Abstract
Tissue engineering is a promising alternative that may facilitate bony regeneration in small defects in compromised host tissue as well as large mandibular defects. This scoping systematic review was therefore designed to assess in vivo research on its use in the reconstruction of mandibular defects in animal models. A total of 4524 articles were initially retrieved using the search algorithm. After screening of the titles and abstracts, 269 full texts were retrieved, and a total of 72 studies included. Just two of the included studies employed osteonecrosis as the model of mandibular injury. All the rest involved the creation of a critical defect. Calcium phosphates, especially tricalcium phosphate and hydroxyapatite, were the scaffolds most widely used. All the studies that used a scaffold reported increased formation of bone when compared with negative controls. When combined with scaffolds, mesenchymal stem cells (MSC) increased the formation of new bone and improved healing. Various growth factors have been studied for their potential use in the regeneration of the maxillofacial complex. Bone morphogenic proteins (BMP) were the most popular, and all subtypes promoted significant formation of bone compared with controls. Whilst the studies published to date suggest a promising future, our review has shown that several shortfalls must be addressed before the findings can be translated into clinical practice. A greater understanding of the underlying cellular and molecular mechanisms is required to identify the optimal combination of components that are needed for predictable and feasible reconstruction or regeneration of mandibular bone. In particular, a greater understanding of the biological aspects of the regenerative triad is needed before we can to work towards widespread translation into clinical practice.
Introduction
Healthy mandibular bone is well vascularised and has a high capacity for regeneration after injury. Whilst recent advances in surgical techniques and biomaterials have improved outcomes of the treatment of mandibular defects, situations still arise when intrinsic regeneration is not possible. Such situations can be categorised into those in which the quantity of bone needed is greater than the intrinsic ability to regenerate it (critical-sized defects), and those in which the ability of the host tissue to regenerate has been compromised. Critical-sized defects of the mandible are routinely encountered secondary to high-energy trauma, the removal of tumour, or congenital abnormalities. In contrast, certain conditions may cause seemingly small insults to progress in large non-healing lesions. Most notably, osteoradionecrosis (ORN) and medication-related osteonecrosis of the jaws (MRONJ) are two iatrogenic diseases that are increasingly reported.
Current methods of reconstruction
Tissue engineering may be defined as the use of cells, techniques of material engineering (such as scaffolds and membranes), and biochemical interactions to improve or restore biological tissues. The unique structure of the mandible, however, poses a specific reconstructive challenge. Functions, including mastication and speech, place it under constant stresses, and these must be considered when planning a suitable reconstruction.
Current methods of mandibular reconstruction include the use of alloplastic materials, non-vascularised reconstructions, and free tissue transfer. In the past, alloplastic materials, such as titanium plates, have been used to re-establish the form of a lost segment. Numerous studies on titanium reconstruction plates have reported high failure rates of up to 52%, with an average retention time of 8-10 months. During functional loading, moment and shear forces produce high concentrations of squeeze and press stress in the mandible, which cause bony resorption and consequently a loss of screw retention. Currently, the use of reconstruction plates is limited to the fixation of bone grafts to the defect site.
Non-vascularised reconstructions can be used in isolated bony defects with minimal loss of soft tissue. The anterior and posterior iliac crest are common sites for harvest, and the latter can provide more particulate bone and considerably larger blocks of corticocancellous bone. Whilst these autogenous grafts are theoretically ideal, they are not suitable for larger defects, and 75% failure has been reported in those that were larger than 12 cm. Other factors such as previous injury from radiation or contamination of the graft by saliva can also contribute to high failure rates.
Currently, the gold standard for reconstruction of large defects is free tissue transfer with microvascular anastomosis. Common donor sites include the fibula, iliac crest, scapula, and radius. Vascularised free flaps have a considerably higher incidence of bony union and subsequent success of dental implants when compared with non-vascularised grafts: 96% (47/49) compared with 69% (18/26) for bony union, and 99% (70/71) compared with 82% (27/33) for dental implants, respectively.
Despite autologous free tissue flaps being the current gold standard, they are not without their complications such as donor-site morbidity. Tissue engineering is a promising alternative that may facilitate bony regeneration in small defects in compromised host tissue, and in large mandibular defects.
