Cellularized Biomaterials Used as Gingival Connective Tissue Substitutes In Vivo : A Systematic Review

Introduction

Periodontal defects, impairing the specialized tissues that both surround and support the teeth to maintain them in the jawbones, are a public health issue. ¹ The most prevalent periodontal defect is gingival recession, affecting more than two-thirds of the population worldwide, according to a recent meta-analysis. ² Gingival recession is described as the apical shift of the gingival margin past the cemento-enamel junction of a tooth, ³ which irreversibly results in exposure of the root surface to the oral environment. Moreover, untreated gingival recession tends to worsen despite good patient motivation. ⁴ Gingival recession has a negative impact on patients’ oral health-related quality of life, ⁵ and requires surgical treatments.

The gold standard for increasing soft tissues around teeth and implants is the subepithelial connective tissue graft (SCTG), which is an autogenous graft. ⁶ However, autogenous grafts present several limitations: increased morbidity, requirement of a large second surgical site in a usually painful area of the oral cavity, and sometimes cannot be performed due to donor site insufficiency. ⁷ Therefore, allografts and xenografts were introduced as alternative solutions. Currently commercialized soft tissue substitutes help avoid a second surgical site and display interesting results. Still, their use is not as clinically efficient as the SCTG.^8,9 Besides, these biomaterials can be expensive, require rigorous procedures, and xenografts involve the use of animals. To go further, it was reported that the stability of the gingival margin is not maintained over time using acellular dermal matrices and collagen matrices from porcine origin compared to autologous connective tissue graft procedures. ¹⁰ These limitations encouraged alternatives from tissue engineering, which combine autologous cells and a substitute matrix mimicking gingival connective tissue to increase the thickness of the missing gingiva. Large viable constructs can be engineered using conventional fabrication techniques (e.g., electrospinning) or using additive manufacturing (e.g., 3D bioprinting) . ¹¹

The hypothesis of this study was that cellularized biomaterials mimicking gingival connective tissue properties may exhibit the best grafting characteristics in vivo, in terms of mechanical properties, vascularization, and extracellular matrix (ECM) remodeling. This systematic review aimed to analyze the current English-language literature and compare the compositions and outcomes of tissue engineered cellularized gingival grafts in vivo. As human trials are not yet widespread, this review specifically focused on animal experiments analyzing gingiva regeneration before any potential human application.

Methods

Results

Study selection and characteristics

The PRISMA flow diagram detailing selection process is presented in Figure 1. Our primary search yielded 631 articles after manual removal of duplicates. Titles and abstract screenings resulted in 58 studies sought for retrieval (kappa inter-observer agreement between the two reviewers was 0.51). One article selected was retracted from publication and consequently excluded. After full text reading, 39 articles out of the 58 assessed for eligibility were excluded for not meeting the inclusion criteria (kappa inter-observer agreement between the two reviewers was 1). In the end, 19 studies were included in this systematic review: 14 developed oral mucosa and gingiva substitutes,^16–29 whereas 5 developed soft tissue substitutes not specific to gingiva but were included because they used cells extracted from gingiva.^30–34 Regarding institutions, five studies were from Spain and three from China, whereas the others were from diverse countries across America, Asia, and Europe. Four^16,18,20,22 out the six Spanish studies and two^27,28 out of the four Chinese studies were from the same team, and thus went further in their work. Most of the included studies were published between 2015 and 2023, whereas the other five were issued between 2007 and 2011.

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FIG. 1.

PRISMA flow diagram of the online selection process. PRISMA, preferred reporting items for systematic reviews and meta-analyses.

Risk of bias assessment

Risk-of-bias assessment ranked from low to high according to the SYRCLE checklist is presented in Figures 2 and 3. Overall, bias associated with baseline characteristics and attrition bias were the lowest (risk of bias domains 2 and 8, respectively). Two studies^21,26 mentioned following the Animal Research Reporting In Vivo Experiments (ARRIVE) guidelines. ³⁵ Nonetheless, most studies lacked details regarding randomization and blinding (risk of bias domains 1, 3, 4, 5, and 6). Corresponding risks of bias were judged “unclear,” as recapitulated in Figure 3. Two studies did not mention the number of animals studied and consequently showed the highest risk of bias.^18,27

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FIG. 2.

