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Establishing a rabbit model with massive supraspinatus tendon defect for investigating scaffold-assisted tendon repair

Biological Procedures Online volume 26, Article number: 31 (2024) Cite this article

Abstract

Background

Shoulder pain and disability from rotator cuff tears remain challenging clinical problem despite advancements in surgical techniques and materials. To advance our understanding of injury progression and develop effective therapeutics using tissue engineering and regenerative medicine approaches, it is crucial to develop and utilize animal models that closely resemble the anatomy and display the pathophysiology of the human rotator cuff. Among various animal models, the rabbit shoulder defect model is particularly favored due to its similarity to human rotator cuff pathology. However, a standardized protocol for creating a massive rotator cuff defect in the rabbits is not well defined. Therefore, the objective of our study was to establish a robust and reproducible model of a rotator cuff defect to evaluate the regenerative efficacy of scaffolds.

Results

In our study, we successfully developed a rabbit model with a massive supraspinatus tendon defect that closely resembles the common rotator cuff injuries observed in humans. This defect involved a complete transection of the tendon, spanning 10 mm in length and encompassing its full thickness and width. To ensure stable scaffolding, we employed an innovative bridging suture technique that utilized a modified Mason-Allen suture as a structural support. Moreover, to assess the therapeutic effectiveness of the model, we utilized different scaffolds, including a bovine tendon extracellular matrix (ECM) scaffold and a commercial acellular dermal matrix (ADM) scaffold. Throughout the observation period, no scaffold damage was observed. Notably, comprehensive histological analysis demonstrated that the regenerative tissue in the tendon ECM scaffold group exhibited an organized and aligned fiber structure, indicating tendon-like tissue regeneration while the tissue in the ADM group showed comparatively less organization.

Conclusions

This study presents a comprehensive description of the implemented procedures for the development of a highly reproducible animal model that induces massive segmental defects in rotator cuff tendons. This protocol can be universally implemented with alternative scaffolds to investigate extensive tendon defects and evaluate the efficacy of regenerative treatments. The application of our animal model offers a standardized and reproducible platform, enabling researchers to systematically evaluate, compare, and optimize scaffold designs. This approach holds significant importance in advancing the development of tissue engineering strategies for effectively repairing extensive tendon defects.

Background

Rotator cuff tendinopathy and tears are a significant cause of shoulder pain and disability. Among these tears, the supraspinatus tendon is particularly susceptible, with a prevalence of 61.9% in men and 38.1% in women [1]. Massive rotator cuff tears involving multiple tendons, full-thickness tears larger than 5 cm, or significant tendon retraction [2], represent a substantial proportion of all rotator cuff injuries (40%) and recurrent tears (80%) [3]. These extensive injuries often result in reduced strength, limited range of motion (ROM), and debilitating pain. Generally, treatment strategies involve surgical repair for younger physically active patients and older patients who do not respond to conservative treatment [4]. However, despite advances in surgical techniques and materials, retear rates remain high, ranging from 40 to 90% [5]. There is thus a need to improve biological augmentation strategies to enhance tendon healing and minimize postoperative degeneration.

With advancements in tissue engineering, tissue scaffolds have emerged as a promising approach to augment rotator cuff repair by providing both mechanical support and favorable biological properties for tendon healing [4]. To evaluate the efficacy of these scaffolds and facilitate translation to clinical applications, rigorous in vivo assessments in preclinical animal shoulder models that simulate human anatomy, physiology, and pathology are essential. Among the available animal models, the rabbit shoulder model is preferred due to its close resemblance to human rotator cuff pathology. Previous studies on rotator cuff tears in rabbits have indicated similar chronic changes to those observed in human patients, including muscle atrophy and fatty infiltration [6]. More importantly, the larger size of rabbit allows for more accurate and reproducible tendon defects compared to other commonly used animal models like rats and mice, and can be performed using standard surgical techniques and equipment [7].

