
==== Front
Animal Model Exp Med
Animal Model Exp Med
10.1002/(ISSN)2576-2095
AME2
Animal Models and Experimental Medicine
2096-5451
2576-2095
John Wiley and Sons Inc. Hoboken

38887851
10.1002/ame2.12439
AME212439
AMEM-2024-0008.R1
Review
Regular Article
Review
Animal models of tendon calcification: Past, present, and future
Li et al.
Li Ruichen 1
Lai Canhao 1
Luo Hong 1
Lan Yujian 1
Duan Xinfang 1
Bao Dingsu 1 2
Hou Zhipeng 1
Liu Huan 1 20016040@163.com

Fu Shijie https://orcid.org/0000-0002-2168-6094
1 fushijieggj@126.com

1 Department of Bone and Joint The Affiliated Traditional Chinese Medicine Hospital, Southwest Medical University Luzhou China
2 Chengdu University of Traditional Chinese Medicine Chengdu China
* Correspondence
Huan Liu and Shijie Fu, Department of Bone and Joint, The Affiliated Traditional Chinese Medicine Hospital, Southwest Medical University, Luzhou 646000, China.
Email: 20016040@163.com and fushijieggj@126.com

17 6 2024
8 2024
7 4 10.1002/ame2.v7.4 Themed Issue: Study on Cardiovascular and Cerebrovascular Diseases 471483
08 1 2024
07 5 2024
© 2024 The Author(s). Animal Models and Experimental Medicine published by John Wiley & Sons Australia, Ltd on behalf of The Chinese Association for Laboratory Animal Sciences.
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc-nd/4.0/ License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made.

Abstract

Tendon calcification is a common clinical condition that frequently occurs as a complication after tendon injury and surgery, or as an expression of fibrodysplasia ossificans progressiva. This condition can be referred to by various names in clinical practice and literature, including tendon ossification, tendon mineralization, heterotopic ossification, and calcific tendonitis. The exact pathogenesis of tendon calcification remains uncertain, but current mainstream research suggests that calcification is mostly cell mediated. To further elucidate the pathogenesis of tendon calcification and to better simulate the overall process, selecting appropriate experimental animal models is important. Numerous animal models have been utilized in various clinical studies, each with its own set of advantages and limitations. In this review, we have discussed the advancements made in research on animal models of tendon calcification, with a focus on the selection of experimental animals, the sites of injury in these models, and the methods employed for modeling.

Various modeling methods for experimental animal models of tendon calcification are shown in the figure: the methods of modeling tendon calcification in experimental animals as shown in the figure can be mainly classified as trauma induced, tissue factor injections, dietary or pharmacological modifications, and gene knockouts.

animal models
review
tendon calcification
the Science and Technology Innovation Cooperation Special Programme of Sichuan Province2022YFS0609‐C1 Industry‐University‐Research Cooperation Foundation2021CXYZ01 Luzhou Science and Technology Plan Project2021‐SYF‐25 China Postdoctoral Science Foundation 10.13039/501100002858 2023M732927 Scientific Research Project of Southwest Medical University2021ZKMS051 2022QN018 source-schema-version-number2.0
cover-dateAugust 2024
details-of-publishers-convertorConverter:WILEY_ML3GV2_TO_JATSPMC version:6.4.8 mode:remove_FC converted:03.09.2024
Li R , Lai C , Luo H , et al. Animal models of tendon calcification: Past, present, and future. Anim Models Exp Med. 2024;7 :471‐483. doi:10.1002/ame2.12439

Ruichen Li and Canhao Lai have contributed equally to this work. Ruichen Li is the first author. Canhao Lai is the co‐first author.
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pmc1 INTRODUCTION

Tendon calcification, characterized by calcium deposition within tendons, primarily involves hydroxyapatite crystals and can occur in various tendons. 1 , 2 The shoulder and hip joints are particularly susceptible to tendon calcification, with the shoulder rotator cuff, supraspinatus muscle, Achilles tendon, and patellar tendon being commonly affected. 3 , 4 Tendon calcification was particularly prevalent in the rotator cuff tendon group, accounting for 25% of the patients with calcific rotator cuff tendinitis and associated tears. Oliva et al. reported shoulder rotator cuff calcification prevalence ranging from 2.27% to 22% in the European population, with a higher incidence among females aged 30–50 years and a bilateral occurrence rate of 10%. 5 Mitsui et al. found that shoulder rotator cuff calcific tendinitis is most prevalent in individuals aged 30–60 years, with a higher incidence among middle‐aged women, and no significant differences in occurrence based on occupation or lifestyle habits; approximately 70% of cases of calcific tendinitis of the rotator cuff involve the supraspinatus tendon, and approximately 20% involve the infraspinatus tendon and rarely affect the subscapularis and teres minor. 6 Calcification of tendons also commonly occurs in the Achilles tendon. Rufai et al. suggested that repetitive motion of the tendon leads to a detachment of the surrounding periosteum, resulting in subperiosteal bleeding and calcification. 7 Knapik et al. proposed that tendon motion can also lead to endochondral ossification and calcification of the subchondral bone. 8 Furthermore, tendon calcification can occur postoperatively. In 2008, Ateschrang et al. reported that the incidence of calcification after percutaneous or open surgery was 14%–62%. 9 A retrospective study in 2017 followed up 81 patients with a median age of 46 years and concluded that the morbidity of tendon calcification after transcosmetically repaired Achilles tendons was 11.1%. 4 In a 2009 study, Huberty et al. found that of 489 arthroscopic rotator cuff repair surgeries, 24 patients (4.9%) experienced postoperative stiffness, with calcific tendinitis identified as an important risk factor for postoperative stiffness. 10 Therefore, the Achilles tendon is often selected as the surgical site. 11 Additionally, endocrine disorders, such as diabetes and hyperthyroidism, can lead to tendon calcification or calcific tendinitis. 12 Ectopic ossification is a major pathological change in calcific tendinitis and is characterized by pathological cartilage ossification in nonbony soft tissues. 13 , 14 Nonhereditary ectopic ossification is mostly caused by trauma and surgical procedures, and the disease can develop at all ages, with a higher prevalence in males, with a male‐to‐female ratio of 3:2. Trauma‐induced cases accounted for 75% of the incidence. 15 , 16 , 17 , 18 , 19 By contrast, hereditary ectopic ossification is rare and primarily includes fibrodysplasia ossificans progressiva (FOP) and progressive osseous heteroplasia.

