Innovative Molecular and Cellular Therapeutics in Cleft Palate Tissue Engineering

Abstract

Clefts of the lip and/or palate are the most prevalent orofacial birth defects occurring in about 1:700 live human births worldwide. Early postnatal surgical interventions are extensive and staged to bring about optimal growth and fusion of palatal shelves. Severe cleft defects pose a challenge to correct with surgery alone, resulting in complications and sequelae requiring life-long, multidisciplinary care. Advances made in materials science innovation, including scaffold-based delivery systems for precision tissue engineering, now offer new avenues for stimulating bone formation at the site of surgical correction for palatal clefts. In this study, we review the present scientific literature on key developmental events that can go awry in palate development and the common surgical practices and challenges faced in correcting cleft defects. How key osteoinductive pathways implicated in palatogenesis inform the design and optimization of constructs for cleft palate correction is discussed within the context of translation to humans. Finally, we highlight new osteogenic agents and innovative delivery systems with the potential to be adopted in engineering-based therapeutic approaches for the correction of palatal defects.

Impact statement

Tissue-engineered scaffolds supplemented with osteogenic growth factors have attractive, largely unexplored possibilities to modulate molecular signaling networks relevant to driving palatogenesis in the context of congenital anomalies (e.g., cleft palate). Constructs that address this need may obviate current use of autologous bone grafts, thereby avoiding donor-site morbidity and other regenerative challenges in patients afflicted with palatal clefts. Combinations of biomaterials and drug delivery of diverse regenerative cues and biologics are currently transforming strategies exploited by engineers, scientists, and clinicians for palatal cleft repair.

Introduction

Natural development of the mammalian palate involves a complex and tightly coordinated series of biological processes involving cell migration, proliferation, growth, and differentiation as well as programmed cell death (Fig. 1). Failures in palatogenesis can occur during the outgrowth, elevation, migration, or fusion of palatal shelves, providing the best evidence that these developmental processes are under strict molecular control.

FIG. 1.

Key developmental stages of palatogenesis. Around the fourth week of human development, cNCCs migrate from the dorsal edge of the rostral neural tube to form the frontonasal prominence and the paired maxillary and mandibular processes that surround the primitive oral cavity. This allows the nasal pits to take form and subsequently develop into paired medial and lateral nasal processes from the frontonasal prominence by week 5. The medial nasal processes then merge with the maxillary processes to form the upper lip and primary palate while bilateral outgrowths from the maxillary processes, termed the palatal shelves, grow vertically along either side of the tongue. At week 7, mandibular growth is evident and results in the descent of the tongue and the elevation of the palatal shelves to a more horizontal position. Further growth leads to formation of the midline epithelial seam and onset of palatal fusion. By week 10 of gestation, the secondary palate fuses with the primary palate and nasal septum, allowing palatal mesenchyme to differentiate into bony and muscular elements. These fusion processes of primary and secondary palate components are complete by week 12 of development, by which time the secondary palate is divided along the anterior/posterior axis into the bony hard palate and the soft palate that is more muscular in nature. cNCCs, cranial neural crest cells.

Clefts of the palate (CP) are classified within a heterogenous group of orofacial cleft disorders, including the subtypes of clefts of the lip alone (CL), and clefts of both the lip and palate (CLP).¹ While the primary and secondary palates have distinct developmental origins, CL and CLP share a defect in the primary palate. Hence, CL and CLP are in a common group: CL with or without CP (CL/P).²

While epidemiological estimates of orofacial cleft disorders vary substantially based on the sample population, surveillance methodology, and clinical classification, the World Health Organization estimates the worldwide oral cleft burden in any form (i.e., CL, CP, or CLP) to be about 1 in every 700 live births.^3–5 In the United States, on average, the incidence of CP is 1 in 1500 (∼2700 cases per year), whereas CLP is 1 in 1000 (∼4400 cases of CLP per year).^6,7 Affected individuals suffer difficulties with feeding, speech, hearing, cognition, and social integration, and endure surgeries and life-long multidisciplinary care.

Etiology of lip and palatal clefting

Environmental factors that are maternal related and associated with pathogenesis of palatal clefts include: smoking, ethyl alcohol, phenytoin, history of diabetes mellitus, increased maternal and/or paternal age, and deficiencies in key vitamins, folic acid, and zinc.⁵ While genetic studies of orofacial clefts date back to centuries, it has been difficult to point to a single etiologic mechanism responsible for this complex trait.⁸ Sequencing Mendelian forms, identifying causative variants, whole-genome sequencing, genome-wide linkage and association studies (GWAS) have collectively increased the understanding of specific genes involved in nonsyndromic cleft lip and/or palate (CLP).⁹ PAX9 (paired homeobox domain-9) along with TGF-β3 and BMP4 are associated with the 14q21-24 locus and have been shown to have genome-wide significance as influencing risk to orofacial clefts.^9,10 Mutational analysis and association studies have confirmed that MSX1 (Msh homeobox 1) plays a role in human nonsyndromic CL and CLP subtypes.^11–13 Despite many successes in GWAS and other approaches that have shown multiple loci associated with CP and CLP, the genetic etiology of these conditions is not fully understood. Weinberg et al.⁸ stated that gene discoveries have outpaced the functional analyses of cellular and molecular mechanisms needed to understand the genesis of clefting. Human genetics reveal that CL, CP, and CLP share common molecular pathways despite developmental differences. As cleft maps describe, neither subtype-specific nor shared genetic effects operate in isolation for this group of orofacial clefts.¹⁴ For example, IRF6 is described primarily as a CL gene, yet it plays a role in CP in humans.^15,16 In CLP, secondary palatal clefts could be related to failure in fusion of the primary palate, but could also be an independent genetic event.¹⁴