Furthermore, the design of the rotator cuff defect model is important and involves several key considerations, including selection of appropriate animal model (e.g., tendon defect site and size) and the strategy for scaffold implantation (e.g., the suturing technique, scaffold implantation method). Currently, there is no established standardized protocol for producing a massive rotator cuff defect in the rabbit model. Past studies have employed different approaches regarding tendon choice [6, 8,9,10], defect lengths [10,11,12], and locations along the tendon. Moreover, multiple techniques exist for scaffold implantation (e.g., augmentation and bridging) and suturing (e.g., Kessler’s, lock loop, Mason-Allen’s suture method) [13,14,15]. These variabilities pose challenges for comparative analysis. Hence, establishing a standardized model would enable more controlled evaluation of scaffold-mediated repair.

Our objective is to develop a robust and reproducible model of a rotator cuff defect for evaluating the regenerative efficacy of scaffolds. Nonetheless, the choice of implantation technique is influenced by the properties of the scaffold itself. Thus, when using a bridging scaffold that is connected in series with the tendon, it is essential for the scaffold to transmit all pulling forces across the bridged tendon ends during muscle activity [16]. Consequently, the scaffold needs to possess high mechanical strength. Considering the severity of tear, loss of tendon length, and the properties of the scaffold, proper bridging technique is critical for implantation. Additionally, proper sutures play a vital role in enabling appropriate force transmission, withstanding the pulling forces generated by muscle contraction, and preventing scaffold dislocation and gap formation under loading [17, 18]. In cases of simple tendon repair involving the tendon-tendon interface, gaps larger than 3 mm can lead to unfavorable clinical outcomes in dogs [19]. However, sutures can potentially interfere with the microcirculations in tendons [17, 20], hindering the blood supply necessary for the formation of fibrovascular tissues and subsequent regeneration [21]. Therefore, it is important to develop an optimal suture strategy that achieves scaffold stabilization while minimizing disruption to native tendon healing.

This study aimed to develop a robust model of a massive supraspinatus tendon defect model in rabbits. The model involved a complete transection of the tendon, spanning 10 mm in length and encompassing the full thickness and width of the tendon. To repair the tendon, we implemented both direct suture repair and scaffold-mediated repair utilizing two distinct scaffolds: the acellular dermal matrix (ADM, a commercially available scaffold for tendon repair) [22] and the tendon extracellular matrix (ECM) scaffold (a polyurethane scaffold enriched with tendon extracellular matrix) [23]. To ensure optimal stability of the scaffold, we employed an innovative bridging suture technique that utilized a modified Mason-Allen suture as a structural support. Tendon healing outcomes were assessed through histological analyses, including Hematoxylin and Eosin (H&E) staining, Picrosirius red staining, and histological evaluation scores.

Materials and methods

Animals

Twelve New Zealand White rabbits (13–16 weeks old) with an average body weight of 4 kg were obtained from the Laboratory Animal Services Centre, the Chinese University of Hong Kong. All procedures, including rabbit surgeries, were conducted using sterile surgical technique in accordance with protocol (No. 18-003-MIS) approved by The Chinese University of Hong Kong Animal Experimentation Ethics Committee in an appropriately equipped room designated for animal surgeries.

Preparation of animals for rotator cuff surgery

  1. 1.

    Surgical instruments were autoclave-sterilized, and sterile gloves were worn throughout the procedure, which was carried out in a sterile operating field.

  2. 2.

    Anesthesia was induced in both female and male New Zealand White Rabbits by administering an intramuscular injection of a ketamine (35 mg/kg; Alfasan) and xylazine (5 mg/kg; Alfasan) mixture.

  3. 3.

    To maintain anesthesia in rabbits during the surgery, a 5 ml syringe containing the ketamine (35 mg/kg) and xylazine (5 mg/kg) mixture was prepared. The rabbit’s ear vein was located, and an indwelling needle was carefully inserted and connected to the syringe containing the anesthetic. The syringe was secured with gauze and medical tape.

  4. 4.

    The anesthetized rabbit was gently positioned in a supine position, ensuring that the surgical area faced upwards. The surgical area was shaved and cleansed by applying three alternating applications of betadine and 70% ethanol, applied in circular motions, starting from the inside and moving outward, using a cotton swab. Surgical drapes were placed to create a surgical window and maintain a sterile environment.

Isolation of the supraspinatus tendon

  1. 1.

