Scaffold-Based Temporomandibular Joint Tissue Regeneration in Experimental Animal Models: A Systematic Review An abstract of this article was presented as a poster,at The Bergen Stem Cell Consortium (BSCC),Annual meeting,Bergen,Norway,September 3–4,2017.

Study design

A review protocol was developed based on the Preferred Reporting Items for Systematic reviews and Meta-Analysis (PRISMA) guidelines, ³⁶ and predetermined inclusion/exclusion criteria.

Search strategy, screening, and study selection

Electronic databases of MEDLINE (through PubMed) and Web of Science were searched for relevant English literature up to and including November 2017. Unpublished literature was searched for through Google and Google Scholar search engines. Bibliographies of the studies selected and relevant review articles were checked for crossreferences. A specific search strategy was developed for MEDLINE and Web of Science (see the Supplementary Table S1; Supplementary Data are available online at www.liebertpub.com/teb). Titles and abstracts of the search-identified studies were screened by two authors (E.H. and S.S.) and full texts of all eligible studies were obtained. Uncertainty in the determination of eligibility was resolved by discussion with the other authors. Two authors (E.H. and S.S.) reviewed the selected full texts independently and final inclusion was based on the aforementioned inclusion criteria. A summary of the screening process is presented in Figure 1.

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FIG. 1.

Flowchart for study screening and selection. n, number of articles; TMJ, temporomandibular joint.

Data extraction

Data were extracted from the full texts of selected articles on: author(s), study design, animal species; strain, age and gender, model type, number of animals (n), observation time(s), cell type, source and number, induction of cells, scaffold biomaterial(s), GF(s), control group(s), outcome(s), method(s) of outcome evaluation, main findings, and conclusions. Descriptive summaries of studies included were entered into tables. Due to high heterogeneity among included studies, a meta-analysis was not performed.

Quality assessment and risk of bias

Reporting quality assessment of all studies was performed based on a modification of the ARRIVE (Animal Research: Reporting In Vivo Experiments) guidelines regarding relevant items^37,38 (Supplementary Table S2). Compliance with the guidelines was evaluated using a predefined grading system applied to each of the 20 items. ³⁹ Reporting quality was judged as “high,” “moderate,” or “low.” Risk of bias (RoB) assessment was performed using a modification of SYstematic Review Center for Laboratory animal Experimentation (SYRCLE) RoB tool for animal studies, and graded as “high,” “low,” or “unclear” ⁴⁰ (Supplementary Table S3). Any disagreement between the reviewers during study selection, data extraction, and quality assessment was resolved by discussion and consensus.

Search results and study characteristics

Of the 613 search-identified studies, 30 studies^41–70 performed in mice (n = 12), rabbits (n = 13), dogs (n = 2), goats (n = 2), and sheep (n = 1), were included in the review. Excluded studies by full-text and reasons for exclusion are reported in the Supplementary Table S4. The included models were either ectopic (n = 12)^{42–44,48,50–52,57–59,62,63} or orthotopic (n = 18)^{41,45–47,49,53–56,60,61,64–70} (Tables 1 and 2). All ectopic models included were subcutaneous implantation on the dorsum of mice. Orthotopic models were performed in the TMJs of rabbits (n = 13),^{41,46,47,49,53–56,60,67–70} dogs (n = 2),^65,66 goats (n = 2),^61,64 and sheep (n = 1). ⁴⁵ Sample sizes ranged from 2 to 25 and 5 to 60 animals in ectopic and orthotopic models, respectively. Observation times varied between species: mice (2–12 weeks), rabbits (3 weeks to 12 months), goats (12–24 weeks), and sheep (4 months).

Risk of bias

Most studies provided adequate information regarding title, abstract, background, study objectives, ethical approval, experimental design, and procedures (Fig. 2). Information about housing and husbandry was generally inadequate and the majority of studies lacked complete information regarding animals' age and/or gender (Tables 1 and 2). No studies reported information on sample-size calculation and half of them provided basic baseline characteristics of the animals. In seven studies, (23%) animals were randomly allocated to different treatment groups to minimize selection bias, although no details of the randomization procedure were reported. Two studies (15%)^45,64 reported blinding of outcome assessors to treatment groups, to minimize detection bias. Most studies reported detailed outcome evaluation, including statistical analyses, and some reported information on adverse reactions or complications. Overall, RoB in most studies was judged to be unclear (Fig. 3). Information regarding study limitations and implications for translation to human models was limited, and none of the studies referred to the principal of the “3Rs” (replacement, refinement, and reduction) for experimental models. ³⁷

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FIG. 2.