Thus far, the discovery of genes responsible for human clefting has been provided through preclinical genetic models in mice and larger animals. In single-gene disorders, these preclinical models have been crucial for development of translatable human therapeutics from bench to bedside. Since morphological events in mice closely resemble that seen in humans, mouse genetic models with secondary palatal defects have been valuable for dissecting signaling pathways that drive palatogenesis and in unraveling the genetic etiology of human clefting.¹⁷ Gene ontology reveals that more than half of the genes associated with palatal clefts in mice are transcription factors, such as Msx1,^18,19 Pax9,^20,21 Osr2 (odd-skipped-related 2),²² Gbx2 (gastrulation brain homeobox 2),²³ Tbx22 (T-box 22),^24,25 Tfap2A (transcription factor AP-2 alpha),²⁶ and Hoxa2 (homeobox A2).²⁷ Growth factors and morphogens such as TGF-βs, Shh (sonic hedgehog), Fgf, and Wnt along with membrane-bound and extracellular matrix (ECM) proteins also play important roles in palate formation.²⁸

Altogether, these studies reveal that a tight interplay of gene networks and environmental risk factors regulate molecular and cellular interactions in palate development. However, much remains to be learned about how these molecules precisely function and how this knowledge can be translated into new treatment strategies for preventing or reversing diverse cleft conditions in humans.

Surgical Procedures for Human Cleft Palate Repair and Associated Complications

Autologous bone grafting has served as the unanimous “gold standard”surgical treatment to repair palatal cleft defects for decades.²⁹ Nearly 600,000 autologous bone grafts are performed in the United States each year³⁰; Of these, at least 6% are performed on the craniofacial complex.³¹ Regardless of technique, cleft palate repair provides both bony continuity and stability of the alveolar ridge as well as soft tissue closure within the primary and secondary palate. Successful repair allows support for the upper lip and nose and provides a foundational path for proper tooth eruption and positioning within the maxillary arch.³² Repair of the palatal muscles enables realignment for proper speech and a pouching in the anterior palate to receive grafting material at the site of the alveolar ridge defect.³³ The overarching goals are to restore both anatomic form and function to the hard palate that provides structural support and functions as the growth center for the maxilla, and, for the soft palate or velum that provides velopharyngeal competence. Hence, cleft surgical repair requires complex combinations of both hard and soft tissue manipulations to yield complex palatal structural restoration also with muscle involvement for producing oral, vocal, and respiratory competence. This complexity sets the bar high for other strategies such as tissue engineering to improve cleft repair outcomes.

A number of factors inform surgical approaches to repair palatal clefts; each technique is tailored to the specific defect length and width and dictated by the nature of the cleft and its Veau classification.³⁴ The Veau classification (I–IV) divides clefts of the lip and/or palate into four groups (Fig. 2) based on severity of cleft defect.^35,36 As midfacial growth and speech development do not occur in unison, disagreement exists as to the timing of palatoplasty most compatible with normal speech development and that does not restrict maxillary growth.^37–42 Some fear that a cleft in the soft palate that persists past 1 year of age may impair normal development of speech mechanisms, therefore, favoring earlier palatoplasty. Conversely, others are concerned that early palatoplasty may impair midface growth, thus arguing for delaying intervention until midface growth is nearing completion.⁴³ Some address this discrepancy by temporally separating soft palate from hard palate repair, effectively uncoupling the perceived deleterious effects of a late soft palate repair from those of an early hard palate repair (see Fig. 3 timeline).

FIG. 2.

Veau classification of cleft lip and palate defects. Group (class) I. Defects of the soft palate only; Group II. Defects involving the hard palate and soft palate; Group III. Defects involving the soft palate to the alveolus, usually involving the lip; and Group IV. Complete bilateral clefts.

FIG. 3.

