Dental manifestations of hypophosphatasia: translational and clinical advances
Hypophosphatasia (HPP) was first identified in 1948 by Dr. John Campbell Rathbun to describe a 3-wk-old boy who lost weight, showed signs of pain, and experienced seizures. Radiographs revealed severe osteopenia and bone deformities and fractures. Low levels of serum alkaline phosphatase activity (ALP) were noted, with normal or elevated serum calcium and inorganic phosphate levels. This patient died at 2 mo of age. Case reports of HPP accumulated in subsequent decades, underscoring ALP deficiency as a primary diagnostic criterion. , In 1988, mutations in the ALPL gene were identified as the genetic basis of HPP; genetic identification of many more cases followed. In 1996, nearly 50 yr after Rathbun’s report, missense mutations were identified in ALPL exon 5 of the mother and exon 9 of the father of the original patient, revealing the child carried compound heterozygous defects. Over the last 2 decades, substantial progress has been made in identifying ALPL mutations contributing to HPP. Genotype-phenotype correlations, functional assays, in silico modeling, and animal models have provided new insights into all aspects of the disease. Research into HPP has expanded, including creation of animal models which have played an important role in the development of new treatments such as enzyme replacement therapy (ERT). These advances have improved our ability to diagnose and manage HPP, offering hope for better outcomes for affected individuals. This review focuses on dental manifestations of HPP, particularly advances in understanding pathological mechanisms, dental care, animal models used to study dental effects of HPP, and effects of ERT on dental tissues. 

Hypophosphatasia (OMIM# 146300, 241500, 241510) is an inherited error in metabolism resulting from loss-of-function variants in ALPL , which encodes tissue-nonspecific alkaline phosphatase (TNAP, TNSALP, or TNALP). , Natural substrates of TNAP include inorganic pyrophosphate (PP i ), pyridoxal 5′-phosphate (PLP), and phosphoethanolamine (PEA), among others. TNAP plays a crucial role in biomineralization of bones and teeth, in part by reducing local levels of PP i , a potent inhibitor of hydroxyapatite crystal growth. Other substrates for dephosphorylation by TNAP include adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP). , Working in concert with other proteins, TNAP can generate extracellular inorganic phosphate (P i ), increasing the P i /PP i ratio and influencing biomineralization and purinergic signaling. The ALPL gene is located on chromosome 1p36.1-34. , , Pathological variants contributing to HPP have been reported in all 12 exons of ALPL , and as of this publication, 465 different disease-causing mutations have been documented ( https://alplmutationdatabase.jku.at ). HPP cases result from missense mutations (79%), small deletions (10%), splicing mutations (4%), nonsense mutations (3%), small insertions (2%), and complex insertion/deletions, large deletions, and mutations in the regulatory sequence (1%). , The large proportion of missense mutations may correlate with the high level of clinical variability that characterizes HPP. Systemic and skeletal effects of HPP are well described in other reviews. , , Low serum ALP activity is the key biochemical indicator of HPP. Elevated levels of serum PLP and increased urinary PEA levels can also indicate HPP. , , The clinical severity of HPP varies greatly between individuals. This has prompted creation of a spectrum of clinical subtypes to describe the onset, severity, and prognosis. , , , Perinatal and infantile forms of HPP are the most severe and can be life threatening. Manifestations include short, bowed long bones, rickets, wide fontanelles and sutures, respiratory complications, craniosynostosis, and vitamin B-dependent seizures (summarized in ). In the c hildhood form of HPP, manifestations emerge after 6 mo and can include rickets, impaired growth, skeletal deformities, and craniosynostosis ( ). In the adult onset form of HPP, individuals may have osteomalacia, chronic pain, stress fractures, pseudofractures, chondrocalcinosis, and loss of mobility. Retrospectively, a history of mild rickets and premature tooth loss is sometimes revealed ( ). Musculoskeletal pain, muscle weakness, and fatigue are increasingly recognized as part of the spectrum of manifestations. , , Odontohypophosphatasia (odonto HPP) primarily affects dental tissues, as described in the next section ( ). The fact that only dental tissues are affected in this subtype, and the observation that dental effects are observed across the spectrum of HPP, suggests these tissues have a special sensitivity to HPP, particularly cementum, which is required for tooth attachment. , While sex differences have been identified in HPP prevalence in a small number of studies, it has been attributed to diagnosis biases and severity of disease has not been noted to differ in males vs females. , Inheritance of HPP is complicated because both autosomal recessive (AR) and autosomal dominant (AD) patterns occur, depending on the nature of the mutation(s). More severe forms of HPP (perinatal and infantile) typically arise from an AR pattern of inheritance wherein the affected individual carries loss-of-function variants in both ALPL alleles. Often these are different pathological variants contributing to compound heterozygosity. Less severe forms of HPP (childhood, adult-onset, and odonto HPP) arise from an AR or AD pattern of inheritance. In the AD inheritance pattern, dominant negative effects have been found as a result of some ALPL variants. , Reported prevalence of HPP varies widely depending on population and HPP clinical type, and estimates have undergone revision in recent decades with additional genetic testing driven in part by availability of ERT. Prevalence of HPP in Europe was estimated at 1:300 000 for severe cases and 1:6370 for mild/moderate cases. Incidence of severe forms has been estimated at 1:100 000 in the general population in North America. Some populations present higher HPP prevalence, for example, 1 in 25 are carriers in the Manitoba Mennonite population, resulting in 1:2500 affected infants at birth. In Japan, prevalence of the most common HPP-associated allele has been estimated at 1:900 000. Importantly, there is a growing consensus that moderate and mild forms of HPP are underdiagnosed. A laboratory-based survey of a population in northern Germany, which used criteria of low ALP and high PLP, found about 1:200 individuals who could be diagnosed with moderate HPP. An in silico and functional analysis employing low ALP and heterozygosity of an ALPL variant as criteria for diagnosis in a population of 424 European individuals estimated moderate HPP prevalence at 1:2430 and mild HPP prevalence at 1:508. This prompted a new genetic-based nosology for HPP including a severe form (AR), moderate form (AR or AD with dominant-negative effects), or mild form (AD due to haploinsufficiency). It remains to be seen if this nosology will be adopted by the clinical community and how dental manifestations will fit into this system. 