In general, tissue engineering of bone is complicated by the interplay between the ground substance, cells and biochemical milieu, and the dynamic process of bony remodelling. In the mandible it currently relies on a combination of techniques that attempt to replicate these uniquely complex features. We may consider these techniques as three distinct categories: scaffolds; bioactive substances; and cell therapy. The use of biomimetic scaffolds that attempt to reconstitute the nascent tissue architecture has been well documented, and a range of materials are now available. Both synthetic and autologous scaffolds, usually extra-cellular matrix components, provide a range of properties, each with its own unique advantages and disadvantages.
The turnover and regeneration of bone is regulated by a plethora of cytokines and growth factors. Most notable are the bone morphogenetic proteins (BMP), a group of pleiotropic cytokines of the transforming growth factor beta (TGFB) superfamily. Different members of this family seem to function at key stages of turnover, with some redundancy. From a tissue engineering perspective, BMPs (amongst other growth factors) have been used, usually in combination with a carrier of some kind, to augment new bony development. A tissue’s response to a given cytokine is not stereotypical, and depends on a number of pharmacodyamic properties, including duration of exposure, dose, and pattern of decay. This is also true of BMPs and their influence on the growth of new bone. The first difficulty when applying these biochemical interactions in tissue engineering is to understand how these different pharmacodynamic properties influence phases of bony growth, and the second is to develop the means by which to reproduce these properties in the engineered material. Different carriers, including biodegradable scaffolds and nanoparticles, may provide the solution.
Bony turnover, under both physiological and pathological conditions, is mediated by the cellular compartment. The concept of a bone-specific stem cell niche is well established, and bone marrow-derived mesenchymal stem cells (BM-MSC) are thought to be fundamental players that contribute to both the normal turnover and repair of bone. Their roles in turnover are initiated through several independent mechanisms, including direct differentiation into osteoblasts, immunomodulation, angiogenesis, and cell recruitment. To this end, tissue engineering has been used to introduce MSCs to a bony defect to aid regeneration. Complexities that arise here are similar to those that arise with the use of cytokines in tissue engineering; delivering the correct phenotype of stem cell at the correct cell density to the correct location within a mandibular defect is no simple task.
Given the potential of tissue engineering to repair mandibular defects, a rigorous review of current evidence is required to identify the challenges that will need to be addressed. We have therefore completed a scoping review with the aim of identifying the characteristics, breadth, and results of existing research on the use of these techniques in mandibular reconstruction.
Methods
This review follows guidelines on the conducting of systematic scoping reviews from the Joanna Briggs Institute. This method summarises the evidence available on a topic to convey its breadth and depth.
Research question
The research question for this review was: “What are the characteristics, breadth, and results of the existing research on the use of tissue engineering techniques in the reconstruction of mandibular defects?”
Information sources and search strategy
A search strategy combining both MeSH and free-text terms for tissue engineering and mandibular reconstruction was developed to retrieve articles of interest from Medline and PubMed Central (supplementary Table 1).
Searches were limited to studies in the English language, and there was no defined time period during which studies must have been conducted. The reference lists of the articles identified from the initial search were screened for further relevant studies.
Eligibility criteria
We considered only full-text papers that reported original data from in vivo studies on mandibular regeneration in animal models. Conference abstracts, review papers, letters to the editor, and opinion pieces, were excluded, as were studies on animal or human tissues in vitro, as these experiments would not reflect the complexity of the architecture and the functional requirements associated with the repair of mandibular defects. Projects that looked at periodontal or alveolar regeneration, dental implants, distraction osteogenesis, autologous bone grafts or free flaps, and the optimisation of fracture healing without tissue loss, were also excluded.
Study selection and data extraction
Titles and abstracts were screened by two reviewers against the agreed inclusion and exclusion criteria. Disagreements between reviewers were resolved by discussion. The reasons for exclusion were recorded only at the full-text stage. A data extraction tool was used for further analysis of the selected full tests – this was initially done by one reviewer, and verified by a second. Extracted items included: article identifiers (author, year, title), study characteristics (sample size, design, population, inclusion and exclusion criteria), setting (animal species, age of animal, type and mechanism of bony defect), and method of engineering (scaffolds, bioactive factors, and cell therapy). Outcome measures were results and conclusions.
Data pertaining only to the in vivo component of a study (when in vitro pre-analyses were conducted) were extracted and included in the present analysis.
Results
A total of 4524 articles were initially identified using the search algorithm. After screening of the titles and abstracts, 269 full texts were retrieved, and a total of 72 studies included ( Fig. 1 ). No further relevant studies were identified after manual searches of the reference lists of the articles included.