Currently, there is a lack of direct clinical evidence to establish a causal link between tendon calcification and the manifestation of specific clinical symptoms. 20 Nevertheless, pertinent research has indicated that calcific tendinopathy may involve calcium deposits within the tendon, accompanied by chronic pain, tenderness, local swelling, and varying degrees of reduced joint mobility. The presence of calcific deposits can exacerbate the clinical indications of tendon disease, augment the probability of rupture, prolong the period of convalescence, and increase the rate of postsurgical problems. 4 The clinical presentation of calcific tendinitis is closely associated with the degree of calcification and disease progression. In cases with inadequate treatment response, calcific tendinitis may result in adhesive capsulitis, rotator cuff tears, ossific tendinitis, and other complications. 21 Nonhereditary hypertrophic ossification (HO) can manifest in various anatomical regions, particularly those prone to injury, and is commonly observed in the shoulder, thigh, elbow, and pelvis. 22 The clinical symptoms associated with nonhereditary HO vary with disease progression. The early symptoms include pain, swelling, and tenderness. At this stage, the volume of the HO increases rapidly, necessitating its differentiation from soft tissue sarcoma. 23 Subsequently, as the new bone tissue matures, the swelling becomes firmer and, if located near a joint, can impede movement. O'Brien et al. proposed that ectopic mineralization can induce tendon weakness, leading to reduced weight‐bearing capacity and tolerance of the affected tendon, thereby affecting an individual's motor function and overall physical condition. 24 In cases of hereditary HO, FOP has been shown to be linked to various inherited malformations, primarily affecting the fingers, toes, and spine, 25 resulting in alterations in posture and gait, as well as restricted joint mobility. 22 FOP or myositis ossificans progressiva is a rare hereditary connective tissue disease, with an incidence of approximately one in 2 million, regardless of race, sex, and region. Congenital big toe deformities and progressive heterotopic ossification are landmark clinical manifestations that seriously affect patients' quality of life. At present, hereditary HO formation in patients with FOP is mostly attributed to the mutation of the activin A receptor type I (ACVR1) gene, which destroys the cell signaling pathway involved in bone formation although retaining the typical endochondral ossification in bones. This damage induces the development of the cartilage and osteoblast lineages. Most primary cells affected by these environmental changes are progenitor cells/stem cells, which have significant potential to differentiate into special cell types (e.g., lymphocytes, muscle cells, osteoblasts, osteoclasts, and adipocytes). Their differentiation ability leads to progenitor cells/stem cells playing a key role regarding tissue damage and subsequent HO formation. These stem cells include hematopoietic, endocrine progenitor, mesenchymal, muscle stem, fibro‐adipogenic, and tendon stem/progenitor cells (TSPC). 26 , 27 Recently, a study found that TSPCs contribute to the formation of cartilage and bone during traumatic HO (tHO). 28 These studies also showed that FOP might have a stronger relationship with tendon calcification. Respiratory difficulties may arise. 29 In conclusion, the current understanding suggests that tendon calcification is linked to numerous tendon and joint symptoms, and indirectly contributes to the development of various diseases. However, no study has definitively confirmed that tendon calcification directly contributes to specific diseases.

Therefore, to enhance patient care and optimize patient outcomes, further investigation of the pathogenesis of tendinous calcification is important. Animal models play a vital role in clinical and fundamental research on these conditions. Efficient utilization of animal models enables researchers to faithfully replicate the pathogenesis of tendinous calcification, thereby facilitating more effective disease studies.

2 TENDON CALCIFICATION (Pathogenesis of tendon calcification)

The etiology of tendon calcification remains unclear, with suggestions that calcification results from active cell‐mediated processes and local compensation for reduced tendon stiffness. Mechanistic studies on calcification include reactive calcification, endochondral osteogenesis, chondrogenesis, and genetic factors. In this section, we outline the current theoretical explorations and pertinent studies on the pathogenesis and factors influencing tendon calcification from these perspectives.