Overview Dental defects occur across the spectrum of clinical subtypes of HPP from mild to severe and represent a substantial contribution to the disease burden ( ). , , , Dental involvement is very common among those with HPP, often one of the most prevalent manifestations according to reports culled from the HPP Global Registry, even exceeding prevalence of skeletal manifestations in some data. , , Genotype-phenotype correlation is notoriously challenging for HPP; , however, case reports and anecdotal evidence have suggested increased prevalence and severity of dental defects in those with infantile and childhood HPP. , , , The Global HPP Registry supports this; for example, 78.8% of adults with pediatric-onset HPP have dental involvement vs 42.6% of those with adult-onset HPP. These results from patient- and doctor-reported surveys are a good start; though more detailed dental histories, qualitative assessments of clinical photos and dental radiographs, and quantitative analyses of dental structures, could provide more precise and insightful data on correlation of dental defects with genetic, biochemical, musculoskeletal, and other manifestations of HPP. Four distinct mineralized tissues comprise the dentoalveolar complex, meaning the tooth and surrounding connective tissues. The tooth is composed of enamel (a highly mineralized tissue covering the crown), dentin (a tough mineralized tissue which composes the bulk of the tooth), and the enclosed dental pulp (housing cells, nerves, and vascular supply). Periodontal tissues surround, connect, and invest the tooth. These include the gingiva (oral epithelium that provides a barrier protecting underlying tissues), cementum (a thin, mineralized tissue covering root dentin), periodontal ligament (PDL) (a soft connective tissue between the tooth root and bone), and alveolar bone (part of the jawbone surrounding teeth and required for their retention). The acellular form of cementum covers the majority of the root and is the type most affected by HPP. Humans have 2 sets of teeth: the deciduous and permanent dentitions. Deciduous teeth begin forming in utero and erupt into the oral cavity in the first few years of life. These teeth are exfoliated and replaced by the permanent dentition between 6 and 12 yr of age. The permanent dentition must last a lifetime. Effects of HPP dentoalveolar tissues span gross observations, radiographic findings, and histological defects (summarized in and , with representative images in ). , , , Clinical observations can include enamel hypoplasia and discoloration (possibly contributing to increased caries prevalence), delayed tooth eruption, tooth mobility, and malocclusion (misalignment of upper and lower teeth due to incorrect positions along the dental arches) ( and ). Premature loss of fully rooted deciduous teeth is pathognomonic for HPP, and loss of permanent teeth in adulthood can also occur. Here, the term “fully rooted” indicates substantial remaining root structure ( ); teeth are lost due to defective PDL-cementum attachment. Radiographic findings include thin dentin, wide pulp chambers, short or deformed tooth roots, and reduced alveolar bone levels around roots ( and ). Histological findings may include dentin mineralization defects, wide predentin, and particularly reduced or absent cementum on root surfaces ( ). Mechanisms of these manifestations are discussed below. 

Dental defects occur across the spectrum of clinical subtypes of HPP from mild to severe and represent a substantial contribution to the disease burden ( ). , , , Dental involvement is very common among those with HPP, often one of the most prevalent manifestations according to reports culled from the HPP Global Registry, even exceeding prevalence of skeletal manifestations in some data. , , Genotype-phenotype correlation is notoriously challenging for HPP; , however, case reports and anecdotal evidence have suggested increased prevalence and severity of dental defects in those with infantile and childhood HPP. , , , The Global HPP Registry supports this; for example, 78.8% of adults with pediatric-onset HPP have dental involvement vs 42.6% of those with adult-onset HPP. These results from patient- and doctor-reported surveys are a good start; though more detailed dental histories, qualitative assessments of clinical photos and dental radiographs, and quantitative analyses of dental structures, could provide more precise and insightful data on correlation of dental defects with genetic, biochemical, musculoskeletal, and other manifestations of HPP. Four distinct mineralized tissues comprise the dentoalveolar complex, meaning the tooth and surrounding connective tissues. The tooth is composed of enamel (a highly mineralized tissue covering the crown), dentin (a tough mineralized tissue which composes the bulk of the tooth), and the enclosed dental pulp (housing cells, nerves, and vascular supply). Periodontal tissues surround, connect, and invest the tooth. These include the gingiva (oral epithelium that provides a barrier protecting underlying tissues), cementum (a thin, mineralized tissue covering root dentin), periodontal ligament (PDL) (a soft connective tissue between the tooth root and bone), and alveolar bone (part of the jawbone surrounding teeth and required for their retention). The acellular form of cementum covers the majority of the root and is the type most affected by HPP. Humans have 2 sets of teeth: the deciduous and permanent dentitions. Deciduous teeth begin forming in utero and erupt into the oral cavity in the first few years of life. These teeth are exfoliated and replaced by the permanent dentition between 6 and 12 yr of age. The permanent dentition must last a lifetime. Effects of HPP dentoalveolar tissues span gross observations, radiographic findings, and histological defects (summarized in and , with representative images in ). , , , Clinical observations can include enamel hypoplasia and discoloration (possibly contributing to increased caries prevalence), delayed tooth eruption, tooth mobility, and malocclusion (misalignment of upper and lower teeth due to incorrect positions along the dental arches) ( and ). Premature loss of fully rooted deciduous teeth is pathognomonic for HPP, and loss of permanent teeth in adulthood can also occur. Here, the term “fully rooted” indicates substantial remaining root structure ( ); teeth are lost due to defective PDL-cementum attachment. Radiographic findings include thin dentin, wide pulp chambers, short or deformed tooth roots, and reduced alveolar bone levels around roots ( and ). Histological findings may include dentin mineralization defects, wide predentin, and particularly reduced or absent cementum on root surfaces ( ). Mechanisms of these manifestations are discussed below. 