    A 3 cm incision was made in the shoulder to expose the rotator cuff tendons. The deltoid muscle was split, and skin and soft tissue retractors were used to create a surgical window, providing a clear view of the rotator cuff tissues. Acromioplasty was performed using a No.15 scalpel (Mingyue). Hemostasis was achieved, and the wound was irrigated with saline solution.

  2. 2.

    The supraspinatus tendon was identified and marked using a 3 − 0 suture (Arthrex FiberWire®). The supraspinatus tendon was sutured using a lock-loop suturing technique before detaching it.

  3. 3.

    A full-thickness defect, measuring approximately 10 × 5 mm, was created in the mid-substance of the supraspinatus tendon using a No.15 scalpel. The distance between the defect and the first lock-loop suture site was approximately 1–2 mm. The opposite end of the supraspinatus tendon was sutured using the lock-loop suturing method.

Implantation of the scaffold

  1. 1.

    To achieve a secure attachment between the scaffold and the tendon, a modified Mason-Allen suture technique was utilized. After completing the lock-loop suture, all subsequent sutures were positioned behind the lock-loop to minimize the risk of suture pull-out. Prior to the surgical procedure, three holes were created in the scaffold. The dimensions of the scaffold were designed to closely resemble the width and thickness of the native rabbit supraspinatus tendon.

  2. 2.

    The first stitch was passed through one-third of the tendon’s width and then secured with a knot, using its own suture as the initial fixation. The second stitch emerged behind the first stitch and connected to one of the scaffold’s holes, forming a stable “cross” structure between the first and second stitches.

  3. 3.

    After the suture was passed through the scaffold, it traversed the tendon from behind the lock-loop suture. At this stage, the Mason-Allen method was employed to tie a “cross” knot at two-thirds of the tendon’s length, while simultaneously connecting the tensioned suture to the second hole of the scaffold. Following the final Mason-Allen knot, an additional knot was tied using the suture itself at the opposing end of the tendon, to enhance fixation of the scaffold-tendon connection. The same suturing technique was employed to secure the scaffold and tendon together at the other end of the tendon.

Wound closure and post-surgery sterilization

  1. 1.

    After the implantation, post-surgery analgesic (0.01-0.05 mg/kg Buprenorphine, Alfasan) was administered subcutaneously to ensure pain relief for the rabbits.

  2. 2.

    To prevent infection, an anti-infective (20 mg/kg Cefalexin, Alfasan) was administered.

  3. 3.

    A suture (3 − 0 PGA) was used to carefully reapproximate the deltoid muscle tissue layer by layer, and the skin was closed with 3 − 0 silk sutures.

  4. 4.

    To sterilize the wound and clean up any blood, betadine was applied in circular motions using a cotton swab, starting from the inside and moving outward.

  5. 5.

    The rabbits were allowed to recover on a heating pad and subsequently allowed cage activity in their cages without immobilization.

Sample harvesting and analysis

  1. 1.

    At the designated time point of postoperative 1 month, euthanasia was performed on the rabbits using a lethal dose of sodium pentobarbital (Alfasan), administered at a dosage of 60 mg/kg. Euthanasia was carried out in strict adherence to institutional animal ethics guidelines to ensure humane treatment of the animals.

  2. 2.

    The harvested samples were immediately immersed in a 4% (w/v) solution of paraformaldehyde (PFA, Sigma) for 48-hour fixation. The tissue samples underwent graded ethanol dehydration and were subsequently embedded in paraffin blocks.

  3. 3.

    The embedded samples were sectioned at 7 μm thickness, deparaffinized and then stained with hematoxylin and eosin (H&E) and Picrosirius red.

  4. 4.

    The stained sections were examined using bright field optics to visualize H&E staining or polarized optics for Picrosirius staining using a Nikon Ni-U Eclipse Upright Microscope, and images captured using a digital camera [Nikon, DSFi3].

  5. 5.

    To quantify the differences among the groups, the H&E-stained sections were evaluated using a semi-quantitative histopathological scale according to a reported grading system [24]. Four parameters, i.e., cellularity, vascularization, feature of inflammation, and collagen alignment, were quantified using a 0–3 grading scale: 0 (normal), 1 (slightly abnormal), 2 (moderately abnormal), and 3 (maximally abnormal). The average scores were used for comparison.