Traffic-light plot of quality assessment for each included study using SYRCLE checklist and robvis tool. SYRCLE, systematic review center for laboratory animal experimentation.

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FIG. 3.

Summary plot of quality assessment for the included studies using SYRCLE checklist and robvis tool. SYRCLE, systematic review center for laboratory animal experimentation.

Discussion

This systematic review aimed to report the state of the art on cellularized biomaterial substituting gingiva tested in vivo and promoting remodeling. To our knowledge, this review is the first focusing on gingival tissue engineering after implantation using cellularized constructs. Our hypothesis could not be confirmed, as most of the approaches achieved vascularization and ECM remodeling of the graft after implantation, without necessarily detailing the mechanical properties. Moreover, most of them used cells and molecules that have been described in the gingival connective tissue.

Besides one retracted article, the search query used for this systematic review did not select human studies. A complementary search allowed us to find ongoing clinical trials using gingival cells loaded in a scaffold for alveolar bone defect treatment for periodontitis without any published outcome (NCT03137979 and NCT03638154) and one with promising published results (Abdal-Wahab et al., 2020, 10.1111/jre.12728). It was not included in the review because this review concentrates on soft tissue regeneration. Cellularized biomaterials tested in humans for maxillofacial application often focus on bone tissue (and not on connective tissue) in the context of periodontitis. However, one ongoing clinical trial is comparing the regenerative and differentiation potential of gingival MSCs from several harvesting sites for future dental tissue regeneration applications (NCT03570333). The use of cellularized biomaterials to mimic and increase gingival connective tissue seems to be a research field that still needs time and effort before human application.

Cell sources were diverse across the included studies. When autologous cells were used, experiments were conducted on big animals (rat, rabbit, dog). Four studies compared cells to find the best vascular remodeling after cellularized engineered scaffold implantation in vivo.^17,18,33,34 Indeed, two models did not find a significant difference in vascularization remodeling after implantation for their gelatin/fibrin construct with human fibroblasts from skin derma and gingiva ³³ or for their silk fibroin construct with human DPSCs and gingival fibroblasts. ³⁴ The article by Blanco-Elices et al. suggests that predifferentiated MSCs from dental pulp and bone marrow have a vascularization potential similar to HUVECs and better than those that are not predifferentiated and predifferentiated MSCs from adipose tissue when added in coculture with gingival fibroblasts in their construct. ¹⁸ To go further, GMSCs and DPSCs compared in vivo showed a similar number of vessels and hemoglobin concentration, but in vitro GMSCs showed better proliferation and migration potentials and a higher ability to form angiogenic tubules compared to DPSCs. ¹⁷ Besides being the cells from the native tissue aimed to be constructed, using gingival cells for tissue engineering offers several advantages. As they are routinely removed and considered biological waste during dental procedures, gingiva is easily accessible and gingival cells culture is well-known. Regarding cell quantities, most studies reported cell numbers in a range between 10⁵ and 10⁶, but they usually did not specify the total volume of the finished construct. Indeed, cells seem to represent 5% of the connective tissue for healthy human gingiva. ³⁷ Thus, comparing the number of cells inside the total volume of the construct could have been interesting to see how well the native cell concentration was reproduced.

The matrices of all included studies were based on naturally derived components, mainly proteins and polysaccharides, to engineer their constructs. The rationale for using natural polymers is to guarantee several requirements of a successful gingival graft, such as biocompatibility, biodegradability, hydrophilicity for soft tissues, and functional similarity to native ECM. The frequent use of fibrin can be explained by its rapid degradation, its minimal foreign body response, and its use as a sealant for wound healing. One of the studies used Matrigel^®, a commercial product mostly composed of laminin and collagen IV, which are derived from a murine tumor called Engelbreth–Holm–Swarm. ³⁸ However, Matrigel^® can present variation in composition even within the same batch, and consequently in its biochemical properties. ³⁹

Technical specifications needed for a cellularized construct to be implanted in vivo include cell viability, biocompatibility, and mechanical properties similar to the native tissue it aims to reproduce. All constructs analyzed in this systematic review were cytocompatible and were tolerated in vivo. Concerning the mechanical properties, comparing the constructs was not possible due to the heterogeneity of measurements. Some studies calculated alternatively the “modulus of elasticity,” the “tensile modulus,” or the “compression modulus.” Simply put, all these terminologies describe how a material can easily stretch and deform. ⁴⁰ Only one study tested the material stiffness, which is a material’s capability to resist elastic deformation. Thus, these measurements are complementary but not similar. Studies measuring mechanical values did not use the same device or settings and therefore could not be compared. Moreover, one major issue was the lack of consensus concerning reference values of human gingival connective tissue mechanical properties. ⁴¹ Indeed, one-third of the studies tried to replicate the original mechanical properties, but they did not use the same parameters.