There are limited numbers of publications on dental management of patients with HPP. Based on complex and variable clinical manifestations, patients with HPP will benefit from multidisciplinary care, potentially involving general or pediatric dentists, periodontists, orthodontists, and prosthodontists. General and pediatric dentists are often the first to diagnose HPP given the clinical presentation of early loss of deciduous teeth. They also help to assist the patient in navigating and coordinating their complex dental needs in addition to routine care. One of the clinical hallmarks of HPP is premature loss of one or more fully rooted deciduous teeth, typically before the age of 5 yr old. This can happen spontaneously or can result from minor traumas that would not normally cause tooth avulsion. Given the initial oral presentation, dentists play a key role in diagnosis. , Additional studies have shown loss of permanent teeth and tooth mobility in patients with HPP. , Loss of TNAP affects the cementum more severely than other dental tissues. Teeth have been reported to exfoliate with dental plaque accumulation along the root. , This results from lack of periodontal attachment, allowing bacterial invasion deep into periodontal tissues. Subgingival plaque accumulation potentially contributes to an inflammatory environment causing additional bone loss and periodontal disease. , Anterior teeth are most frequently reported to exfoliate prematurely in children with HPP; however, prevalence by tooth type is not well understood. Incisors may be more prone to exfoliation because of their smaller, conical-shaped roots, whereas canines may be lost less frequently because of their relatively larger roots, and molars may fall out least frequently because of multiple roots that are more difficult to displace from surrounding bone. Many reports that examine teeth affected by HPP have described only incisors. , However, Van den Bos and colleagues observed 19 deciduous incisors and 12 deciduous canines from 7 individuals with HPP and reported uniform absence of cementum across tooth types and no apparent dentin defects. Luder also described a canine tooth affected by HPP where acellular cementum was absent but dentin was normal. Wei and coauthors examined one deciduous canine and found absence of cementum. Kramer et al. analyzed 8 deciduous teeth, including incisors, canines, and molars, from a child with odonto HPP, finding cementum present on some canines, which were nonetheless lost prematurely. Wolfel and colleagues found dentin and cementum hypomineralization in 11 deciduous teeth from a female with HPP, but results were not segregated by tooth type. This points to more complex concepts of cementum defects other than simply cementum aplasia; possibly cementum can be hypomineralized, lack PDL insertions, and/or be dysfunctional in other aspects. Periodontists, as dental specialists who focus on the function of tooth root cementum, PDL, and alveolar bone in health and disease, play a key role in monitoring presence of inflammation, bone loss, and managing oral hygiene. Early loss of deciduous teeth occurs due to cementum developmental defects which compromise tooth attachment. , HPP also impairs alveolar bone mineralization and reduced alveolar bone may arise from both direct effects on bone and indirect effects from compromised periodontal attachment. Compromised cementum and bone can increase the risk of gingival recession and the formation of periodontal pockets, spaces that form between tooth root surfaces and gingival tissues which can contribute to accumulation of bacteria, increased inflammation, and development of periodontitis, an inflammatory and destructive condition which compromises the periodontal tissues. Similar to the general population, cumulative loss of periodontal attachment from HPP leads to greater periodontal destruction. Overall, HPP illustrates the critical importance of periodontal attachment and the consequences when one or more components of this system are defective. Periodontal manifestations and treatment were described for a 15-yr-old boy with HPP in Japan. The boy exhibited premature exfoliation of deciduous teeth and permanent dentition was beset by alveolar bone loss and mobility. Severely affected teeth were extracted, and remaining teeth were treated by periodontal surgical treatment, scaling and root planning with open flap. This procedure is a form of “deep cleaning” that involves gently lifting the gum to access the tooth roots and underlying bone for the removal of subgingival plaque and calculus. Remaining permanent teeth showed stabilized periodontal status. In another case report, long-term periodontal therapy was described for male identical twins with odonto HPP who suffered from premature loss of deciduous teeth, short roots, reduced alveolar bone height, and tooth mobility (Patients 1 and 2 in ). Both patients began losing permanent teeth as teenagers. Supportive periodontal therapy was initiated, including frequent visits every 3 mo, nonsurgical mechanical debridement of plaque, and oral hygiene instructions. Signs of worsening periodontal conditions in their 20s prompted a more proactive regime of visits every 2 mo and scaling and root planning. This approach stabilized the periodontal condition over the course of 2 yr, preparing for orthodontics and dental implants. Orthodontists’ main role is to address teeth crowding and create adequate spacing for implant placement or restorative work, as needed. Additionally, orthodontists can ensure a proper occlusion, that is, positions and alignment of upper and lower teeth. Orthodontic treatment was performed on the twin brothers with odonto HPP described in the previous section (Patients 1 and 2 in ). This phase of dental treatment was initiated after tooth stability