2.1 Reactive calcification

Uhthoff and Sarkar and Uhthoff and Loehr 29 , 30 delineated three phases of tendon calcification of the rotator cuff: precalcification, formation, and reabsorption. During the precalcification phase, fibrocartilaginous metaplasia occurs within the tendon, and proteoglycan formation creates conditions for calcification. Calcification can be divided into three stages: formation, resting, and resorption. In the formative phase, calcium crystals are deposited in the matrix vesicles and aggregate to form large calcified foci with a chalky appearance. During the quiescent phase, deposition ceases, and mature calcification foci are formed. During the resorptive phase, vascular tissue forms around the calcified deposits. Macrophages and multinucleated giant cells surround, infiltrate, and phagocytose the calcified vascular endothelial cells, removing some calcified foci. These foci appear like thick, milky‐white toothpaste and may penetrate the nearby joint capsule, causing capsule thickening and chemical irritation, leading to severe impingement pain. During the later stages of calcification, new vascular tissue promotes the formation of type III collagen, which remodels the space previously occupied by the calcified foci. Subsequently, type III collagen is replaced by type I collagen, and the tendon gradually heals as the calcified foci dissolve. These pathophysiological processes lead to various types of shoulder pain. The staging of the calcific tendinitis of the rotator cuff is shown in Figure 1. It is now more widely accepted that tendon calcification is mostly caused by tendon resident cells or exogenous mesenchymal stem cells. The formation phase is distinguished by the deposition of calcium sediments. The subsequent reabsorption phase entailed macrophages engulfing the calcium deposits. This theory posits that tendon calcification is a dynamic process that is regulated by cells in an appropriate environment.

FIGURE 1 Stages of tendon calcification:Uhthoff et al. delineated three phases of tendon calcification of the rotator cuff: precalcification, formation, and reabsorption. The precalcification phase involves fibrocartilaginous chemotaxis in tendon tissues. The formation phase is distinguished by the deposition of calcium sediments. The subsequent reabsorption phase entails macrophages engulfing the calcium deposits. This theory posits that tendon calcification is a dynamic process regulated by cells in an appropriate environment. The calcification phase can be further divided into resting and resorption phases.

Shen et al. 31 induced heterotopic ossification in rat Achilles tendons through injury and observed that the growth hormone‐releasing hormone agonist impedes tenocyte‐derived proteoglycan matrix degradation and osteogenic differentiation. Patients typically remain asymptomatic during the precalcification and calcification phases. However, in the advanced stages of calcification, painful manifestations may emerge as granulation tissue transforms into fully developed collagenous tissue, and thus the tendon undergoes reformation.

2.2 Endochondral osteogenesis and chondrogenesis

Previous studies have indicated that the calcification process involves not only reactive calcification but also cartilage or chondrocytes. Calcification in the Achilles and patellar tendons typically follows an endochondral ossification process, accompanied by osteoblast‐ and osteoclast‐mediated bone remodeling and reshaping. Benjamin et al. 32 reported an animal model of tendon ossification at the insertion site in rats and suggested that the fibrocartilage at the bone junction can progress to calcified bone at the insertion site. This process represents typical endochondral ossification, with calcification initiating at the tendon–bone intersection and gradually extending into the tendon substance. In this process, fibrocartilage cells originate from tenocytes, leading to fibrous cartilage destruction and invasion by blood vessels, ultimately resulting in a bone spur. Notably, no microdamage to the tendon or involvement of inflammatory cells was observed.

Lui et al. 33 conducted histological investigations on a model of collagenase‐mediated calcification of Achilles tendons in rats, revealing a substantial presence of chondrocytes around the calcified tissue, but no inflammatory cells were present. Additionally, positive labeling for types I and X collagen was observed in chondrocytes and their surrounding scaffolds, with a notable absence of type I collagen staining. Type X is a reference marker for endochondral ossification and typically has a high level of this protein in calcified cartilage or is indicative of hypertrophic chondrocytes. In the same tendon calcification model, an increase in collagen subtype expression and the ratio of subtype I to type I collagen was observed. Such alterations are typically associated with a disordered tendon fiber arrangement, tendon thinning, and reduced biomechanical strength.

Some researchers believe that tendon calcification results from the transformation of tendon cells into osteoblasts. Previous research has linked tendon calcification to the presence of chondrocytes or osteoblasts. However, evidence supporting its origin from the surrounding cartilage tissue is scarce, and numerous research teams have conducted relevant studies to gain a better understanding of this phenomenon. Previously, tendons were believed to consist of only tendon cells. However, in 2007, Bi et al. 34 extracted a cell from tendons that expressed mesenchymal stem cell markers and had multipotent differentiation characteristics, and they named it the “tendon progenitor cell.” This finding provides theoretical support for future research. Some researchers have suggested that tendon calcification may result from the misdifferentiation of tendon precursor cells. 35 However, tendon calcification involves multiple factors, necessitating further research to identify the primary cause. Bone morphogenetic protein (BMP) is currently the most probable factor that directly influences tendon calcification. In a study by Hashimoto et al., 36 BMP‐2 was injected into rabbit tendons, resulting in varying degrees of aggravated tendon ossification in a rabbit model, suggesting a direct association between BMP and tendon calcification/ossification. Lin et al. 37 developed a rat model of HO by severing the rat Achilles tendon under aseptic conditions. Radiographic and histological examinations were performed at different time points to study HO formation. We found a significant increase in the expression of BMP‐4 and BMP‐7 at different stages of HO formation. Some researchers have discovered that a BMP pathway inhibitor can effectively inhibit tendon calcification, indicating that the BMP signaling pathway could be a focal point for the research and treatment of tendon calcification. 38 , 39