Concerning all the tissue engineering techniques encountered in this review, 3D bioprinting offers advantages when cells are needed. Indeed, constructs can be customized to fit the defect in clinical application. The defect geometry can be digitalized with optical impression (intraoral scanner) or imaging segmentation (computed tomography-scan, cone beam computed tomography, magnetic resonance imaging). Thanks to automatization, the construct production is reproducible and fast. Another advantage of bioprinting is the differential cell distribution within layers of the cellularized construct. Each bio-ink can exhibit a specific cell type, and cell density can be controlled in the printed construct.

Most of the constructs included in this review needed additional chemical factors for crosslinking. Because they could modify the properties of the construct, new technologies present promising potential. For instance, the use of nucleotide lipids can give auto-assembly properties to a gel and thus produce crosslinker-free scaffolds. One example in the literature tested their nucleotide lipid-based hydrogel with gingival fibroblasts to show cytocompatibility. ⁴²

Vascularization was mostly explored by histological analysis, and few studies quantified the number of vessels and the hemoglobin concentration after implantation. However, it was surprising that none explored the branching length per field, the number of junctions per field, or the number of meshes per field. Those characteristics are typically explored to assess vascularization remodeling.

Various limitations were met during this systematic review. A meta-analysis of the studies was not relevant due to the restricted number of studies and the heterogeneity in materials and methods. Another limitation was the low number of studies focusing exclusively on connective tissue, leading to the inclusion of studies on bone-gingiva constructs. Research on gingival connective tissue engineering can expand to these types of multilayered constructs for periodontology surgery. In these cases, the extraction of results concerning strictly the connective tissues was not always possible. An additional limitation was the risk-of-bias judgement using the SYRCLE checklist. Most items were judged unclear due to a lack of clear statements in the included studies, even for research teams declaring adherence to the ARRIVE checklist. Thus, it is possible that some studies do not report all technical details important for compiling results. As each approach combined multiple protocol specificities, correlations between one composition parameter and one specific outcome were difficult to isolate. Researchers working on cellularized biomaterials used as gingiva substitutes should be more rigorous in their reporting of animal studies and should more closely follow the ARRIVE guidelines. In addition, stiffness of the biomaterial is usually a limitation for handling and was not systematically reported. Mechanical characterization should be more complete to better report the chemicophysical properties of the material used. For now, research on cellularized biomaterials used as gingiva substitutes aims to show proof of concept. In doing so, there is usually no study of the biomolecular aspect. Indeed, articles report only graft integration, vessel formation, and sometimes ECM remodeling.

Nonetheless, gingival cells as a cell source in sufficient numbers, natural components with a solution to add stiffness to better replicate the native gingival connective tissue, and 3D bioprinting as a tissue engineering technique all seem like interesting parameters to combine to obtain a graft that may be used for human application one day. Further studies seem necessary to validate which cellularized biomaterial is the most suitable to human application to compensate for gingival loss. In the literature, several hydrogels not yet tested in vivo showed potential for increasing gingival thickness. For instance, Oliveira et al. developed an extrudable hydrogel composed of methacrylated-collagen and methacrylated-HA, ⁴³ an interesting lead to use gingival cells and go a step further toward human application. Other promising candidates should arrive in a couple of years in the literature, offering new perspectives on the subject.

Conclusion

This systematic review showed the multiplicity of cellularized biomaterial used to engineer large viable connective tissue constructs tested in vivo. Various cell types from several cell sources could be used to do so. Few studies integrated coculture in their constructs to promote vascularization. Vascularization and ECM remodeling of the graft after implantation occurred in most of the included studies. Remodeling after implantation could be achieved using several tissue engineering technologies to engineer constructs made of naturally derived components. These results will enable us to set up experiments based on all the parameters, the diversity of which shows that there is a certain amount of freedom.