was improved through periodontal therapy, and with the goal of improving occlusion, the way the upper and lower teeth come together. While orthodontic treatment has undoubtedly been performed on additional individuals affected by HPP, no detailed case reports have yet been published with which to guide clinical approaches. Those with HPP have difficulty finding specialists because of limited exposure to such complex cases, increased risk and liability if treatments are unsuccessful, and lack of publications to provide guidance on treatment planning. Prosthodontists assist with the replacement of missing teeth through crowns, bridges, or implant prostheses. In addition to periodontal changes outlined above, enamel and dentin defects can create challenges for successful dental rehabilitation, including crowns and bridges that require adhesive bonding to the tooth. Thin, hypomineralized dentin, and short roots contribute to tooth mobility, exacerbating instability due to defects in cementum and alveolar bone. The majority of HPP-centered dental case reports describe prosthetic approaches to tooth loss. A report from Ireland outlined placement of 7 implants to support fixed partial dentures following tooth mobility and loss. The work took place over 6 yr, while the patient was in his 20s. One implant failed, likely due to insufficient bone quantity. In another report, prosthetic rehabilitation for a 16-yr-old woman with HPP was challenging due to loss of nearly all anterior teeth and severe reduction in alveolar bone height. A maxillary overdenture and mandibular partial denture were prepared. Reduced bone height was also a limitation for an 18-yr-old woman with HPP who had lost numerous maxillary and mandibular permanent teeth. Insufficient bone for implant retained prostheses, leading to interim removable partial dentures. A recent case report followed a 24-yr-old female who was diagnosed with HPP as a child (Patient 3 in ). She experienced early loss of all deciduous and several permanent teeth. Despite excellent oral hygiene, she was diagnosed with periodontal disease due to alveolar bone loss secondary to HPP. Full mouth prosthetic rehabilitation was planned over several steps, which required approximately 2 yr. This approach involved extraction of several teeth due to substantial mobility and use of guided bone regeneration with autogenous bone and bone graft material to prepare for implant-retained fixed partial prostheses ( ). Follow-up included oral hygiene instructions and regular cleanings. After 7 yr, implants were reported to be stable. The above cases highlight the need for a multidisciplinary dental care team. Active periodontal disease must be resolved prior to restorative and orthodontic interventions. It is critical to have adequate imaging to evaluate bone levels prior to orthodontic tooth movement or implant placement. Notably, dental implants may be contraindicated in young children until jaw bone growth is completed. Excellent oral hygiene is vital to successful outcomes. Patients with HPP should be seen more frequently for cleanings and dental exams. Treatment plans may extend much longer to account for more extensive rehabilitation and/or conservative approaches to treatment planning. In addition to the dental management, it is also necessary to consult with the managing endocrinologist to verify ALP levels and determine if the patient is receiving ERT (described in more detail below) prior to starting intervention. Additional research is needed to create specific guidelines for management of the changing dental needs from childhood to adulthood for patients with HPP. As more patients start ERT at younger ages, we will gain a better understanding of dental effects and the impact on bone quality. If patients can avoid early tooth loss and periodontal disease then dental management becomes less complex. Older patients who have experienced loss of teeth at an early age, but now have increased ALP due to ERT, may be good candidates for orthodontics or dental implants. Understanding the impact of ERT on the alveolar structures and dental tissues will help guide diagnosis and treatment planning thus creating the best outcomes for patients. 

Animal models of HPP have provided key insights into the role(s) of TNAP in dentoalveolar tissues and served as preclinical models for development of therapies. TNAP is expressed in dental cells and tissues TNAP expression in dentoalveolar tissues has largely been explored in mice. The dentoalveolar complex, that is, the tooth and supporting periodontia, includes 4 unique hard tissues: enamel, dentin, cementum, and alveolar bone. Gene expression and protein localization experiments demonstrated TNAP in hard tissue cells during odontogenesis, suggesting important roles in tooth formation and mineralization. , The first evidence of increased TNAP in the oral cavity is during jawbone mineralization ( ). During crown formation, TNAP is abundant in both the enamel organ and odontoblasts that make dentin ( ). , , TNAP shows a specific pattern of expression in the enamel organ, localized in maturation stage ameloblasts, aligning with their role in mineralization of the enamel. Pre-ameloblasts and secretory stage ameloblasts do not express TNAP, whereas the adjacent stratum intermedium shows intense TNAP staining. The role of TNAP in the stratum intermedium, a supportive but nonmineralizing tissue, remains unclear. During root formation, TNAP is abundant in the forming periodontium, including cementoblasts, osteoblasts, and in the wider PDL region ( ). , , Mouse models of HPP Several mouse models of HPP have been engineered over the past 3 decades, allowing experiments into mechanisms of HPP as well as therapies. Mice engineered to globally delete Alpl (Formerly Akp2 −/− , now Alpl −/− ) exhibit skeletal