2.3 Association between inflammation and tendon calcification

Kan et al. 39 , 40 developed transgenic mouse models that overexpressed BMP4 (NSE‐BMP4) or the BMP inhibitor noggin (NSE‐noggin) using the NSE promoter to investigate the function of BMP signaling in brain development. This meant that the BMP‐4 transgenic mice developed severe postnatal heterotopic ossification. To investigate this further, the researchers crossed NSE‐BMP4 and NSE‐noggin transgenic mice and screened their progeny for double transgenics to obtain mice expressing both BMP4 and noggin. They found that the macrophage response to tissue injury stimulates local stem/progenitor cell differentiation into bone and that elimination of macrophages inhibits tendon calcification. Additionally, prostaglandin (PG), an important inflammatory mediator, may be important in the process of tendon calcification. Zhang and Wang 41 , 42 showed that PGE2 43 was significantly elevated in a mouse model of myofibrillar fatigue and found that PGE2 led to the secretion of BMP‐2 by tendon progenitor cells in an in vitro study. These results also suggest the involvement of inflammation in the formation of tendon calcification. In clinical practice, nonsteroidal anti‐inflammatory drugs (NSAID) may have positive effects on tendon calcification. Preclinical studies have demonstrated that NSAIDs inhibit tendon calcification, as shown in the study by Zhang et al., 44 who were the first to prove that celecoxib could be used to slow calcification in an Achilles cutting model in SD rats. However, some studies have reported that NSAIDs hinder tendon repair. 45 , 46 Li et al. 46 used punctured mouse Achilles tendons to induce HO. Celecoxib was ineffective in reducing the progression of HO in both humans and mice, and further research is necessary to investigate the clinical efficacy of NSAIDs in the treatment of tendon calcification. Nevertheless, these findings still suggest that NSAIDs have an inhibitory effect on tendon calcification and more clearly highlight the correlation between inflammatory response and tendon calcification. The inflammatory response is an important direction for future clinical research on tendon calcification.

2.4 Other pathogenic factors associated with tendon calcification

In addition, metabolic disorders such as diabetes and hyperthyroidism can impact the process of tendon calcification. 47 Previous studies have shown that over 30% of patients with insulin‐dependent diabetes exhibit tendon calcification. 12 Renal function is also associated with ectopic calcification. Common sites of metastatic calcification include heart valves, blood vessels, joints, vertebrae, and skin. 48 , 49 , 50 , 51 , 52 These calcifications are caused by persistent hyperphosphatemia and hyperparathyroidism. 52 A hypoxic environment, 53 immobilization, 54 and other factors are also related to the development of tendon calcification and ectopic ossification. According to a previous study, 55 the pathological progression of tendon calcification was influenced by age. Tendon stem cells (TSC) derived from older mice exhibit a significantly slower proliferation rate than those derived from younger mice. The reduced expression of stem cell markers indicates a decline in tendon regeneration with age. 56 However, moderate exercise attenuated the adverse effects of aging on stem cells, indicating that moderate exercise has a positive effect on aging tendons. 57

3 ANIMAL MODELS OF TENDON CALCIFICATION

3.1 Choice of experimental animals

Currently, mice and rabbits are commonly used as experimental animals in the field of tendon calcification research, with mice being the most used. Male mice are often selected for modeling because reports suggest a higher success rate of calcification in male mice than in female mice during trauma‐induced tendon calcification or HO animal modeling. 58 To ensure that the experimental process is not influenced by gender differences, researchers typically choose a single sex of mice for modeling and comparative analysis. Other research teams have utilized sheep, 59 , 60 , 61 zebrafish, and other animals as experimental models. Kakkar et al. 61 used a collagen protein derived from sheep tendons to induce both mineralization and demineralization reactions in an in vitro system to study the effects of different fluoride concentrations on the mineralization and demineralization reactions induced by collagen. In the study by Shea et al. 59 using sheep models, a significant similarity in mineral content was observed between calcified fibrocartilage and bone in humans and sheep, with no statistical difference found when compared with rats. Conversely, the mineral concentrations in rats were lower. There was also a greater similarity in bone structure and pore density between humans and sheep than between humans and rats. We believe that the use of tendon insertion in sheep is a more suitable model for assessing fibrous cartilage calcification in older hip fractures than the use of rats.

