The Need for Integrated Research Autopsies in the Era of Precision Oral Medicine

Abstract

Background:

The autopsy has benefitted the practice of medicine for centuries; however, its use to advance the practice of oral health care is relatively limited. In the present era of precision oral medicine, the research autopsy is poised to play an important role in understanding oral-systemic health, including infectious disease, autoimmunity, craniofacial genetics, and cancer.

Types of Studies Reviewed:

The authors reviewed relevant articles that employed medical and dental research autopsies to summarize the advantages of minimally invasive autopsies of the dental, oral and craniofacial tissues and to outline practices for supporting research autopsies of the oral and craniofacial complex.

Results:

We provide a historical summary of research autopsy in dentistry and provide a perspective on the value of autopsies for high-resolution multiomic studies to benefit precision oral medicine. As the promise of high-resolution multiomics is becoming realized, there is a need to integrate the oral and craniofacial complex into the practice of autopsy in medicine. Furthermore, the collaboration of autopsy centers with researchers appears will accelerate the understanding of dental, oral, and craniofacial tissues as part of the whole body.

Conclusions:

Autopsies must integrate oral and craniofacial tissues as part of biobanking procedures. As new technologies allow for the high-resolution, multimodal phenotyping of human samples using optimized sampling procedures will allow for an unprecedented understanding of common and rare dental, oral, and craniofacial diseases in the future.

Practical Implications:

The COVID-19 pandemic highlighted the oral cavity as a site for viral infection and transmission potential; this was only discovered via clinical autopsies. The realization of integrated autopsy’s value in full body health initiatives will benefit patients across the globe.

1. Reframing a Role for Research Autopsies in Modern Medicine (Words: 543):

As the mouth is the window to the body, the autopsy is the window opening to understand the relationship between health and disease states. While the autopsy has benefitted the practice of medicine for centuries, its use to advance the practice of oral health care is relatively limited. Most conventional autopsies procedures employ surgical methods for forensic, clinical, or academic purposes¹; though outside of forensics (i.e., forensic odontology), autopsies involving oral and craniofacial tissues are uncommon². However, even in medicine, conventional autopsies are widely known to be on the decline over the last 50 years—especially in hospital settings^{3, 4}. For example, in the US, conventional autopsies have decreased from 50% of all hospital deaths to less than 5% since the 1970s^{5, 6}; this is comparable globally^{7, 8}. The reasons for the decrease in autopsies include 1) cultural and/or religious objections, 2) high costs for time and limited personnel, and 3) growing opinions on the lack of an autopsy’s usefulness compared to other diagnostic techniques^{9, 10}.

Despite these beliefs, conventional autopsies still provide enormous value for guiding treatment decisions, understanding trends as to the cause of death, and providing context for healthcare policy prioritization, and research¹¹. For example, the conventional autopsy used for research purposes—sometimes called the research autopsy¹²—has proven incredibly useful for understanding cardiovascular diseases¹³, stroke¹⁴, multiple sclerosis¹⁵, cancer biology and chemotherapeutic failures^{12, 16}, and viral pandemics such as H1N1¹⁷ and COVID-19^{18, 19} and epidemics caused by Zika virus²⁰. Again, despite this utility, autopsies have been limitedly used in research of the oral and craniofacial complex, and few of the most widely productive autopsy centers in the world include tissues and fluids of the oral and craniofacial complex as part of their inspection, sampling, and biobanking procedures (Figure 1). There remains a need to apply new techniques and promote the wider recognition of dental, oral, and craniofacial tissue relevance for global initiatives that aim to promote precision medicine as well as oral and overall health^21–24.

Figure 1 |. Opportunities to Collaborate with High-Volume Autopsy Centers.

While conventional autopsies have decreased across the globe, there are still major autopsy centers in North America (United States), South America (Brazil), Europe (UK, Germany, the Netherlands), and Asia (China). Since there remains a need for studies that incorporate autopsy as a valuable tool for understanding human disease, there is an opportunity to collaborate with these and other sites that perform an autopsy to advance initiatives in precision oral medicine. For example, there are current collaborations between the University of São Paulo and many local and international partners. Considering this is a highly productive autopsy center (~15,000 cases per year), collaborations for dental, oral, and craniofacial research are possible here because this center importantly includes oral tissues as part of autopsy procedures and invites dental professionals to participate as part of the autopsy team.