and dental features consistent with severe infantile HPP. The research group of Dr. José Luis Millán developed the Alpl −/− mouse line in 1997 which has been used for the majority of studies in the ensuing decades. These Alpl −/− mice exhibit defective cranial vault and base mineralization, abnormal cranial shape, and craniosynostosis, consistent with severe, infantile HPP ( ). Molars and incisors of Alpl −/− mice displayed defective enamel mineralization and disrupted ultrastructure ( ). Histologically, Alpl −/− ameloblasts showed abnormal and disorganized morphology. In a separate study, the first molar germ of Alpl −/− mice showed a thin, uneven, enamel layer on postnatal Day 5, linked to disrupted enamel matrix proteins. While it is clear that global deletion of TNAP disrupts amelogenesis, the exact function(s) of TNAP in enamel development and mineralization remain incompletely understood. Defective root dentin mineralization was described in the Alpl −/− mice. , , Defects varied from minor delays to severe disruptions in root dentinogenesis ( ). Delays were documented in mantle dentin mineralization; mantle dentin is the initial dentin matrix secreted and a form which depends on matrix vesicles to initiate mineralization. , The defects were correlated with disordered odontoblasts and altered expression of osteocalcin ( Bglap /OCN) and dentin sialophosphoprotein ( Dspp /DSP and DPP). , Further characterization of dentin in molars and incisors of Alpl −/− mice demonstrated wider mantle dentin was associated with defective mineralization of the adjacent circumpulpal dentin. Alpl −/− mice also exhibit acellular cementum hypoplasia responsible for impaired periodontal attachment ( ). , , Interestingly, the absence of acellular cementum was commonly observed in HPP teeth with mantle dentin defects, indicating a possible link between these phenotypes. Alpl −/− mice phenocopy many aspects of severe infantile HPP; however, they die before weaning age and their disease severity and lethality hinder analysis at advanced ages. This limitation prompted efforts to develop alternative HPP mouse models to better understand the enzyme’s role in development and explore strategies for rescuing dental defects. The first mouse model of odonto HPP was engineered by knocking in a human HPP mutation (c.346G>A) into Alpl exon 5, resulting in an A116T substitution. This variant was associated with a large kindred of individuals with primarily dental manifestations. Alpl +/A116T mice exhibited 50% reduced ALP, with minimal impact on skeletal and dental mineralization. The A116T mutation showed hypomineralization of mouse alveolar bone and cellular cementum, with a trend of reduced acellular cementum. However, the defects were so unexpectedly mild as to make it difficult to pursue additional studies of interventions. Additional mouse models of later-onset HPP with extended life spans were developed employing the Cre/loxP system to engineer conditional Alpl deletion. Alpl fl/fl mouse models, when crossed with with mice harboring Cre recombinase driven by either the Col1a1 promoter (to target Alpl in osteoblasts and dental cells) or Prx1 promoter (to target Alpl in limb buds, chondrocytes, osteoblasts, and craniofacial mesenchyme), phenocopied key aspects of human juvenile and adult forms. These conditional knockout (cKO) models exhibit 75% decreased ALP at 24 wk and pronounced skeletal anomalies including rachitic changes, deformations, and fractures. Col1a1 cKO of Alpl caused dental defects including thin dentin, absence of acellular cementum, alveolar bone hypomineralization, and periodontal breakdown ( ). Sheep model of HPP HPP mouse models have significantly contributed to advancing our knowledge of disease mechanisms and therapeutic strategies. Despite showing reduced cementum and detachment of PDLs, HPP mouse models do not exhibit the hallmark of premature tooth loss, which is a key diagnostic criterion in HPP. This disparity may be attributed to differences in growth and physiology between mice and humans; mice are monophyodonts with only one set of teeth. Mouse appendicular bones are also imperfect models for human skeletal diseases, as mice lack osteonal bone remodeling and load bearing is substantially different. Sheep, unlike mouse models, have a diphyodont dentition (2 sets of teeth: deciduous and permanent) and osteonal bone remodeling similar to humans, making them a better model for studying the pathophysiological mechanisms underlying HPP dental and skeletal defects. CRISPR/Cas9 was used to develop a sheep model of HPP by introducing a missense mutation (c.1077C>G; p.I359M) into the ALPL gene. This mutation was based on a human patient with childhood HPP. Longitudinal analysis revealed a spectrum of musculoskeletal, craniofacial, and dental abnormalities in sheep, including premature tooth loss, hypomineralization, muscle weakness, and metaphyseal flaring of long bones. , Interestingly, compound heterozygous sheep ( ALPL -/I359M ) exhibited the most severe defects, followed by homozygous ( ALPL I359M/I359M ) and heterozygous ( ALPL +/I359M ) mutants, correlating with ALP levels. Key dental findings include early defects in cementum and alveolar bone, possibly leading to poor periodontal attachment and premature tooth loss, with fully rooted teeth being shed ( ). Reduced crown dentin thickness and severe mantle dentin hypomineralization were observed in an HPP sheep model, along with genotype-associated cementum hypoplasia. These dental manifestations in sheep closely resemble those observed in a human patient carrying the same mutation. While a previous study reported a mutation in Karelian Bear Dogs resembling HPP, dental abnormalities were not observed. This makes sheep the first animal model to replicate premature deciduous tooth loss seen in HPP. 