The human ACVR1 gene (Ensembl: ENSG00000115170), also known as ALK2, is located on chromosome 2q23‐q24 and compiles the 509 amino acid proteins. ACVR1 compiles the TGF‐β receptor superfamily of BMP type I receptors, which are implicated in many biological processes, including skeletal, cardiac, cartilage, neural, and reproductive development and regulation, and some studies have suggested that ACVR1 is more closely related to FOP. 62 In 2017, LaBonty et al. developed transgenic zebrafish expressing heat shock–induced constitutively active ACVR1 (ACVR1/Q204D) labeled with mCherry. 60 The phenotypes of both juvenile and adult zebrafish were examined, and adult zebrafish expressing ACVR1/Q204D, induced by heat shock, exhibited phenotypes comparable to those associated with human FOP, such as HO, spinal deformity, vertebral fusion, and pelvic in addition to pelvic fin anomalies. The researchers concluded that transgenic zebrafish expressing heat shock–induced mutations in ACVR1/Q204D could be used as human FOP models. In a follow‐up study conducted in 2018, researchers analyzed various injury models in zebrafish expressing ACVR1/Q204D. Some animals were injected with cardiotoxin, and their caudal fins were cauterized, which resulted in HO in remote regions such as the spine. 63 In 2022, Wentworth et al. 64 conducted a functional study in zebrafish. They found that knockdown of wild‐type BMPR1A and ACVR2A reduced ACVR1R206H signaling, whereas ACVR2AV173I unexpectedly increased ACVR1R206H‐mediated signaling in zebrafish. In addition, there is evidence that ACVR2A has a direct adverse effect on osteoblast bone density. 65 This study offers a practical method and platform for functionally testing genes and genetic variants that affect the BMP signaling pathway. Sclerotinia (Scx) is closely associated with normal tendon development and pathological changes in tendons. 66 Zebrafish have two mammalian homologues of Scx, 67 and one research group observed that both Scxa and Scxb have high levels of amino acid similarity to Scx in mice and humans. 68 The aforementioned literature and experimental results indicate that zebrafish can be used as an animal model for studying tendon calcification, particularly in HO. In summary, when selecting experimental animals for tendon calcification‐related research, in addition to mice and rabbits, sheep and zebrafish can be chosen, as these animals have certain advantages over traditional mouse and rabbit models. Commonly used animal model modeling methods are shown in Figure 2.

FIGURE 2 Various modeling methods for experimental animal models of tendon calcification are shown in the figure: the methods of modeling tendon calcification in experimental animals as shown in the figure can be mainly classified as trauma‐induced (transection or insertion of the Achilles tendon and injury to the patellar tendon), tissue factor injections (including injections of collagen type I, TGF‐β, and BMP‐4), dietary or pharmacological modifications (statins, high‐phosphatidylinositol or low‐Mg diets), and gene knockouts (ANK, TGF‐β).

3.2 Selection of modeling methods for animal models of tendon calcification

3.2.1 Animal models of tendon overload

With the accumulation of chronic injury, catabolism within the tendon tissue exceeds anabolism, weakening the self‐repair mechanisms within the tendon. Molecular damage responses, including inflammatory cellular regulators and collagen alterations, contribute to failed tendon healing, leading to degenerative lesions in collagenous tissues and potentially resulting in tendinopathy or calcification due to inflammatory stimulation or reactive hyperplasia. Animal models of fatigue load can be used to simulate tendon calcification to replicate this pathological process and better simulate the fatigue state associated with chronic exercise. This can be achieved by treadmill running, weight‐bearing swimming, sleep deprivation, or other methods that induce fatigue in animals. Each modeling method has its own advantages and disadvantages. Animal models for exercise‐induced fatigue can be categorized as either an acute or a chronic exercise fatigue model. 69 , 70 , 71 However, tendon fatigue modeling is not commonly used to design models of tendon calcification or calcific tendinitis. In current research on tendon calcification, animal models of fatigue loading are commonly prepared using treadmill‐running and weight‐bearing swimming methods. Zhang and Wang 41 investigated the effects of PGE2 on TSCs in vitro using a treadmill‐running mouse model to simulate repetitive mechanical loads on the tendons. The researchers found significantly higher PGE2 levels in tendons of treadmill‐running mice compared to the control group. When TSCs were treated with PGE2, decreased cell proliferation and increased adipogenesis and osteogenesis were observed. This indicates that the treadmill‐running model could be used to study tendon calcification and ossification. The treadmill‐running method is suitable for constructing animal exercise fatigue models because of its simplicity, adjustability of training intensity, broad applicability to various animals, and cost effectiveness. However, its disadvantages are also evident: the treadmill's large footprint and the potential difficulty in integrating it with other experimental devices, such as high‐pressure or high‐temperature equipment. In addition, this modeling method has been used less frequently in studies related to tendon calcification, and its reproducibility requires further investigation.

