examining the effects of CAPE on cartilage at the molecular level and in the context of chronic degeneration. These studies have demonstrated the inhibitory effects of CAPE on inflammatory cytokines and its ability to reduce the expression of enzymes that degrade extracellular matrix components [23]. Furthermore, studies have reported that CAPE has a chondroprotective effect in the context of chronic degeneration [22,24]. In agreement with the literature, our study demonstrates the positive effects of CAPE on cartilage healing in cases of acute cartilage damage. What distinguishes our study is that the effects of CAPE in acute damage have not been previously investigated in the literature. In summary, CAPE appears to exert its positive effects through a combination of anti-inflammatory, anti-fibrotic, and regenerative mechanisms that contribute to the healing of acute cartilage defects. By reducing inflammation, promoting cellular preservation, stimulating matrix production, and supporting bone-cartilage integration, CAPE enhances tissue repair and improves histological outcomes.

Our study has several limitations. One significant limitation is that it was conducted on animal models rather than human cartilage, primarily due to the use of a compound intended solely for experimental purposes in histopathological planning. Additionally, the lack of administration of different doses prevented the observation of dose-dependent effects of CAPE. However, biologically, rat cartilage exhibits similar characteristics to human cartilage. Furthermore, the anatomical similarities between the rat knee and the human knee make the limitations of our study relatively negligible. We also believe there is room for improvement in our research. In our study, surgical methods other than microfracture were not employed for the treatment of cartilage defects. Consequently, we were unable to compare the effects of CAPE when used in conjunction with these methods or independently. We anticipate that future studies will explore the effects of CAPE alongside other treatment modalities apart from microfracture.

Conclusions

Our findings indicate that the application of CAPE, either alone or in combination with microfracture, results in improved outcomes in macroscopic and histopathological parameters for acute cartilage defects in the knee joint, leading to a healing tissue that resembles hyaline cartilage. This study could serve as a foundation for further research on the effects of CAPE or other antioxidants in cartilage repair under acute conditions. Clinical studies could elucidate their effects and help determine the optimal dosage, improving prevention of post-traumatic osteoarthritis.