Statistical analysis

All data were expressed as mean ± standard deviation (SEM) and compared by the Kruskal-Wallis test followed by post hoc pair-wise comparison using the Mann-Whitney U test [25]. Statistically significant differences are indicated by asterisks (*, p ˂ 0.05; **, p ˂ 0.01; and ***, p ˂ 0.001).

Results

Establishing a rabbit model of massive rotator cuff tendon defect

To evaluate the tendon healing efficacy of different scaffolds (i.e., ADM and tendon ECM scaffold), a massive tendon defect model was created on the supraspinatus tendon of New Zealand White rabbits (Fig. 1A). As demonstrated in Fig. 1B, the supraspinatus tendon was identified following a 3 cm incision in the shoulder. The surrounding tissue was carefully isolated, and the supraspinatus tendon was sutured with a lock-loop suturing technique. The suture site was positioned 1 cm apart, and a full-thickness defect measuring 5 mm in length was created between the suture sites to simulate a significant tendon defect (Fig. 1B). To bridge the tendon defect using different materials, the scaffold was implanted between the defect site and connected to the two tendon ends via a modified Mason-Allen’s suture technique (Fig. 2). To ensure a tight connection between tendons and scaffold, the combination of lock-loop suture and Mason-Allen’s suture method was applied. This approach ensured a secure connection between the tendons and the scaffold. The modified Mason-Allen’s suture method was performed between tendon and scaffold, while the lock-loop suture method was employed on the tendon ends below the Mason-Allen’s suture knots. This design resulted in a tight and robust connection between the scaffold and tendon, significantly reducing the risk of suture pull-out.

Fig. 1
figure 1

Schematic surgery diagram of repairing a massive rotator cuff tendon defect. A Diagram illustrating the creation of a massive rotator cuff tendon defects. B Representative images depicting surgical procedure steps

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Fig. 2
figure 2

Schematic diagram illustrating the suture technique used for scaffold implantation. The highly stable implantation model was achieved by combining the modified Mason-Allen method with the locking loop suture method

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Assessment of tendon healing outcomes using histological analysis

To assess the efficacy of the designed scaffold (tendon ECM scaffold) in comparison to a commercially available scaffold (ADM), four experimental groups were established: a healthy tendon control group, a group undergoing direct suture repair, a group undergoing ADM-mediated repair, and a group undergoing tendon ECM scaffold-mediated repair (Fig. 3A). At 1 month, the rabbits were euthanized, and samples were collected for analyses, including H&E staining, Picrosirius red staining, and scoring analysis. Histologically, the H&E staining images demonstrated that both the ADM and tendon ECM scaffold groups displayed complete bridging of the 1-cm tendon defect and exhibited increased cellularity when compared to the intact control group. Additionally, in comparison to the suture repair and ADM-mediated repair groups, the regenerative tissue in the tendon ECM scaffold group exhibited a more aligned fiber structure, indicating a greater degree of tendon-like tissue regeneration.

Fig. 3
figure 3

Histological analysis using H&E staining to assess healing outcome at 1 month after surgery. A Schematic diagram illustrating the experimental design. B H&E analysis revealing the evaluation of tendon healing outcomes. The tendon extracellular matrix (ECM) scaffold group demonstrated the presence of regenerative tissue with a well-aligned structure resembling that of a healthy tendon. In contrast, in both the suture repair and acellular dermal matrix (ADM)-mediated repair groups, a disorganized tissue structure with increased cellularity was observed, compared to the organized architecture seen in the healthy tendon group

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To evaluate the alignment and thickness of collagen fibers, picrosirius red staining observed with polarized light microscopy was utilized. When viewed under polarized optics, the tendon ECM scaffold-mediated group exhibited a more pronounced birefringence compared to both the suture repair and ADM-mediated repair groups. Additionally, the tendon ECM scaffold group displayed a higher abundance of orange-to-red fibers, indicating thicker fibers, while the suture repair and ADM-mediated repair groups had a greater prevalence of yellow fibers, suggesting thinner fibers (Fig. 4). However, it is noteworthy that the fiber thickness in the tendon ECM scaffold-mediated groups remained lower than that observed in the healthy tendon group.