3.2.2 Animal model of trauma‐induced tendon calcification

Among the various modeling methods that have been used, trauma‐induced modeling, particularly surgery‐based modeling, is the most frequently employed. However, there is no recognized gold standard method for establishing animal models of trauma. tHO animal models are mainly classified into the following categories: joint fixation model, 72 ectopic implantation of bone matrix or osteoprogenitor protein model, 73 muscle injury combined with local osteoprogenitor protein implantation model, hip injury model, 74 , 75 limb exploding amputation model, 76 and simple Achilles tendon total amputation model. 11 Of these, the Achilles tendon is the most used injury site for modeling. The procedure for this model is relatively straightforward, involving exposure and transection of the midsection of the Achilles tendon, followed by suturing of the skin to complete the modeling process. This rat model was initially developed by Buck in 1953. 11 In 1986, Brown et al. 77 further refined a rat model of paraphyseal osteoarthropathy by anesthetizing male and female Sprague–Dawley rats weighing 51–120 g, making an incision in the Achilles tendon, resuturing it, and then conducting collagen analysis to study ectopic bone formation. However, despite the abundance of modeling methods, except for the simple Achilles tendon total cut model, the rest are considered to lack controllability and reproducibility and have not been widely adopted. As mentioned previously, although the Achilles tendon total cutoff model is currently a commonly used animal model, it has disadvantages, such as longer heterotopic bone formation time, smaller volume, and inability to accurately reflect the systemic inflammatory state commonly observed in clinical cases of tHO. In 1983, McClure 78 repeated this experiment and showed that the incidence of tHO at 5 weeks postoperatively was only 60% and that 100% of the cases did not develop until 10 weeks postoperatively. Therefore, several researchers have improved and expanded the Achilles tendon cutting model. For instance, in their 2018 study, Lin et al. 79 enhanced the widely used Achilles tendon amputation model by examining the changes in the contralateral tendon after modeling from various perspectives, including radiological and histological, using transmission electron microscopy on mouse specimens after unilateral tendon amputation. This study confirmed the development of calcific Achilles tendinopathy in nonoperated Achilles tendons 12 weeks after unilateral tendonotomy. Additionally, intratendinous cartilage ossification and Achilles deformities were observed in the nonoperated Achilles tendon, along with the occurrence of Achilles tendon ossification–associated tissues. These changes were also observed in nonoperated limbs that did not undergo Achilles tendon transection. This modeling method not only is simple to perform but also generates new research ideas related to HO. Some researchers have also established a method for total Achilles tendon severance combined with skin burns to induce tHO. 80 This method not only meets the local bed conditions for tendon heterotopic calcification but also better simulates systemic inflammation, and it is more strongly associated with the pathogenesis of calcific tendinitis, especially HO, seen in the human body. In 2023, Wang et al. 81 compared the simple Achilles tendon severance method with the Achilles sound cutoff combined with the skin scald method in a comparative study. They found that the total Achilles tendon cutoff combined with skin scalding could induce tHO in mice earlier than the traditional total Achilles tendon cutoff alone, had a higher success rate, and resulted in larger‐sized ectopic bone formation. This formed the basis for early diagnosis, especially for determining the degree of ossification activity, and was more advantageous than the traditional Achilles tendon cutoff alone. However, a few of these studies considered the sex of the modeled animals, which should be considered when selecting animals for future research on traumatic tendon calcification. Additionally, O'Brien et al. 82 designed an animal model of tendon calcification induced by needle injury, which involved inserting a 23G needle 2 mm distal to the Achilles bone–Achilles tendon junction after exposing the Achilles tendon and sewing up the skin immediately after the needle punctured the tendon. All the samples underwent endochondral osteogenesis. This modeling method was found to have good reproducibility and is simple to operate but requires a lengthy 20‐week postoperative follow‐up, which is time consuming and expensive. If the follow‐up time is shortened, ensuring the success rate of tendon calcification modeling may be difficult, necessitating further research. Furthermore, some teams have used the Achilles tendon and gastrocnemius muscle in mice combined with a mild traumatic brain injury model commonly used in traumatic brain injury (TBI) to model tendon pathology. This model suggests that hypoxia at the injury site in TBI mice creates an environment that promotes pericyte accumulation and activation, leading to the formation of endochondral osteoblasts. 83 , 84

3.2.3 Animal models of chemically induced tendon calcification

In addition to animal models of surgical trauma, researchers have explored methods for inducing tendon calcification using oral or injectable drugs, extracts, or tissue factors. Lui et al. first reported an animal model where patellar tendon defects were created by inducing a defect at the patellar tendon and then suturing the skin, representing a straightforward modeling approach. However, during the postoperative follow‐up, only 50% of the samples developed tendon calcification, 33 suggesting that this model lacked reproducibility and may not be widely adopted as a standard modeling method to address this issue. The team developed a model of tendon calcification induced by injecting collagenase in a study published in 2008. 85 This model entailed exposing the patellar tendon and flexing the knee joint at 90°. Subsequently, 20 μL of 0.015 mg/μL of type I collagenase was injected into the tendon. At weeks 2, 4, 8, 12, and 16 in the collagenase‐injected group, the rats were killed, and the patellar tendon was obtained for routine histology, immunohistochemical staining, and von Kossa staining. Two rats injected with collagenase were euthanized at week 32 for viva computed tomography imaging. All samples developed tendon calcification at postmodeling follow‐up, indicating the reproducibility of the modeling method. However, there is no direct evidence of a link between collagenase and tendon calcification, necessitating further research to establish the clinical relevance of collagenase injection in the treatment of tendon calcification. Hannallah et al. 86 conducted a related study in 2004. They introduced BMP‐4‐transduced stem cells into the hind limbs of mice and implanted varying numbers of noggin‐expressing muscle stem cells in the treatment group. After 4 weeks, the mice were euthanized to detect HO. In addition, the hind limbs of severely combined immunodeficient mice were implanted with 80 mg of artificial human demineralized bone matrix. The study concluded that the administration of noggin through muscle‐derived stem cells could prevent abnormal bone growth by inducing BMP‐4, developing a demineralized bone matrix, and preventing physical injury in a laboratory animal model.