Furthermore, as the research and clinical communities begin to realize the promise of high-throughput and high-resolution, multiomic assays—so-called “deep phenotyping”—using genomic, epigenomic, transcriptomic, proteomic, and metabolomic sequencing—to usher in the era of precision medicine for oral and overall health²⁵, there is a need to integrate the oral and craniofacial complex into the practice of autopsy in medicine (Figure 2). Consider recent efforts to understand the bidirectional relationship of the oral cavity in the context of extraoral diseases such as cardiovascular disease²⁶, type I and type II diabetes^{27, 28}, inflammatory bowel diseases²⁹, and among many others³⁰—that could benefit from integrated research autopsy, biobanking, and downstream multiomic assays^{31, 32}.

Figure 2 |. Sampling the Integrated Anatomy of the Aerodigestive Tract Through Autopsy.

(A) The oral cavity is both the beginning of the airways and digestive tract. There are several common—and especially rare---diseases that affect the oral cavity in addition to more internal niches of these tracts. New advanced sequencing and imaging technologies, which are foundational for the new single-cell and spatial biology, are laying the foundation for new ways to conceptualize the tissue niches by considering cellular and molecular spatial organization, cellular neighborhoods, and structural motifs—the so-called rules or “core components” that construct a niche. These concepts may provide important and novel biological readouts for precision medicine. For example. considering integrated oral, nasal, and airway barrier niches—the so-called “inhalation interface” which displays unique niches from external to internal sites-may be a potential accelerator for these efforts. (B) This integrated sampling approach could be leveraged for curated biobanking, single-cell and spatial multiomic studies combined with genomic sequencing, and non-invasive sampling of RNA/proteins for disease biomarker discovery.

It is important to emphasize that the oral cavity is contiguous with the skin and also a part of the aerodigestive tissues at the axis of breathing and digestion. As such, several rare diseases affect these shared epithelial barriers such as epidermolysis bullosa³³, Job’s syndrome (Hyper-IgE syndrome)³⁴, acanthosis nigricans³⁵, recurrent respiratory papillomatosis³⁶ as well as systemic diseases of the liver, kidneys, and hematopoietic, immune, and endocrine systems that can present lesions in the oral cavity^{37, 38}. To address these concepts and argue for increased collaboration, we 1) provide a historical overview of the research autopsy in dentistry, 2) describe the advantages of minimally invasive autopsies (MIA) in the oral and craniofacial complex, and 3) provide a perspective on the value of autopsies for high-resolution multiomic studies to benefit the era of precision medicine.

2. A Brief History of Autopsies in Medicine and Dentistry (Word Count: 2107):

An Overview of Autopsy in Medicine

In the 20^th century, the autopsy has been able to clarify, elucidate, or outright reveal disease etiology for an estimated 75 medical conditions and growing³⁹. Some medical disciplines have been quick to employ the autopsy to understand relevant diseases in their field; one of the highest impacts of the autopsy can be seen for cardiovascular diseases such as congenital heart disease, atherosclerotic diseases, coronary artery disease, and myocardial infarction^40–43. For example, the embolus triggered by the rupture of a coronary plaque was first elucidated by postmortem examinations and histopathological studies of heart vessels in patients that died from cardiovascular diseases^{44, 45}. This deeper, nuanced understanding of atherosclerotic disease enabled scientific discoveries that resulted in advances in cardiovascular disease treatment. Furthermore, the evolution of histological and biomolecular methodology using autopsy samples elucidated the cell and molecular immune response of cardiovascular diseases, changing the use of medications to prevent the formation of new plaques^{46, 47}. Other important conditions that have significantly benefitted from autopsy include tuberculosis⁴⁸, HIV⁴⁹ as well as cytomegaloviruses and herpesviruses⁵⁰. These and many other findings that have been facilitated by convention autopsies are well-reviewed elsewhere (see references above).