Fig. 4
figure 4

Histological analysis using picrosirius red staining and polarized light microscopy to assess the healing outcome at 1 month after surgery. In the tendon ECM scaffold group, a distinctly wavy and more aligned ECM structure was observed, characterized by a pronounced birefringence. Furthermore, a more abundant presence of thicker collagen fibers was observed compared to both the suture repair and ADM-mediated repair groups. However, the fiber thickness in the tendon ECM scaffold group remained lower than that observed in the healthy tendon group, suggesting an ongoing regenerative stage at the 1-month time point

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Additionally, to evaluate tendon healing, a grading system [24] was implemented to assess the levels of cellularity, vascularization, inflammation, and collagen alignment in each group (Fig. 5A). The suture repair and ADM-mediated repair groups demonstrated significantly higher cellularity and inflammatory response, as well as less organized collagen, when compared to the healthy tendon group (Fig. 5B). In contrast, the tendon ECM scaffold group exhibited no significant differences in terms of cellularity, vascularization, and inflammation compared to the healthy tendon group (Fig. 5B). However, fiber alignment in the healthy tendon group remained significantly higher than that observed in all the repair groups, with the suture repair group displaying the least fiber alignment when compared to the ADM-mediated repair and tendon ECM scaffold repair groups (Fig. 5B).

Fig. 5
figure 5

Histological evaluation and grading of tendon repair on the basis of Hematoxylin and Eosin (H&E) staining. A Grading system for tendon healing. B Scores assigned for cellularity, vascularization, inflammation, and collagen alignment. Both the suture repair and ADM-mediated repair groups displayed significantly higher cellularity and inflammatory response, as well as less organized collagen, compared to the healthy tendon group. In contrast, the tendon ECM scaffold group exhibited no significant differences compared to the healthy tendon group regarding cellularity, vascularization, and inflammation. n = 3, biological replicates; mean ± SEM; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001

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These findings suggest that the implantation of the tendon ECM scaffold resulted in the formation of tendon-like tissue with improved organization and fiber thickness compared to the suture repair and ADM-mediated repair groups. Nonetheless, it is important to emphasize that despite the improved alignment and fiber thickness observed in the tendon ECM scaffold-mediated group, these parameters still fell short of reaching levels comparable to those observed in a healthy tendon. These findings highlight the ongoing regenerative process of the regenerated tendon tissue at the 1 month post-surgery time point.

Discussion

Rotator cuff injuries are a significant cause of pain, functional limitation, and morbidity [26]. The high prevalence of rotator cuff pathologies underscores the urgency for the development of innovative strategies or therapeutics to address these conditions [26]. Animal models have contributed greatly to the advancements in researching and alleviating the burden of rotator cuff injuries, owing to their capacity to replicate anatomical, biomechanical, cellular, and molecular aspects of the human rotator cuff [7]. Among animal models, rabbits are commonly utilized for studying rotator cuff tendon repair [7]. Compared to smaller animal models such as rats and mice, rabbits exhibit less spontaneous tendon healing following tendon injury [27]. Additionally, the supraspinatus muscle of rabbits’ experiences fatty degeneration after tendon detachment, which closely resembles the condition observed in humans [27]. The larger size of rabbits also allows for the ready use of surgical models and techniques, enhancing the accuracy and reproducibility of experimental procedures. Similar to larger animal models such as goats, sheep, and dogs, rabbits possess the advantages of accommodating standard-of-care surgical techniques, robust mechanical loading, and scaffold-based repair strategies [28]. Additionally, their more upright posture closely resembles human anatomy compared to larger animals, which often have limited overhead reaching abilities [28]. Moreover, rabbits offer a cost-effective option for research due to lower purchase and housing expenses [27]. To provide more specific details, we have summarized representative studies that utilized various animal models to investigate acute and chronic rotator cuff tears in Tables 1, 2, 3, 4, 5 and 6.