Frequency distribution (%) of the scores assessed for each item of the modified ARRIVE guidelines in all studies included. Items 2, 3, 5, 7–10, 12, 13, and 15–20 were scored 0, 1, or 2 (clearly inadequate, possible adequate, or clearly adequate). All other items scores 0 or 1 (no or yes).

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FIG. 3.

Frequency distribution (%) of the risk of bias assessment for each item of the modified SYRCLE RoB tool in all studies included. Items 1–8 were judged as “yes,” “no,” or “unclear”; items 9 and 10 were judged as “yes” or “no” (risk of bias; yes = low, no = high, unclear = unclear). SYRCLE RoB, SYstematic Review Center for Laboratory animal Experimentation Risk of Bias.

Characteristics of animal models

Characteristics of TE strategies

Outcome measures

Animal models

In the present review, the majority of the studies reported the use of small animal models. In musculoskeletal research, small animal models primarily include two species—rodents (i.e., rats and mice) and rabbits. ⁷³ Significantly lower costs, easier housing and handling, and minimal social concerns are some of the reasons why they are preferred. ⁷⁴ The possibility to define and control the genetic background of the experimental animals creates less variation and need of fewer animals to achieve statistically valid data. ⁷⁵ Thus, small animals usually constitute a starting point for proof of principle studies ³² using ectopic models, which evaluate tissue regeneration in a distant site, most commonly subcutaneous, as was observed in the present review. The lack of endogenous chondrogenic and/or osteogenic cells, cytokines, and mechanical factors (all of which can potentially stimulate cartilage and bone formation) within the local intradermal environment, ensures that, theoretically, any resulting cartilage or bone formation after cell and/or scaffold transplantation is of exogenous origin. ⁷⁶ In addition to their low cost, immunodeficient animals allow xenograft-based experiments, and their lax skin permits large-volume implants. ⁷⁶ However, separating the implant from surrounding tissue, and migration of small implants in the lax skin, can be challenging in subcutaneous implantation models. ⁷⁶ Ectopic models in the included studies were performed in immunocompromised mice and aimed for disc,^48,51,59 synovial membrane, ⁵⁰ or condyle^{42–44,52,57,58,62,63} regeneration. However, a major drawback using mice in TMJ TE studies is the physical difficulty of an orthotopic approach, due to limitations in size of the joints.^77,78 Rabbits provide the same advantages as rodents, in terms of their small size and easy handling, but are large enough to allow experimental TMJ surgery.^41,49,55,79 A majority of included studies reported the use of orthotopic models in rabbits. Orthotopic models are performed in an environment with the presence of tissue-specific endogenous factors and cells. ⁷² Rabbit TMJ discs share many similarities to humans, given their biochemical composition, morphology, collagen fiber organization, and attachments.^80–82 They may therefore more closely reflect the human clinical situation when compared with ectopic models in rodents. At the same time, results of (osteo)chondral regeneration in rabbit models should be interpreted with caution, due to the high potential for spontaneous healing reported in orthopedic experimental models, and considerable differences in size and loading conditions, as compared with the human situation.^83–85

Although no single species can provide the optimal experimental model for TMJ research, ⁸⁶ large animal models are reported to more closely mimic the clinical situation and predict therapeutic efficacy. ⁷⁵ High costs, ethical issues (e.g., in the case of dogs), and handling difficulty (e.g., in the case of pigs) are some limitations of large animals. ⁷⁹ In context, mini pigs represent a more suitable model because of more easily handling than farm pigs ⁸⁶ and morphological similarities to human bone and TMJ structures than other large animal models.^34,87 There has been an increase in preclinical in vivo publications on TMJ-related research along with a considerable change in species used, ⁷⁸ which may partly be explained by the high costs and pressure from animal protection groups. ⁸¹ In the present review, orthotopic models in large animals were used for regeneration of the TMJ disc in dogs,^65,66 and condyle, in either sheep ⁴⁵ or goats.^61,64 The TMJ of the dog has a slight laterotrusion movement, ⁸⁸ although it is a hinge joint and mainly rotate. Considering that dogs are carnivores, ⁸⁶ their TMJ loading is reportedly greater than that of both rodents and primates, ⁷⁸ and may more closely mimic the strain in a human joint. Sheep TMJ disc and condyle anatomy have similarities to humans, in addition to easy surgical access, and have therefore been proposed as a prime model for TMJ research, ⁸⁹ despite the fact that both sheep and goats are herbivores and mainly have translational TMJ function. ⁸⁶ No studies reported TMJ models in pigs or mini pigs, which have been reported to most closely resemble human TMJ disc morphology,^80,87 anatomy, and function.^87,90,91 However, additional drawbacks to high cost, as difficult surgical access through lateral prearuicular approaches, due to the zygomatic arch anatomy, might be the explanation. ⁸⁷