The Research Autopsy in Oral Health Research

A literature search of dental or oral autopsy primarily reveals the role of oral and craniofacial biology in forensic applications^{2, 51–54}. Simply put, autopsy in dental, oral, and craniofacial research has been historically limited outside of oral pathology training or to support the work of forensic odontologists (Table 1). While this may have been a missed opportunity, there is still an opportunity to break down artificial barriers between oral health care and medicine by normally including oral tissues as part of research supported—even in part—by autopsies. Importantly, there are still high-volume autopsy centers in North America (United States), South America (Brazil), Europe (United Kingdom, Germany, Netherlands), and Asia (China; Figure 1); this is not to overlook many other autopsy centers or hospitals with their own active autopsy services⁵⁵. Even among these high-volume centers, there is little coordination between medical and oral health researchers (Figure 1).

Table 1 |. A Recent Historical Summary of Autopsy in Oral Health Research.

Dental/oral autopsies may often be thought of in the context of forensics; however, there is also a limited history of using autopsy in dental, oral, and craniofacial research. This list—while not comprehensive—demonstrates research projects over the last four decades that have used autopsy to support their findings—this includes recent projects that have helped define the oral axis of infection by SARS-CoV-2 in the early days of this COVID-19 pandemic.

Authors	Title	Sample type	Samples	Date	Discipline	Location
Wright and Fenwick	Candidiasis and atrophic tongue lesions	Tongue	46	1981	Pathology/Oral Medicine	JK
Vander val and Van der Val	Candida Albicans In Median rhomboid glossitis: a post mortem study	Tongue	100	1986	Pathology/Oral Medicine	Netherlands
Hashimoto, et al.	Pathological characteristics of metastatic carcinoma in the human mandible	Mandible	62	1987	Pathology/Oral Medicine	Japan
Whittaker et al.	Histological response and clinical evaluation of heterograft and allograft materials in the elevation of the maxillary sinus for the preparation of endosteal dental implant sites. Simultaneous sinus elevation and root form implantation: an eight-month autopsy report	Maxilla	1	1989	Perio/Oral Surgery	JSA
Takeda and Yamamoto	Iron deposits in the human labial minor salivary glands: a postmortem study	Minor Salivary Glands	195	1989	Pathology/Oral Medicine	Japan
Tanimoto, et al.	Comparison of computed with conventional tomography in the evaluation of temporomandibular joint disease: a study of autopsy specimens	Temporomandibular Joint	15	1989	Radiology	Sweden
Tanimoto, et al.	Comparison of computed with conventional tomography in the evaluation of temporomandibular joint disease: a study of autopsy specimens	Temporomandibular Joint	15	1990	Radiology	Sweden
Pedersen, et al.	Tooth displacement analysed on human autopsy material by means of a strain gauge technique	Mandible	3	1991	Orthodontics	Denmark
Moskow and Poison	Histologic studies on the extension of the inflammatory infiltrate in human periodontitis.	Mandible/Maxilla, Gingiva	350	1991	Perio/Oral Surgery	JSA
Landini	Videodensitometrical study of the alveolar bone crest in periodontal disease	Alveolar Bone	25	1991	Radiology	Japan
Donath	Pathogenesis of bony pocket formation around dental implants	Alveolar Bone	1	1992	Perio/Oral Surgery	German
Moskow and Poison	A histomorphologic study of the effects of periodontal inflammation on the maxillary sinus mucosa	Mandible/Maxilla	20	1992	Perio/Oral Surgery	JSA
Jystrom, Kahnberg, and Albrektsso	Treatment of the severely resorbed maxillae with bone graft and titanium implants: histologic review of autopsy specimens	Maxilla	1	1993	Perio/Oral Surgery	Sweden
Vacek, et. al	The dimensions of the human dentogingival junction	Mandible/Maxilla, Gingiva	10	1994	Perio/Oral Surgery	JSA
Widmalm, et. al	Temporomandibular joint pathosis related to sex, age, and dentition in autopsy material	TMJ	224	1994	Pathology/Oral Medicine	JSA
Wehrbein, Bauer, and Diedrich	Gingival invagination area after space closure: a histologic study	Maxilla	1	1995	Orthodontics	German
Wehrbein, Fuhrmann, and Diedrich	Human histologic tissue response after long-term orthodontic tooth movement	Maxilla	1	1995	Orthodontics	German
Wehrbein, Bauer, and Diedrich	Mandibular incisors, alveolar bone, and symphysis after orthodontic treatment. A retrospective study	Mandible	1	1996	Orthodontics	German