Table 1 Established mouse models of tendon defect
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Table 2 Established rat models of tendon defect
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Table 3 Established rabbit models of tendon defect
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Table 4 Established canine models of tendon defect
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Table 5 Established ovine models of tendon defect
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Table 6 Established primate models of tendon defect
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To replicate massive rotator cuff tears observed in humans, a 10 mm segment of the rabbit supraspinatus tendon was used in this study, representing approximately 50% of the total tendon length [98]. The purpose was to simulate tendon retraction and atrophy following a supraspinatus tear [98]. Similar approaches have been employed in studies conducted by Yokoya et al. and Zheng et al. [10, 12]. Different repair strategies have been employed. In some studies, the torn tendon was directly pulled back into its original position before being anchored with sutures, resulting in excessive tension on the repaired tendon [99]. This increased traction force during augmentation can weaken the initial fixation and increase the risk of retear [100]. Conversely, other studies completely transected the tendon without removing any tendon tissue and directly bridged it with a scaffold, leading to a postoperative tendon length that exceeds 150% of the normal length [101, 102]. However, this method may result in insufficient mechanical stimulation within the defective tendon, ultimately leading to poor functional recovery. In our study, we utilized scaffolds specifically designed to match the dimensions of the 10 mm defect and bridge the tendon stumps. This design allowed us to achieve a tension level that closely mimics a normal tendon in the repair construct. By employing this approach, our aim was to enhance the accuracy and effectiveness of our experimental model.

An essential aspect that influences the success of tendon repair is the choice of a suitable suture strategy, which is crucial in ensuring the stability of the scaffold and preventing suture rupture or the formation of gaps under significant mechanical stress [103]. In our study, we primarily employed the modified Mason-Allen suture method, known for its high load-to-failure and superior resistance to gap formation [104]. However, given the specific conditions of our repair site, the modified Mason-Allen suture alone was deemed insufficient. Previous literature has introduced the concept of incorporating a row of medial fixation that runs perpendicular to the tendon fibers, serving as a rip-stop structure [105, 106]. Consequently, we opted to utilize the Ford interlocking suture technique to establish a rip-stop barrier. This approach reinforced the modified Mason-Allen suture and further minimized the potential for gap formation. By combining these two suturing approaches, we aimed to provide immediate mechanical support to the defective tendon until functional recovery occurs. It is worth noting that our strategy may result in an increased surface area of suture exposure on the tendon, which could potentially promote adhesion formation. However, this drawback can be overcome by implementing passive or active mobilization of the joint and tendon during the rehabilitative phase [17]. By doing so, we can effectively counterbalance the potential disadvantages associated with the use of additional rows of interlocking stitches. Ensuring the reproducibility of results in experimental animal research faces challenges [107]. The issue of poor reproducibility in animal research primarily arises from biological variation and experimental design [107].

To address the challenge of biological variance, we selected rabbits as our animal model due to their resemblance to human rotator cuff pathology. Regarding experimental design variance, we implemented several measures. Firstly, we carefully selected rabbits within the age range of 13–16 weeks, with an average weight of 4 kg, to minimize the influence of age-related factors. This selection aimed to reduce variations associated with different developmental stages. While a large proportion of massive rotator cuff tears are reported in elderly patients, it is important to recognize that adolescents [108] and adults under 40 years old working as manual labourers or actively serving in the military [109] are also affected and experience challenges not typically observed in older patients. These include unsatisfactory rates of return to physical activity or work at preinjury levels [110]. When defining the biological parameters for our rabbit model, both age and body weight were used since innate physiological and biomechanical differences in humans and rabbits made considerations based on age alone challenging. Specifically, the age and body weight of rabbits used herein was chosen to strike an appropriate balance for modelling both young and adult patients as 91% of mature adult body length is achieved 16-weeks postnatally while our rabbits’ mean body weight was 4 kg and about 90% similar to 1.5- to 2.5-year old mature adult rabbits [111]. Furthermore, numerous studies involving rabbit rotator cuff animal models have been reported with similar age (12–16 weeks old) [112] and body weights (2 to 2.5 kg [113] or 3 to 4 kg [112, 114]). Secondly, we included both male and female rabbits in the study to account for potential sex-based differences. Additionally, we provided a clear and detailed step-by-step description, along with schematic illustrations, of the surgical procedure used to create a massive supraspinatus tendon defect in rabbits. By doing so, we aimed to minimize potential variations arising from diverse surgical techniques. By taking these measures to mitigate biological and experimental design variances, we aimed to enhance the reproducibility of our findings and promote consistency in future studies utilizing our model.