Additionally, the team induced tendon calcification through dietary means. 87 The asj‐20J mice were subjected to an “accelerated diet” with a twofold increase in phosphate and a 2% decrease in magnesium, whereas the mice were fed either a standard diet or the accelerated diet. Researchers inspected both groups of mice for ectopic mineralization and conducted blood analyses to determine the moment of mineralization onset. This study shows that ENPP1 influences the regulation of calcification in soft and bone tissues, and an increasing number of clinical investigations have identified a positive association between statins and tendon disorders. de Oliveira et al. 88 reported that an oral human equivalent dose of statins promoted Achilles tendon rupture in rats, and a subsequent study by the same team 89 demonstrated a notable decrease in noncollagenous proteins and collagen I in Achilles tendon cells with statin treatment. These results suggest that statins induce an imbalance in the extracellular matrix components, which may lead to tendon microinjury. Ferda Kaleağasıoğlu et al. 90 further explored the correlation between statins and tendon calcification by administering statins to rats via tube feeding at 20 and 40 mg/kg daily for 3 weeks before dissecting the Achilles tendon and measuring. In this study, simvastatin, atorvastatin, and resveratrol were used, and all the statins tested were associated with the risk of calcific tendinopathy. The Achilles tendons of all experimental rats showed varying degrees of calcification, indicating that statin use is associated with the formation of tendon calcification and can be used to model tendon calcification. Although the injection or oral administration of statins can promote the formation of calcification in animal models of tendon calcification, the small sample size and limited related studies make them unsuitable for independent‐wide applications at present. Perhaps the injection or oral administration of statins in addition to Achilles tendon amputation accelerates calcification.

3.2.4 Genetic models of tendon calcification

As previously discussed, the initiation of tendon calcification, particularly in HO, is genetically determined. To emulate the genetic regulation of calcification, some research groups have used gene deletions in animals to replicate tendon calcification more accurately. With the ongoing advancements in gene editing and related technologies, this modeling approach has seen an increase in experimentation and applications. In the subsequent sections, we primarily present various transgenic animal models associated with tendon calcification, including ANK‐deficient, twy, and TGF‐β type II receptor‐deficient mice.

ANK‐deficient mice: the ANKH gene, located on human chromosome 5p near the ANKH gene region at D5S1954, encodes a 492‐amino acid transmembrane protein responsible for the intra‐ and extracellular transport of inorganic pyrophosphate. 53 , 56 ANKH is known for its anticalcification properties. 91 Mice with a mutated ANKH gene exhibit progressive ankylosis of the limb joints and spine, reduced joint space, ligament ossification, and intervertebral disc alterations, resembling those observed in postnatal ankylosing spondylitis in humans. In 2000, Ho et al. 92 used positional cloning to identify mouse ANK genes. The authors examined the exon and intron structures of a rodent gene and partially sequenced a human expressed sequence tags clone. Furthermore, researchers have determined the complete coding tails of ANK human genes. Direct homologues of mouse ANK have also been identified in rats, cattle, and zebrafish. Although this mouse model has been utilized in studies related to HO, the region of ossification is primarily in the blood vessels and ligaments. Further investigations are required to determine its full and precise applicability to tendons. 93 , 94 , 95

TGF‐β type II receptor‐deficient mice: tendon calcification/HO was found to be highly correlated with inflammatory responses and macrophages. TGF‐β is produced by M2 macrophages expressing the cell surface marker CD206, 96 and it plays an important role in regulating the process of HO via the promotion of cartilage‐forming differentiation. It is currently a popular target for drug studies. Abnormal activation of TGF‐β is generally thought to be associated with skin fibrosis and metastasis of malignant tumors in kidney, liver, lung, and other organs. 97 However, some studies have found that high‐level activation of TGF‐β can increase the number of mesenchymal stromal cells/precursor cells, thus promoting the formation of ectopic ossification. 98 In a related study, a team of researchers designed a mouse model of ectopic ossification/tendon calcification by inhibiting or knocking down TGF‐β. 99

Transgenic/knockout mice have the advantages of a short culture cycle and are a better fit for the pathogenesis of tendon calcification by precisely introducing or knocking out relevant genes. This modeling method is increasingly adopted in tendon calcification‐related studies. However, it also has drawbacks such as high preparation cost, low embryo survival rate, and possible silencing of the transgene, which require further research. The molding method can be referred to in Table 1.

TABLE 1 Main applied animal models of tendon calcification and their modeling methods.