Rautemaa and Meri	Protection of gingival epithelium against complement-mediated damage by strong expression of the membrane attack complex inhibitor protectin (CD59)	Gingiva	2	1996	Perio/Oral Surgery	Finland
Lindh, Petersson, and Rohlin	Assessment of the trabecular pattern before endosseous implant treatment: diagnostic outcome of periapical radiography in the mandible	Mandible	7	1996	Radiology	Sweden
Jden, Ganatra, Reinhardt, and Pat	Histology near periodontitis osteoclasts	Periodontium	13	1998	Perio/Oral Surgery	JSA
Kuramitsu, Qi, Kang, and Chen	Role for periodontal bacteria in cardiovascular diseases	Fibrous Cap	381	2001	Perio/Oral Surgery	JSA
Riviere, Riviere, and Smith	Molecular and immunological evidence of oral Treponema in the human brain and their association with Alzheimer’s disease	Brain	34	2002	Perio/Oral Surgery	JSA
Rocha, et al.	Expression of Secretory Leukocyte Proteinase Inhibitor in the submandibular glands of AIDS patients	Salivary Glands	36	2008	Pathology/Oral Medicine	Brazil
Ohyama, et al.	An unusual autopsy case of pyogenic liver abscess caused by periodontal bacteria	Biliary Tract, Portal Cein, and Hepatic Artery	1	2009	Perio/Oral Surgery	Japan
Otani, et al.	Polymorphisms of the formylpeptide receptor gene (FPR1) and susceptibility to stomach cancer in 1531 consecutive autopsy cases	Stomach	1531	2011	Pathology/Oral Medicine	Japan
Silva, et al.	PLUNC protein expression in major salivary glands of HIV infected patients. Oral Disease	Salivary Glands	45	2011	Pathology/Oral Medicine	Brazil
Mikko J Pyysalo, et al.	The connection between ruptured cerebral aneurysms and odontogenic bacteria	Brain	7	2013	Pathology/Oral Medicine	Finland
Louhelainen, et al.	Oral bacterial DNA findings in pericardial fluid	Pericardial Fluid	22	2014	Pathology/Oral Medicine	Finland
Gondak, et al.	Decreased CD1a, CD83 and factor XIIIa dendritic cells in cervical lymph nodes and palatine tonsils of AIDS patients	Tongue	53	2014	Pathology/Oral Medicine	Brazil
Fonseca, et al.	Neuroepithelial structures associated with neurogenous subgemmal plaque of the tongue: an autopsy finding	Tongue	1	2015	Pathology/Oral Medicine	Brazil
de Paula, et al.	The expression of water channel proteins during human salivary gland development: a topographic study of aquaporins 1, 3 and 5	Salivary Glands	20	2017	Pathology/Oral Medicine	Brazil
Kim, Hu, and Jung	Reosseointegration After Regenerative Surgical Therapy Using a Synthetic Bone Substitute for Peri-implantitis: Human Autopsy Study	Alveolar Bone	1	2018	Perio/Oral Surgery	China
de Mello Gomes, et al.	Apoptosis and proliferation during human salivary gland development	Tongue, Salivary Glands	50	2019	Pathology/Oral Medicine	Brazil
Bertoldo, et al.	Lingual salivary gland hypertrophy and decreased acinar density in chagasic patients without megaesophagus	Tongue	27	2019	Pathology/Oral Medicine	Brazil
Matuck, et al.	Periodontal tissues are targets for Sars-Cov-2: a post-mortem study.	Gingiva	8	2020	Pathology/Oral Medicine	Brazil
Bandou, et al.	Utilization of oral check-up data of autopsy cases	N/A	403	2021	Epidemiology	Japan
Gameiro, et al.	Individualization of the three-piece base arch mechanics according to various periodontal support levels: A finite element analysis	Maxilla	1	2021	Orthodontics	Denmark
Sowmya, et al.	Histopathological Changes in Oral Tissues Induced by Pesticide Poisoning: A Pilot study	Tongue, Buccal Muosa	10	2021	Pathology/Oral Medicine	India
Zarpellon, et al.	Oral lesions and SARS-CoV-2: A postmortem study.	Tongue, Gingiva, Mucosa	30	2021	Pathology/Oral Medicine	Brazil
Matuck, et al.	Salivary glands are a target for SARS-CoV-2: a source for saliva contamination.	Major and Minor Salivary Glands	25	2021	Pathology/Oral Medicine	Brazil
Huang, Pérez, Kato, Mikami, et al.	SARS-CoV-2 infection of the oral cavity and saliva.	Minor Salivary Glands, Tongue, Gingiva, Mucosa	18	2021	Pathology/Oral Medicine	JS
Sakashita, et al.	Lewy pathology of the submandibular gland in Lewy body disease: A report of autopsy cases	Submandibular Salivary Gland	64	2021	Pathology/Oral Medicine	Japan
Wong, et al.	Multisystemic Cellular Tropism of SARS-CoV-2 in Autopsies of COVID-19 Patients	Salivary Glands	8	2021	Pathology/Oral Medicine	German
Sørensen, et al.	Entrapment of drugs in dental calculus - Detection validation based on test results from post-mortem investigations	Dental Calculus	10	2021	Forensics	Denmark