TNAP expression in dentoalveolar tissues has largely been explored in mice. The dentoalveolar complex, that is, the tooth and supporting periodontia, includes 4 unique hard tissues: enamel, dentin, cementum, and alveolar bone. Gene expression and protein localization experiments demonstrated TNAP in hard tissue cells during odontogenesis, suggesting important roles in tooth formation and mineralization. , The first evidence of increased TNAP in the oral cavity is during jawbone mineralization ( ). During crown formation, TNAP is abundant in both the enamel organ and odontoblasts that make dentin ( ). , , TNAP shows a specific pattern of expression in the enamel organ, localized in maturation stage ameloblasts, aligning with their role in mineralization of the enamel. Pre-ameloblasts and secretory stage ameloblasts do not express TNAP, whereas the adjacent stratum intermedium shows intense TNAP staining. The role of TNAP in the stratum intermedium, a supportive but nonmineralizing tissue, remains unclear. During root formation, TNAP is abundant in the forming periodontium, including cementoblasts, osteoblasts, and in the wider PDL region ( ). , , 

Several mouse models of HPP have been engineered over the past 3 decades, allowing experiments into mechanisms of HPP as well as therapies. Mice engineered to globally delete Alpl (Formerly Akp2 −/− , now Alpl −/− ) exhibit skeletal and dental features consistent with severe infantile HPP. The research group of Dr. José Luis Millán developed the Alpl −/− mouse line in 1997 which has been used for the majority of studies in the ensuing decades. These Alpl −/− mice exhibit defective cranial vault and base mineralization, abnormal cranial shape, and craniosynostosis, consistent with severe, infantile HPP ( ). Molars and incisors of Alpl −/− mice displayed defective enamel mineralization and disrupted ultrastructure ( ). Histologically, Alpl −/− ameloblasts showed abnormal and disorganized morphology. In a separate study, the first molar germ of Alpl −/− mice showed a thin, uneven, enamel layer on postnatal Day 5, linked to disrupted enamel matrix proteins. While it is clear that global deletion of TNAP disrupts amelogenesis, the exact function(s) of TNAP in enamel development and mineralization remain incompletely understood. Defective root dentin mineralization was described in the Alpl −/− mice. , , Defects varied from minor delays to severe disruptions in root dentinogenesis ( ). Delays were documented in mantle dentin mineralization; mantle dentin is the initial dentin matrix secreted and a form which depends on matrix vesicles to initiate mineralization. , The defects were correlated with disordered odontoblasts and altered expression of osteocalcin ( Bglap /OCN) and dentin sialophosphoprotein ( Dspp /DSP and DPP). , Further characterization of dentin in molars and incisors of Alpl −/− mice demonstrated wider mantle dentin was associated with defective mineralization of the adjacent circumpulpal dentin. Alpl −/− mice also exhibit acellular cementum hypoplasia responsible for impaired periodontal attachment ( ). , , Interestingly, the absence of acellular cementum was commonly observed in HPP teeth with mantle dentin defects, indicating a possible link between these phenotypes. Alpl −/− mice phenocopy many aspects of severe infantile HPP; however, they die before weaning age and their disease severity and lethality hinder analysis at advanced ages. This limitation prompted efforts to develop alternative HPP mouse models to better understand the enzyme’s role in development and explore strategies for rescuing dental defects. The first mouse model of odonto HPP was engineered by knocking in a human HPP mutation (c.346G>A) into Alpl exon 5, resulting in an A116T substitution. This variant was associated with a large kindred of individuals with primarily dental manifestations. Alpl +/A116T mice exhibited 50% reduced ALP, with minimal impact on skeletal and dental mineralization. The A116T mutation showed hypomineralization of mouse alveolar bone and cellular cementum, with a trend of reduced acellular cementum. However, the defects were so unexpectedly mild as to make it difficult to pursue additional studies of interventions. Additional mouse models of later-onset HPP with extended life spans were developed employing the Cre/loxP system to engineer conditional Alpl deletion. Alpl fl/fl mouse models, when crossed with with mice harboring Cre recombinase driven by either the Col1a1 promoter (to target Alpl in osteoblasts and dental cells) or Prx1 promoter (to target Alpl in limb buds, chondrocytes, osteoblasts, and craniofacial mesenchyme), phenocopied key aspects of human juvenile and adult forms. These conditional knockout (cKO) models exhibit 75% decreased ALP at 24 wk and pronounced skeletal anomalies including rachitic changes, deformations, and fractures. Col1a1 cKO of Alpl caused dental defects including thin dentin, absence of acellular cementum, alveolar bone hypomineralization, and periodontal breakdown ( ). 

HPP mouse models have significantly contributed to advancing our knowledge of disease mechanisms and therapeutic strategies. Despite showing reduced cementum and detachment of PDLs, HPP mouse models do not exhibit the hallmark of premature tooth loss, which is a key diagnostic criterion in HPP. This disparity may be attributed to differences in growth and physiology between mice and humans; mice are monophyodonts with only one set of teeth. Mouse appendicular bones are also imperfect models for human skeletal diseases, as mice lack osteonal bone remodeling and load bearing is substantially different. Sheep, unlike mouse models, have a diphyodont dentition (2 sets of teeth: deciduous and permanent) and osteonal bone remodeling similar to humans, making them a better model for studying the pathophysiological mechanisms underlying HPP dental and skeletal defects. CRISPR/Cas9 was used to develop a sheep model of HPP by introducing a missense mutation (c.1077C>G; p.I359M) into the ALPL gene. This mutation was based on a human patient with childhood HPP. Longitudinal analysis revealed a spectrum of musculoskeletal, craniofacial, and dental abnormalities in sheep, including premature tooth loss, hypomineralization, muscle weakness, and metaphyseal flaring of long bones. , Interestingly, compound heterozygous sheep ( ALPL -/I359M ) exhibited the most severe defects, followed by homozygous ( ALPL I359M/I359M ) and heterozygous ( ALPL +/I359M ) mutants, correlating with ALP levels. Key dental findings include early defects in cementum and alveolar bone, possibly leading to poor periodontal attachment and premature tooth loss, with fully rooted teeth being shed ( ). Reduced crown dentin thickness and severe mantle dentin hypomineralization were observed in an HPP sheep model, along with genotype-associated cementum hypoplasia. These dental manifestations in sheep closely resemble those observed in a human patient carrying the same mutation. While a previous study reported a mutation in Karelian Bear Dogs resembling HPP, dental abnormalities were not observed. This makes sheep the first animal model to replicate premature deciduous tooth loss seen in HPP. 