To assess the outcomes of tendon repair, we conducted histological analysis using H&E staining (bright field optics) and Picrosirius red staining (polarized optics). Additionally, we implemented a scoring system to evaluate various parameters, including cellularity, vascularization, inflammation, and collagen alignment. Our findings demonstrated that the group receiving scaffold implantation exhibited superior healing outcomes in terms of collagen alignment compared to the group undergoing suture repair alone. Furthermore, we observed that different scaffold types yielded distinct results. Specifically, the tendon ECM scaffold group showed improved outcomes in terms of vascularization, inflammation, and collagen alignment when compared to the ADM group. These results indicate that the healing effect is not solely dependent on the suture techniques employed but is also strongly influenced by the specific implanted scaffold utilized.

Our rabbit rotator cuff tear model more closely resembles clinical acute rotator cuff injuries, rather than chronic injuries, which are more clinically predominant [115]. Acute rotator cuff injuries are generally defined as those occurring within 2 weeks to 6 months, with a “traumatic” onset following a shoulder trauma [116]. These acute injuries are commonly encountered in clinical orthopedics, with a reported incidence of 8% [117] and a prevalence of up to 40% of all rotator cuff tears [118]. Moreover, acute tears are a common cause of morbidity in the elderly, with an estimated incidence of 2.5 per 10,000 patients aged 40–75 years [119]. Particularly, acute rotator cuff tears after shoulder dislocation are particularly common in older patients, with rates of 54% in those aged 40–87 years [120] and 49% in those aged 60–89 years [121]. However, traumatic rotator cuff tears can occur in patients of all ages and lead to short- and long-term disability if not appropriately managed [115]. A literature review found that 37.6% of rotator cuff tears were attributed to trauma, with the majority caused by simple falls [122]. For young and healthy adults, the forces during falls can be great enough to tear even a tendon without degenerative changes [122], while the risk is higher for the elderly due to their greater risk of falling and poor tendon quality [122, 123]. The commonly involved tendons in acute rotator cuff tears are supraspinatus (84%), infraspinatus (39%), and subscapularis (78%) [26]. Interestingly, a prospective study showed that 50% of patients initially diagnosed with full-thickness rotator cuff tears and receiving conservative treatment had enlargement of tear size after 1 year [124], suggesting the need for early surgical repair, which correlates with previous follow-up studies [125, 126]. Patients with symptomatic rotator cuff tears typically experience pain, weakness, loss of function, and cascading effects on sleep, work, leisure, and psychosocial functioning, including depression and anxiety [127], necessitating early surgical repair. In contrast, patients with chronic full and partial thickness tears due to tendon degeneration and attrition are not always referred to the hospital unless they have substantial problems [128]. Therefore, further study on acute rotator cuff tear models is needed before progressing to more complicated chronic rotator cuff tear models.

One major drawback of using animals to model human rotator cuff tears is that most animals rely on their limbs for support, and their forelimbs have more weight-bearing functions than humans [27]. Rabbits, rats, dogs, and sheep lack the blending of individual flat tendons to form a single insertion, which is a defining feature of the human rotator cuff anatomy [129]. However, animal models still serve as practical means to understand the cellular and molecular pathways and pathology of rotator cuff tears and to develop new technologies to improve existing treatments. Ideal animal models of rotator cuff repair should lack spontaneous tendon healing after injury, have tendon sizes that allow for suture repair techniques like those used in humans, and exhibit irreversible muscular atrophy, stiffness, and fatty accumulation after injury [27]. A systematic review found that the rat model is most used (53.56%), followed by the rabbit model (25.67%), with the supraspinatus tendon being the most common injury site (62.10%), and acute full-thickness tear being the most common injury type (48.41%) [130]. The most common research purposes were testing the repair effect of patches (24.94%), observing pathophysiological changes after rotator cuff injury (20.78%), and testing the intervention effect of drugs (11.00%).