Since there remains a need for studies that incorporate dental, oral, and craniofacial tissues, there is also to establish networks and secure funding for these studies. If possible, the usefulness of the “integrated” autopsy could benefit both oral and overall health. For example, an integrated biopsy design could include removing entire structures from multiple sites like whole alveolar processes, mandibles, TMJ, tonsils and the tissues comprising Waldeyer’s ring, and tongues samples that include neurovascular complexes. There is a need to collect samples from healthy individuals both young and old for the possibility of designing research studies that can age-, sex-, and ancestry-match samples whenever possible. To achieve this will take various investigative teams to coordinate and share samples as part of a global biobanking network.

While there are numerous examples of autopsy benefitting various dental, oral, and craniofacial projects throughout the 20^th century (Table 1), it remains unknown which challenges are the most impactful to address to increase its utility. Conventional autopsies typically use invasive surgical tools to completely remove organs and tissues, but when planning for including dental, oral, and craniofacial tissues, some specifically tailored protocols need to be created and followed. Furthermore, for ethical or legal reasons, it is not possible to remove all tissues in every case as some deceased bodies will be prepared for burial at the family’s discretion.

Due to the gross sampling strategies discussed, one objection to autopsies including dental, oral, and craniofacial tissues is the disfigurement that can occur when recovering these tissues in conventional “en masse” sampling methods⁵⁶. The development of minimally invasive autopsies (MIA) using magnetic resonance imaging (MRI) and computed tomography (CT) to guide sample acquisition could overcome that (Figure 3)^57–59. If sustainable partnerships can form across academic hospitals, public and private research institutions, and industry, it would be expected that autopsies including dental, oral, and craniofacial would increase to support discoveries across disciplines. To highlight these opportunities, this review will focus on the use of autopsy in periodontal disease and salivary gland disease research, though other head and neck cancer⁶⁰ and temporomandibular joint disorder have also benefitted^61–63 (Table 1).

Figure 3 |. Employing New Autopsy Techniques to Increase Utility in Oral Health Research.

(A) One objection to autopsy is the disfigurement that can occur when recovering tissues from the oral and craniofacial complex. The development of minimally invasive autopsies (MIA) using magnetic resonance imaging (MRI) and computed tomography (CT) to target tissue sample recovery may be an important way to overcome this objection to increase dental, oral, and craniofacial sample acquisition. If sustainable partnerships can form across academic hospitals, public and private research institutions, and industry, it would be expected that autopsies including dental, oral, and craniofacial would increase to support discoveries across disciplines.