Setting
Most of the articles (44/47) included at full-text review were from just three countries: the United States of America, Germany, and China. The rest were from a number of other global institutions. This distribution is a reflection of the total number of mandibular defects from these three geographical regions compared with elsewhere (1148 defects from USA, Germany, and China, compared with just 533 from other regions) ( Table 1 ).
| Country | No. of studies | Total No. of defects in analysis | Reference |
|---|---|---|---|
| China | 18 | 357 | |
| USA | 17 | 412 | |
| Germany | 9 | 319 | |
| Japan | 6 | 56 | |
| United Kingdom | 6 | 46 | |
| South Korea | 4 | 123 | |
| Iran | 2 | 16 | |
| Spain | 2 | 29 | |
| Sweden | 2 | 100 | |
| Brazil | 1 | 36 | |
| Egypt | 1 | 16 | |
| Finland | 1 | 24 | |
| France | 1 | 6 | |
| Israel | 1 | 25 | |
| Turkey | 1 | 56 |
The frequency of publications increased progressively from 1994 to the present time, reflecting growing interest in the field ( Fig. 2 ).
Study design, type of defect, and mandibular models
Study design was categorised according to the type of mandibular defect, either critical or osteonecrosis (of any cause), and the intervention used. Interventions were broadly categorised into three primary groups: use of a tissue scaffold, use of cell therapy (irrespective of cell identity), or use of a bioactive substance.
Just rest involved the creation of a critical defect. Most reported a combination of the three interventions (39 (54%) combined use of a scaffold with a bioactive marker, while 14 (19%) combined use of a scaffold with some form of cell therapy) ( Fig. 3 ).
All the papers, except four observational studies that lacked control groups, were designed with both intervention and control arms. There was considerable variation in complexity and scale. The modal sample size, defined here as the number of mandibular defects/treatment or control group (the smallest sample size was taken when sample heterogeneity was identified), was six (range 1–12) defects.
Only four studies alluded to blinding in the study design. They blinded examiners to the histological or stereological analysis of repaired defects after extraction from the models. Twelve studies specified the randomisation of animal models into initially predefined treatment and control groups, or randomisation of selection for sacrifice (if staggered end points were used). None, however, mentioned the method of randomisation.
The duration of follow up (the time at which the mandibles were analysed for progress – either using imaging if the animal was to remain alive, or at the time of sacrifice) varied widely, and depended strongly on the animal used. This was also true for the size of the critical defect, though this also seemed highly heterogeneous even within one animal model ( Table 2 ). A total of 24 studies exploited staggered end points to allow for the sequential monitoring of mandibular repair. This involved serial imaging if animals were to remain in the study, or sequential sacrifice with subsequent analysis of the extracted mandibular graft.
| Animal model | No. of studies | Mean (SD) critical defect (mm) | Mean (SD) follow up (weeks) | Reference |
|---|---|---|---|---|
| Dog | 12 | 24.1 (9.9) | 26.7 (32.0) | |
| Mini-pig | 4 | 31.25 (19.3) | 36.0 (28.5) | |
| Monkey | 8 | 27.0 (3.5) | 36.3 (31.7) | |
| Mouse | 1 | 10.0 (na) | 4.0 (na) | |
| Pig | 1 | 35.0 (na) | 8.0 (na) | |
| Rabbit | 13 | 15.0 (4.7) | 9.0 (3.9) | |
| Rat | 25 | 4.8 (0.5) | 9.9 (6.0) | |
| Sheep | 8 | 32.6 (10.6) | 21.7 (15.3) |
Scaffolds
A number of different biomaterials were used as scaffolds (supplementary Table 2).
Although 75% of the studies used scaffolds, comparison of their function was considered the primary variable of investigation in only 11. , , All the other studies used a scaffold for cell seeding or to distribute growth factors (discussed further in the section on outcome measures). Of those that used a scaffold, whether as a primary variable or as a delivery vector, there was considerable heterogeneity in the materials used. Most were synthetic 3D porous scaffolds that used some synthetic polymer, such as beta-tricalcium-phosphate. Two studies reported the use of coral as a biological alternative.
Bioactive substances
Bioactive molecules were used in 53 studies, either alone or in combination with a scaffold or cell therapy, or both ( Fig. 4 ). The most common growth factor was BMP2, which was used alone in 28 studies and in combination with other growth factors in a further six (including one study that used a “bone growth factor mixture”). Ten studies included treatment groups with varying dosages of growth factor, which allowed for comparison of the influence of variable dosages on bony regeneration.