TE strategies

Future design of preclinical models

Overall, the RoB of included studies was considered to be unclear. When designing future in vivo studies, increased focus on the ARRIVE^37,38 and SYRCLE ⁴⁰ guidelines, would be preferable to lower the RoB, establish consistency, and minimize discrepancy. Reporting of species age, gender, strain, weight, source, and housing and husbandry, is important for comparing methodology and outcome, and was frequently inadequately reported in the present review. Sample size and power calculations should be reported, to detect and confirm statistically significant differences between experimental groups and to justify the number of animals used. Randomization and blinding procedures, should be applied to limit selection bias and detection bias, respectively. Standardization of animal models would decrease the heterogeneity observed and improve translation to clinical applications. ¹³⁶ A recent review ⁸⁶ suggests the possibility of exploring naturally occurring TMJ disorders in companion animal models, that is, dogs and cats. This could more closely mimic the “true” progression of OA than surgically created defects, in addition to reflect the genetic diversity and variations seen in humans. ¹³⁷ Although, it might be difficult to standardize such models, it could potentially benefit both animals and humans in the future.

Selection of appropriate controls is essential. Several studies^{41,65,66,68,69} used the contralateral joint as a control which could be efficient in reducing the number of animals used. No included studies reported contralateral healthy controls, which may bias the results in terms of contralateral overload, ¹³⁶ and therefore a bilateral intervention may be favorable. Quantitative measures of outcome to evaluate success, for example, histomorphometry and radiography, would ease the comparison between studies and increase the validity of the results. Only two included studies^51,65 reported biomechanical assessment of TMJ implants to detect clinically relevant differences in functional performance. However, the first study (published in 1994) ⁵¹ compared compressive load of ectopically regenerated fibrocartilage to native rib and glenohumeral hyaline cartilage, which do not represent the ideal controls. More recent studies have contributed to increased knowledge about differences between tissue-specific characteristics of cartilage. ¹¹⁶ Since the human TMJ is a highly functional ginglymoarthrodial joint with a capacity for multidirectional activity, that is, rotation and translation,^86,138 assessment of biomechanical parameters, for example, compressive modulus and ultimate strength, ⁵⁵ may be important indicators of functional performance. Furthermore, inclusion of biochemical assessments of organic content of in vivo regenerated tissues, for example, collagen and GAG's, ⁶⁵ would support standard histological and radiographic data, and strengthen the structural comparison to native tissues.

Clinical relevance

When diagnosing symptomatic clinical TMDs through arthroscopy and imaging, ¹³⁹ favorable treatment options are currently limited. Discectomy of unsalvageable discs may trigger a more degenerative state of the disease, whereas replacement with an interpositional graft material, of temporal muscle, dermis, or fat grafts, would result in extensive donor-site morbidity and scar tissue formation. Treatment options for more severe TMDs consist of autogenous grafts or TMJ reconstruction prosthesis.^14,16 Costochondral or sternoclavicular grafts are associated with donor-site morbidity, technical sensitivity, and delayed functional loading with unpredictable results such as high failure rates due to graft overgrowth and risk of reankylosis.^17,140 TMJ prosthesis allow the patient to return to function sooner, result in lower postoperative pain scores, avoid donor-site morbidity, and offer better replication of functional anatomy and earlier postoperative rehabilitation. ¹⁴¹ However, the high costs preclude their applicability among all socioeconomic groups of patients, in addition to concerns regarding potential material wear/failure, long-term stability, and function. ¹⁴² Distraction osteogenesis of the posterior border of the ramus is another approach of condyle regeneration. However, the method is technically difficult due to surgery and vector control, and the prolonged consolidation period makes the method prone to infections. ¹⁴³ Thus, due to the limitations of current treatment modalities, TE may offer promising alternatives for treatment of TMDs.^{11,23,122,144} While successful clinical outcome of TE for knee meniscus regeneration has been reported,^145–147 it is unclear whether these results can be translated to the TMJ. To our knowledge only one study ¹⁴² so far has reported the use of HA scaffolds loaded with BM aspirates for condyle regeneration in seven pediatric patients (mean age 9.71 years) with high success in terms of clinical (mouth opening) and radiographic (CT) outcomes, after 1 year follow-up. However, considering their young age and short follow-up, the long-term results may still be unpredictable.