An Overview of Autopsy in Periodontal Medicine

Historically, periodontal disease researchers have more often utilized autopsy to make discoveries about local and systemic disease mechanisms—dating back to the early 19^th century as the disease was still being defined. As recently redefined, periodontitis (a type of periodontal disease) is a complex and multifactorial chronic inflammatory disease that has been widely studied in the 20^th century. What has been clear for many decades is that periodontitis is caused by dysbiosis that elicits a destructive immune response of the alveolar bone in susceptible individuals⁶⁴; however, for some individuals, periodontal lesions can be the result of diseases that either originate in other parts of the body or are systemic in their involvement⁶⁵. Many questions remain about the etiology and pathophysiology of periodontal diseases in the oral cavity, and importantly, the relationships between the mucosal immunology of the overlying gingiva, osteoimmunology of the local alveolar bone, and systemic conditions remain mostly unresolved. This is because systemic conditions can modify the periodontal status and sometimes periodontal pathogens can contribute to the establishment of systemic disease⁶⁶ as well as numerous well-known host-modifying factors such as genetics, smoking, diabetes, and obesity, among others⁶⁷.

In classic studies to understand the pathophysiology of periodontitis, there is considerable evidence of the autopsy’s role in clarifying disease mechanisms and outcomes. For example, at the turn of the 20^th century, periodontal tissues were examined at the time of autopsy which informed further research into the classification of periodontal disease⁶⁸. While many examinations of periodontal tissues in healthy and diseased sites were examined over the next fifty years as the classification and definitions of periodontal diseases continued to be refined⁶⁹. At the same time—while not technically an autopsy study—, Waerhaug added his take on the concept of “traumatic occlusion” using skulls to study occlusal relationships and site-specific bone loss⁷⁰. Furthermore, in an influential study from 1960⁷¹, Gargiulo, Wentz, and Orban published an important study on the dentogingival junction in health using integrated measurements of autopsy blocks from this and past studies⁷². This study established that there are some common measurements of this structure that are still referenced to this day⁷³.

The classic physiopathology of periodontitis was further explored in parallel. For example, in the 1970s, Waerhaug further supported the hypothesis in the field that subgingival plaque could contribute to attachment loss in an observational study of six autopsied patients. In this study, a correlation was drawn using the biofilm thickness related to site-specific bone loss. A classic study realized in the 90’s also observed the periodontitis progression using tissues from pediatric tissues and complemented with spleen and lymph node tissue from deceased bodies to better understand the extent of host inflammatory infiltration⁷⁴. However, the challenge with these studies is often a lack of well-annotated medical histories that would substantiate these claims.

Through these types of studies over a 120-year history, hypotheses were supported or challenged using what we would now call “research autopsies”. Though it can be rare, conventional autopsies have also been used in some case reports to create a link between fatal diseases and periodontal findings. There are few publications like this, likely due to the limited number of dentists and oral pathologists working in autopsies centers around the world^{75, 76}. These findings have been reported on periopathogens in distant body sites such as infected pharyngeal⁷⁷ and liver⁷⁸ tissues, supporting the long history of the oral-systemic link. The bidirectional phenomenon of disease-disease (poly-immune) associations has been discussed in the literature as “periodontal medicine”⁷⁹; this idea has been termed a “rediscovery” as it has existed for over a century after work by William Hunter published his work on oral microbes and their role in systemic diseases⁸⁰. This early work was only possible by building off of oral-systemic work from W.D. Miller at least a decade before Hunter^{81, 82}. Despite these early efforts, the true mechanisms of causation—as opposed to simply association—remain another opportunity for integrated research autopsies as some important questions, such as how periodontal diseases differ and those disease subtypes can influence inflammatory signatures in body fluids (saliva, urine, blood) and at distant organs such as the kidneys and heart⁸³.

While there is much to be understood about the oral-systemic links for the >60 systemic diseases associated with periodontitis, one such link with Alzheimer’s disease (AD) is nearly impossible without using research autopsy as a tool. Building off a seminal report that found evidence for periopathogen virulence factor LPS in AD brains⁸⁴, one such periopathogen P. gingivalis was found in the brains of patients with AD⁸⁵. Recent studies, using autopsies samples, also found P. gingivalis itself in the amyloid plaque formation in Alzheimer’s disease (AD) patients. This discovery utilized brain specimens obtained in autopsies procedures but not from gingival tissues from the same patients⁸⁵. Of course, the idea to test the AD-periodontal disease link became justified after several observational and association studies. Not surprisingly, other several studies previously published had suggested that gingipain inhibitors or periodontal treatment could mitigate the appearance of beta-amyloid in brain tissues of AD patients^{86, 87}. Another oral-brain axis is also possible considering aneurysms where an autopsy study found endodontic and periopathogens DNA in aneurysm clipping and autopsy material⁸⁸. While this is an emerging field and the literature is sparse, the collaboration between oral health researchers and neurobiologists will continue to be critical for the next advances.