Prior to the advent of the ERT, treatment of HPP patients was largely limited to supportive care, including pain management, orthopedic surgeries for rickets and fractures, and respiratory support for severely affected infants. Early attempts to treat HPP included transfusion of ALP-rich blood and bone marrow cell transplantation; benefits were transient and lasting improvements were not achieved. Bisphosphonates, drugs that inhibit osteoclast function, have in some cases been administered to HPP patients, usually because of misdiagnosis or misunderstanding of the etiopathology of HPP. These drugs worsen the hypomineralization caused by HPP and are contraindicated for use in HPP patients. Teriparatide (TPTD), a recombinant peptide based on parathyroid hormone, is an anabolic agent used to treat osteoporosis. Case reports indicate potentially positive effects on reducing osteomalacia and accelerating bone healing in those with HPP. , Relative success of TPTD therapy may depend on the clinical subtype and biochemistry of each individual HPP patient as the increased numbers and activity of osteoblasts will still produce defective TNAP enzyme. 

Based on prior unsuccessful attempts to treat HPP, a new goal was set to develop a recombinant ERT. Dr. Philippe Crine and scientists at Enobia Pharma engineered a soluble form of TNAP via removal the hydrophilic GPI anchor and adding the Fc region of human IgG, allowing column purification and increasing the protein’s half-life in circulation. A highly negatively charged deca-aspartate (D 10 ) tail was added in order to target delivery to mineralized tissues ( ). Preclinical studies of TNAP-D 10 (Asfotase alfa) were conducted by Dr. José Luis Millán and colleagues using the Alpl −/− mouse. Starting with newborns, daily subcutaneous injections of 8.2 mg/kg TNAP-D 10 prevented seizures, increased lifespans, and ameliorated skeletal defects and fractures ( ). , Following preclinical studies, clinical trials for TNAP-D 10 in infants and young children with perinatal or infantile HPP began in 2008. Over the next decade, additional clinical trials conducted by Alexion Pharmaceuticals, Inc., enrolled infants, children, juveniles, and adults. The drug substantially improved bone mineralization, leading to improvement in respiratory function and survival, resolution of rickets and skeletal deformities, and improved fracture healing in neonates and infants with severe HPP. In 2015, Asfotase alfa Strensiq was approved in Japan, Canada, the European Union, and the United States for pediatric-onset HPP. Asfotase alfa is delivered 3 or 6 times weekly as a subcutaneous injection. ALP levels following treatment dramatically increase as high as ~24 000 IU/L within weeks (about 50-fold higher than normal, age-adjusted ALP levels) and remain elevated at 3000-6000 IU/L after years of therapy. , Clinical trial results showed good safety profiles, with the most common adverse effects being injection-site reactions in approximately 75% of treated individuals, including edema, pain, irritation, and in rare cases, an anaphylactic response. , , An unusual, localized lipohypertrophy reaction occurs in some patients and appears to persist for several years, even after discontinuation of ERT. , , 

Responses of craniofacial and dentoalveolar tissues to asfotase alfa were evaluated in the Alpl −/− mouse model. ERT prevented craniosynostosis and normalized cranial shape and mineralization ( ). , , ERT prevented enamel defects in Alpl −/− mice. The same regiment also dramatically reduced dentin mineralization defects and prevented cementum hypoplasia and alveolar bone defects, restoring periodontal attachment and function ( ). , , Preclinical experiments in Alpl −/− mice indicated tremendous potential for ERT to prevent craniofacial and dental manifestations of HPP. One caveat was that ERT was initiated at early postnatal ages, prior to onset or before maximum impact of dental manifestations. Because the skeleton is in a constant state of remodeling, introduction of TNAP-D 10 to mice or humans with HPP would be expected to replace poorly mineralized osteoid with improved bone. In contrast, enamel, dentin, and cementum of teeth do not remodel, and dentin and cementum have limited ability for repair. Deciduous and permanent dentitions in humans are forming and mineralizing in utero and during early months to years after birth. Early therapeutic intervention is critical to maximally improve formation, mineralization, and function of the teeth. No detailed prospective, randomized studies of the effects of ERT on dental tissues in patients with HPP have been conducted, making it difficult to assess the real clinical impact. However, some insights can be gained from clinical trials and case reports. In an early open-label study, ERT was administered to 10 infants and young children (aged 7.5-36 mo) with life-threatening HPP. All treated patients experienced eruption of deciduous teeth, with only one female patient experiencing premature tooth loss. This patient had already lost 9 deciduous teeth before beginning ERT treatment at 36 mo of age, and the remaining 6 exhibited mobility. After 6 mo of treatment, the remaining 6 teeth became less mobile, and 4 additional teeth erupted. Another 7-mo-old female patient in the same study showed improved tooth mineralization after 1 yr of ERT. The long-term results showed improvements in skeletal mineralization, respiratory function, growth, and cognitive and motor function. Four patients (36%) experienced tooth loss. Case studies and series offer mixed assessments regarding the effects of ERT on oral tissues. Conclusions about efficacy of ERT are often obfuscated by different subtypes and severity of HPP and age at initiation of ERT. One case report focusing on the impact of ERT on dental features described a patient with infantile HPP that initiated ERT 1 d after birth. During examination at 41-mo-old, enamel and dentin were hypomineralized and permanent tooth development appeared delayed, but no prematurely lost deciduous teeth were noted. However, by 47-mo-old, this patient lost 3 incisors due to trauma. The exfoliated teeth were examined and showed hypomineralization of enamel, dentin, and cementum, and hypoplastic cementum. In a case