Species	Model mechanism	Type of tendon	Method	Timeline	Outcomes	Referenced here	
C57BL/6J mice	Tendon overload	Achilles tendon	Treadmill running	The treadmill protocol included 1 week of training (15 min/day, 5 days/week), followed by running 13 m/min until the mice were exhausted. Immediately after treadmill running, the mice were euthanized, and patellar and Achilles tendons were harvested	Mouse tendons produced higher levels of PGE2 than tendons of cage control mice, and PGE2 decreased TSC proliferation and induced adipogenic and osteogenic differentiation of TSCs in an apparent dose‐dependent fashion	[37]	
C57mice/SD rat	Traumatic injuries	Achilles tendon	Achilles tendon cut	Mice were killed at 1, 4, 6, and 10 weeks after surgery, and the right calf was removed, washed with PBS, and fixed in paraformaldehyde overnight	Ten weeks after surgery, μCT revealed that tendon calcification occurred in all Achilles tendonectomized mice, mainly at the insertion of the tendon, that is, at the bone–tendon junction and at the tendon–muscle junction	[63, 64, 71, 72]	
		Achilles tendon amputation combined with skin burns	The mice were executed at 8 weeks after surgery, and the limbs below the calf were sampled after the knee joint was isolated. Image‐J 1.8 image analysis software was used to calculate the ectopic bone area	All mice developed traumatic heterotopic ossification, with a distinct round‐like hyperdense shadow at the Achilles tendon site. Total Achilles tendon dissection combined with skin scald was able to induce traumatic heterotopic ossification in mice earlier and with a higher success rate than traditional total Achilles tendon dissection alone, and the volume of heterotopic bone formed in the model was larger	[73, 74]	
C57Bl/6 mice		Achilles tendon	Needlestick injury	Mice underwent LH injury (I, n = 11) and were euthanized after 20 weeks along with noninjured controls (C, n = 9). All hind limbs were examined using μCT followed by biomechanical testing (I = 7 and C = 6)	No differences were found in the biomechanical responses of injured tendons compared with controls. However, the RH tendons contralateral to the LH injury exhibited greater static creep strain and total creep strain compared with those LH tendons (p ≤ 0.045) and RH tendons from controls (p ≤ 0.043). RH limb lesions of injured mice were thrice larger compared with controls (p = 0.030)	[75]	
SD rat	Patellar tendon	Excision of the central third of the patellar tendon (1 × 4 mm)	At weeks 2, 4, and 12 after injury, rats were killed, and both patellar tendons were harvested (n = 6 for each group) for histology as well as immunohistochemical staining and analysis of collagen type X, collagen type II, Sox 9, collagen type I, collagen type III, decorin, biglycan, fibromodulin, and aggrecan	Ectopic chondrogenesis and ossification were observed inside wound in 50% samples at week 12	[30]	
Rabbit	Vastus intermedius muscles	Forcible flexion of immobilized knees	Rabbits were killed at 1–21 days after the daily forcible flexion of immobilized knees	All but one pharmacologically treated animal exhibited an absence of HO at 3, 4, 5, or 6 weeks	[72]	
C57B/6 mice	Quadriceps femoris muscle	Muscle impaction injury	A one‐time muscle impaction injury was induced by dropping a stainless steel ball weighing 16.3 g (15.6 mm in diameter) from a height of 100 cm onto the right quadriceps muscle immediately after BMP‐2 injection.

The intact thighs, including the femur and quadriceps, were scanned in vivo at 1, 2, 4, and 8 weeks after treatment using a Scanco Viva CT 40 μCT

	After trauma, the expression of BMP‐2, BMP‐4, BMP receptor 1, SOX9, and RUNX2 increased in muscle. Although little or no HO was observed in mice receiving 1 μg of BMP‐2, combining this dose with muscle trauma produced an abundance of HO. At higher doses of BMP‐2, trauma did not augment mineral deposition	[68]	
SD rat	Hamstrings and quadriceps	Trauma‐induced blast and extremity injury	Rats (n = 55) were anesthetized with 2%–4% isoflurane and then exposed to blast overpressure (120 ± 7 kPa) by a pneumatically driven shock tube, followed by controlled femoral fracture, a 1‐min soft tissue quadriceps crush injury, limb amputation through the zone of injury, and inoculation of the wound with 1 × 106 colony‐forming units of methicillin‐resistant Staphylococcus aureus as previously described		[70]	
SD rat	Chemically induced	Patellar tendon	20 μL of 0.015 mg/μL of type I collagenase was injected into the tendon	At weeks 2, 4, 8, 12, and 16 in the collagenase‐injected group, the rats were killed, and the patellar tendon was obtained for routine histology, IHC, and von Kossa staining	All samples developed tendon calcification at the postmodeling follow‐up	[78]	
Abbreviations: BMP, bone morphogenetic protein; HO, hypertrophic ossification; IHC, immunohistochemical; LH, left hind; RH, right hind; SD, Sprague–Dawley; TSC, tendon stem cell; μCT, microcomputed tomography.

4 CONCLUSIONS

Currently, trauma‐induced calcification, such as Achilles tendonotomy, is the primary method for studying tendon calcification. The most innovative modeling methods related to tendon calcification have been chosen to improve this basis to achieve better success rates and reproducibility in modeling. With further studies on the pathogenesis of tendon calcification and the continued development of transgenic and other technologies, the selection of animal models will no longer be limited to mice. Instead, animal models will be selected based on their ability to accurately simulate human tendon calcification rather than relying only on the trauma‐induced models used in rats and rabbits.

AUTHOR CONTRIBUTIONS

All authors contributed to study conception and design, with Ruichen Li as the first author and Canhao Lai as the co‐first author. Literature search, data collection, and analysis were performed by Ruichen Li and Canhao Lai, and the final version of this paper was reviewed by Shijie Fu and Huan Liu. The first draft of the manuscript was written by Ruichen Li, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

CONFLICT OF INTEREST STATEMENT

The authors declare that research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

ACKNOWLEDGMENTS

This work was supported by the China Postdoctoral Science Foundation (grant number: 2023M732927), the Science and Technology Innovation Cooperation Special Programme of Sichuan Province (grant number: 2022YFS0609‐C1), the Luzhou Science and Technology Plan Project (grant number: 2021‐SYF‐25), the Industry‐University‐Research Cooperation Foundation (grant number: 2021CXYZ01), and Scientific Research Project of Southwest Medical University (grant numbers: 2021ZKMS051 and 2022QN018).
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