An Overview of Autopsy in Salivary Gland Diseases

Among the barrier niches, salivary glands are one of the most distinct tissues in the craniofacial complex; however, they also share features with other exocrine glands across the body⁸⁹. There is now an emerging ruleset for related tissue structures across the body⁹⁰, and the formation of these oral glandular secretory structures depends on diverse cell migration from the neural cord to the ectomesenchyme where epithelial-mesenchymal interactions are coordinated is necessary for maturation and secretion of saliva⁹¹. The importance of this maturation process is essential during human facial development as well as aging where they play an important role in dental, oral, and craniofacial homeostasis through the regulated flow of saliva secretions^{92, 93}.

While the morphology and maturation of salivary glands were determined in ex vivo culture⁹⁴; these fundamental processes of salivary gland biology were further confirmed utilizing the research autopsy⁹⁵. Furthermore, the branching morphogenesis necessary for salivary gland development was first described in animal models, and then validated in autopsy samples acquired from different gestational ages⁹⁶. The evolutionary conservation of some salivary gland development is also shared between other exocrine glands such as submucosal glands of the nasal cavity and lower airways, and the relationship between salivary glands and systemic health has been discussed with growing frequency due to their role in infectious diseases such as COVID-19⁹⁷ and polyautoimmune involvement in diseases such as rheumatoid arthritis⁹⁸, multiple sclerosis⁹⁹, and Sjögren’s disease¹⁰⁰. The use of salivary glands tissues obtained by research autopsy procedures not only has illuminated human embryology¹⁰¹ and physiology¹⁰² related to glandular biology but also has provided a fundamental understanding of viral interactomics, of which the glands are known hotspots of infection^{103, 104}. This understanding has led to research to understand salivary viromes and saliva as a viral transmission medium^{97, 105, 106} as well as salivary tests for diseases such as COVID-19¹⁰⁷.

It can now be argued that the research autopsy became a foundational tool in the understanding of the oral infection axis in COVID-19. For example, viral invasion in epithelial cells is a common mechanism of pathogenicity, but it was unknown if oral epithelial barrier cells expressed the requisite membrane molecules to encourage host-viral interactions to permit productive infection. As described by Huang, Perez, Kato, and Yu, et al.⁹⁷ and Matuck, et al.¹⁰⁸, SARS-CoV-2 is one the first virus shown using in situ approaches to invade and replicate inside acinar and duct cells of the salivary glands; this was learned using research autopsy material in collaboration with autopsy groups at the National Institutes of Health and São Paolo, respectively, and allowed for advanced spatial biology methods to be applied in research autopsy samples of major and minor salivary glands obtained in patients that died due to COVID-19-related complications. Due to the historic divide between oral health care and medicine, this kind of sample collection is challenging to curate, as major salivary gland tissues have limited sampling access using extraoral approaches common in conventional autopsies; however, integrating sample collection across the body is an important endeavor considering pandemic preparedness measures to combat future viral pandemics¹⁰⁹.

3. The Value of Integrated Autopsies in the Era of Multiomics (Word Count: 806):

New Methods to Benefit Integrated Research Autopsies

While conventional autopsies have generally decreased in medicine, the advent of virtual autopsies (‘virtopsies’), which only use magnetic resonance imaging (MRI) and computed tomography (CT), are on the rise⁵⁷, especially in combination with advanced diagnostic tools assisted by artificial intelligence (AI) and machine learning (ML)^{110, 111}. Furthermore, minimally invasive autopsy, which combines MRI and CT with targeted microsurgery has become an important new technique for even more directed post-mortem studies of human disease⁵⁹. Recent studies are also beginning to combine clinical metadata (i.e., sex, age, ancestry) with AI- and ML-assisted analysis of virtopsy and classical histology assays as a modern way to advance disease-oriented research from transdisciplinary teams¹¹².