of an infantile HPP in Japan, a 20-mo-old boy experienced early exfoliation of deciduous teeth. ERT was initiated at 21-mo-old, and the patient demonstrated stable periodontal conditions of deciduous molars afterwards. In another report from Japan, an 11-yr-old boy with childhood HPP had premature loss of 2 deciduous incisors. ERT appeared to positively affect mandibular bone density and periodontal stability. Larger studies have provided more incisive insights. A nationwide survey conducted in Japan gathered dental findings from 52 HPP cases, including 16 individuals with odonto HPP and 36 with other forms of HPP. Some dental manifestations appeared resistant to ERT. Two patients with childhood and infantile HPP treated with ERT experienced loss of permanent teeth. Four patients with various HPP forms showed severe periodontal disease and early exfoliation of deciduous dentition. In this study, early exfoliation of deciduous teeth and treatment with ERT were not correlated. In perhaps the most conclusive study to date, a Canadian open-label clinical trial for ERT collected data on the development and loss of deciduous and permanent teeth in a group of 11 children under the age of 5 with infantile HPP was collected. The study found that children who were treated with ERT at an early age experienced significantly fewer lost deciduous teeth compared to those who started treatment at a later age. Results from both preclinical studies and human case reports and clinical trials suggest starting ERT early and maintaining consistent treatment may ameliorate some dentoalveolar defects. However, the effects of ERT are somewhat limited in many cases, likely due to varying severity of disease and variable age at initiation of ERT. Clinicians should remain cautious and expectations should be tempered by the complexities of HPP. Earlier intervention should be considered if maintenance of the dentition is a goal of ERT. 

Shortcomings of current ERT have driven research into alternative treatment strategies. Limitations include high frequency of administration (3 or 6 times weekly), injection site reactions and lipohypertrophy ( ), costly drug prices, and lifetime requirement. For more than a decade, gene therapy strategies to deliver RT have been tested in the Alpl −/− mouse model of severe HPP. These approaches typically employ single injections of TNAP-D 10 -expressing viral vectors. In the first study, a lentiviral vector expressing TNAP-D 10 was injected intravenously into newborn pups, resulting in vector integration in liver and 10-fold higher ALP levels than control mice. Treatment eliminated seizures, normalized body size, improved skeletal mineralization, and extended lifespans in Alpl −/− mice. Subsequent gene therapy approaches employed a variety of adeno-associated viral (AAV) vectors. Expression of TNAP-D 10 under the tissue-nonspecific CAG promoter and delivered in an AAV8 vector promoted phenotypic correction of Alpl −/− pups injected shortly after birth. Fetal gene therapy was successfully accomplished using transuterine intraperitoneal injection of AAV9-expressed TNAP-D 10 in a pregnant dame carrying Alpl −/− mouse fetuses on embryonic day 15 (about 4 d before birth). ALP levels were increased in pups after birth, preventing seizures, normalizing weight and growth rate, and ameliorating skeletal defects. A strategy was pursued to deliver a muscle-directed AAV8 construct expressed TNAP-D 10 under the muscle creatine kinase (MCK) promoter. Muscle-driven production of TNAP-D 10 increased ALP levels in Alpl −/− to more than 10-fold over normal levels, extending lifespans, increasing body size, and improving skeletal mineralization. Skeletal rescue was incomplete, as indicated by reduced bone lengths, abnormal cortical and trabecular bone parameters, and hypomineralization. This was the first preclinical gene therapy study to include dentoalveolar analyses. In treated Alpl −/− mice, jawbones showed improved mineralization, though molars exhibited thin and hypomineralized root dentin and periodontal detachment suggested defective cementum. As an alternative to gene therapy approaches outlined above, Shimada and colleagues attempted ex vivo gene therapy using lentiviral expressed TNAP-D 10 transduction of BM cells (BMCs), which were then transplanted into irradiated Alpl −/− mouse pups. Transduced BMCs elevated ALP levels to 10-fold higher than controls, prolonged survival, and improved bone parameters. However, limitations were evident in disorganized growth plates and reduced bone lengths. There was little evidence for improvement in dentin, cementum, or periodontal attachment and function, possibly reflecting different origins of craniofacial tissue precursors and inability of the elevated ALP to reach these tissues. Most recently, a refinement of this approach used a single intramuscular dose of AAV8 encoding TNAP-D 10 . This increased ALP levels 800-fold over normal, extended lifespan, and essentially normalized skeletal and dentoalveolar development in Alpl −/− mice, a dramatic result ( ). The same vector improved skeletal mineralization in mouse models of adult-onset HPP and osteomalacia. ERT gene therapy approaches have the potential to revolutionize therapy for those with HPP and ameliorate the disease burden for many affected individuals. While a clinical trial for ERT gene therapy is yet to begin, it is important for dental clinician-researchers to be included in this work because of their key roles in diagnosis and treatment of HPP and missed opportunities of past clinical trials. 

Research in recent decades has yielded critical insights into HPP, including mechanisms underlying dental and periodontal defects. However, more work is required on multiple fronts. First, additional genotype-phenotype studies of HPP in different clinical types are required to understand the sensitivity of enamel, dentin, cementum, and alveolar bone to reduced TNAP function. Second, dental researchers and clinicians should be involved in preclinical studies and clinical trials for any new treatment. Third, more case report publications on dentoalveolar manifestations and responses to ERT are necessary to increase awareness and provide guidance for dental clinicians to treat HPP patients. 