Conclusions

In summary, this study successfully developed a massive rotator cuff tendon defect model in rabbits. Furthermore, a scaffold-mediated approach utilizing a modified Mason-Allen suture technique was specially designed as the repair strategy. This technique ensured a secure and tight connection between the damaged tendon and the implanted scaffold. The defect model and repair strategy developed here represent a highly practical animal model for conducting a wide range of preclinical studies aimed at evaluating the efficacy of tissue engineering-based tendon repair methods. This comprehensive protocol provides a powerful tool for studying massive rotator cuff tendon defects and facilitates the development of novel, tissue engineering based therapeutic strategies for tendon repair.

Availability of data and materials

Data sharing is not applicable to this article as no datasets were generated or analyzed during the current study.

Data availability

No datasets were generated or analysed during the current study.

Abbreviations

ADM:

Acellular dermal matrix

ECM:

Extracellular matrix

H&E:

Hematoxylin and Eosin

PFA:

Paraformaldehyde

ROM:

Range of motion

SEM:

Standard deviation

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Acknowledgements

Not applicable.

Funding

We express our gratitude to the funding support, including The Research Grants Council of Hong Kong SAR (14121121, DMW; 14118620, DMW; 24201720, DFEK), National Natural Science Foundation of China/RGC Joint Research Scheme (N_CUHK409/23, DMW), The Innovation and Technology Commission of Hong Kong SAR Health@InnoHK (DMW, DFEK, RST), National Natural Science Foundation of China (No. 81974328, No. 82372358, DX), and Natural Science Foundation of Guangdong Province for Distinguished Young Scholars (2022B1515020044, DX).

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Authors and Affiliations

  1. School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong SAR, China

    Shuting Huang, Ming Yik Tam, Wai Hon Caleb Ho, Hong Ki Wong, Rocky S. Tuan & Dan Michelle Wang

  2. Institute for Tissue Engineering and Regenerative Medicine, The Chinese University of Hong Kong, Hong Kong SAR, China

    Shuting Huang, Rocky S. Tuan & Dan Michelle Wang

  3. Center for Neuromusculoskeletal Restorative Medicine, Hong Kong Science Park, Hong Kong SAR, China

    Shuting Huang, Dai Fei Elmer Ker, Samuel KK. Ling, Rocky S. Tuan & Dan Michelle Wang

  4. Department of Orthopaedics and Traumatology, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong SAR, China

    Meng Zhou, Samuel KK. Ling, Rocky S. Tuan & Dan Michelle Wang

  5. Department of Orthopedic Surgery, Center for Orthopedic Surgery, Guangdong Provincial Key Laboratory of Bone and Joint Degeneration Diseases, The Third Affiliated Hospital of Southern Medical University, Guangzhou, China

    Chun Zeng & Denghui Xie

  6. Department of Biomedical Engineering, Faculty of Engineering, The Hong Kong Polytechnic University, Hong Kong SAR, China

    Dai Fei Elmer Ker

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  1. Shuting Huang
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  2. Ming Yik Tam
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  3. Wai Hon Caleb Ho
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  5. Meng Zhou
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  10. Rocky S. Tuan
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  11. Dan Michelle Wang
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Contributions

SH: methodology, formal analysis, investigation, data curation, writing-original draft, writing-review & editing. MYT: writing-review & editing. WHHC: writing-review & editing. HKW: writing-review & editing. MZ: methodology, investigation. CZ: manuscript editing; DX: manuscript editing and funding acquisition; DFEK: methodology, manuscript editing, funding acquisition. RST: manuscript editing, funding acquisition. DMW: conceptualization, writing-review & editing, supervision, funding acquisition.

Corresponding author

Correspondence to Dan Michelle Wang.

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All animal experiments were performed in accordance with a protocol approved by The Chinese University of Hong Kong Animal Experimentation Ethics Committee (18-003-MIS).

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The authors declare no competing interests.

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Huang, S., Tam, M.Y., Ho, W.H.C. et al. Establishing a rabbit model with massive supraspinatus tendon defect for investigating scaffold-assisted tendon repair. Biol Proced Online 26, 31 (2024). https://doi.org/10.1186/s12575-024-00256-z

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Biological Procedures Online

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