Minimally invasive autopsies (MIA) can contribute to solving the problem of removing samples from head and neck tissues without creating damage to facial recognition or ethical problems. The focused use of advanced imaging tools, like ultrasonography, tomography, and nasofibroscopy tubes that can be attached to a monitor—or even a smartphone—make it more possible to reach internal body niches for sampling specific tissues from the respiratory tract and adjacent tissues at the same time as dental, oral, craniofacial, and oral and nasal cavity tissues (Figure 2,3). Refined approaches like this will allow for more collaborative projects to understand the cell types that are shared and unique between related sites, especially considering the emerging concept of the inhalation interface of the oral and nasal cavities, pharyngeal tissues, and lower airways (Figure 2) in addition to the oral-gut and oral-brain axes referenced in this text³⁷.

While “rapid autopsy” or “warm autopsy” programs, which collect targeted tissues within hours after confirmed death, continue to be popular in academic medical centers^{55, 113}, there is a need to include oral and craniofacial specialists such as oral pathologists, oral surgeons, oral medicine, periodontists, radiologists, geneticists as well as basic science research teams focused on oral-systemic disease research to advance whole body health initiatives. For example, because larger amounts of tissue can be preserved and processed from multiple sites, rapid autopsy studies have revealed new knowledge on tumor evolution within the same patient^{114, 115}. The ideal team of the future would have educated patients and their families about the value of an integrated, minimally invasive, and rapid autopsy procedure well before the need to consent, collect and process tissues.

Biorepositories for Multiomics Assays

In an ideal world, high-volume and dedicated autopsy sites would be funded to build out comprehensive biorepositories in which de-identified, well-annotated, and barcoded patient samples are logged in searchable databases for research teams. This is especially important for the ~7000 defined rare diseases in which any samples are challenging to come by. As there is the ability to sample and subdivide tissues, downstream analysis optimized biobanks would be able to make the same sample available to multiple researcher teams or different samples available to multiple groups for multiple assays such as host single cell and spatial multiomics (combined genomics, epigenomics, transcriptomics, proteomics, and metabolomics in parallel with bulk and emerging single-cell microbiomics techniques^{12, 116, 117}. The field of host-microbe interactions (i.e., interactomics) is nascent but again could directly benefit from these approaches¹¹⁸.

The presence of oral health professionals in the rapid autopsy team could contribute to a deeper understanding of these diseases by integrated biobanking of oral tissues with whole-body samples. This inclusion will be important because the rapidity of the collection can ensure that rapid autopsy materials are of the highest quality and even comparable to freshly collected surgical biopsies as has been reported¹¹⁹. Considering all these factors in the context of a branched, international autopsy network where sample data is shareable and searchable could further facilitate future studies on severe chronic diseases with oral manifestations and may advance the field of craniofacial genetics in ways not previously possible.

Future Directions

The partnership between oral health research centers, dental schools, academic hospitals, and associated autopsy centers is highly important for the future of oral and overall health initiatives. Among all participants of these new transdisciplinary teams, it will be important to normalize a research culture where autopsies are a useful tool for the rapid understanding of disease pathophysiology. As samples grow within these partner networks, more equitable inclusion of samples considering age, sex, and ancestry will be possible with appropriate statistical power to shorten the distance between preclinical and clinical research. Additionally, considering the recent COVID-19 pandemic, emerging viral diseases are predicted to be continually encountered and are like a burden that humanity will continue to endure. In the last two decades, pandemics are becoming more common¹²⁰. Evidence now supports that the oral cavity plays a more important role in viral diseases than may have previously been appreciated, and thus, the described establishment of new autopsies centers, participants, and practice must be a goal for global pandemic preparedness and biosecurity of the future.

Abbreviation Key:

AI

Artificial Intelligence

CT

Computed Tomography

MIA

Minimally Invasive Autopsy

ML

Machine Learning

MRI

Magnetic Resonance Imaging

H1N1

Influenza A virus subtype H1N1

COVID-19

Coronavirus Disease of 2019

TMJ

Temporomandibular Joint