Surgical treatment staging timeline of cleft repair. (A) Lip closure through muscular alignment typically occurs by about 3 months of age; Following the “Rule of 10’s”: Hb = 10, 10 lbs body weight, 10 weeks of age. If the cleft lip is particularly wide, often adhesion is performed before lip closure. (B) In the range of 9–18 months of age, the soft palate is typically closed, as recovery of speech (consonant formation) is the primary concern around this age. If soft palate is closed before 9 months of age, the patient may be at risk for midfacial hypoplasia. (C) By 5–7 years of age, surgical repair of velopharyngeal incompetence is performed, often through superior-to-inferior pharyngeal flap elevation. Also, at this stage the surgeon can correct the nasal and/or lip deformities resultant following primary lip repair. (D) In the range of 7–12 years of age, the alveolar cleft defect is repaired, with exact timing dependent upon eruption of dentition (preferably performed during the mixed stage, when canine root is 2/3 formed). Approximately 95% of maxillary growth is completed by age 8 years. Following preoperative maxillary expansion, grafting procedure (either iliac crest bone graft or allograft) is performed to regenerate palatal bone. (E) As the patient progresses into adolescence and early adulthood, orthognathic corrective surgery (dependent on sex and gender) is often undergone. Males typically experience complete mandibular growth by age 21–23, while females typically are fully developed in the craniofacial complex by 2 years postpuberty. Corrective rhinoplasty is typically performed at this stage in conjunction with maxillary advancement to correct midface hypoplasia and/or dental malocclusion. Hb, hemoglobin.

Adding to the literature controversy is the large variability in the timing of surgical interventions. Depending on institutional protocols and surgeon preference, patients may undergo palate correction between 3 months to 2 years of age for velum, and 6 months to adolescence for hard palate. Rohrich and colleagues detected a statistically significant speech deficit with delayed (48.6 months) versus early (10.8 months) hard palate closure and no effect on maxillofacial growth associated with later repair.^44,45 Furthermore, Robertson and Jolleys saw no difference in occlusion or midface growth between patients undergoing palatoplasty from 12 to 15 months of age and those who underwent palatoplasty at 5 years of age.⁴⁶ A more recent analysis by Botticelli et al. compared patients with unilateral complete cleft lip/palate who underwent either early (12 months) or delayed (36 months) intervention and found that delayed repair resulted in significantly shallower three-dimensional (3D) cross-section measurement in middle and posterior palate regions.³⁸ This more recent analysis is likely more pertinent to consider for patients with isolated cleft secondary palate, as the midface is unlikely to be affected given the absence of a primary palatal bony defect. Therefore, the current literature evidence on delayed intervention is likely not sufficiently convincing to justify sacrificing the opportunity to correct soft palate anatomy and facilitate normal speech development with early palatoplasty.

Palate repair is ultimately a surgery for “speech and nutritional improvement’,” as the palate structure and function are essential for velopharyngeal competence that leads to proper speech development and feeding.⁴⁷ Failure to sufficiently close the velopharyngeal valve during secondary palate repair can lead to a common complication, velopharyngeal insufficiency (VPI).⁴⁸ VPI is defined as the inability to completely close the velopharyngeal sphincter that separates the oropharynx and nasopharynx; this closure is required for normal production of all but the nasal consonants.⁴⁹ VPI is seen in a wide range of patients with secondary palate defects (between 5% and 40%), leading to nasal air escape, hypernasality, misarticulation, and low-speech volume.⁵⁰ Also, secondary consequences of VPI can include nasal regurgitation of liquids, compensatory misarticulations, and facial grimacing. VPI may significantly impact the child's confidence, social development, and overall quality of life.⁵¹ Surgeons aim to restore velar anatomy so to optimize eustachian tube function and support hearing, thus minimizing the risk for development of recurrent otitis media secondary to insufficient tubal dilation and impaired drainage. In the meta-analysis cited above, the authors identified an overall incidence of oronasal fistulae to be 8.6%. No significant difference was detected in fistula incidence based on continent of origin of each study or the repair technique used. Furthermore, the incidence of fistula in cleft lip–cleft palate was 17.9%, which was significantly higher than in cases of cleft palate alone (5.4%).⁵² Interestingly, this meta-analysis of cleft palate repair outcomes concluded that the Furlow palatoplasty approach was associated with less risk of fistula formation than any other approach analysed (von Langenbeck/Veau/Wardill/Kilner techniques). Lower incidence of VPI compared with the Bardach palatoplasty was also reported.⁵³ Furthermore, they identified that one-stage repair was associated with less risk of fistula formation and VPI than two-stage repair. In the United States, the vast majority of surgeons performing cleft repairs utilize either the Furlow palatoplasty (double-opposing Z-plasties comprising 2 oral flaps and 2 nasal flaps) or the Bardach palatoplasty (with an intravelar veloplasty), the two approaches together comprising ∼87% of all cases. The most common time of intervention is between 9 and 12–18 months of age (74% of all cases).⁵⁴

Cleft palate defects can be corrected surgically in either a single operation or by way of two-staged interventions. The first is typically performed when the patient is ∼12 months of age, and the second operation timing can vary, as discussed above. Proponents of the single operation strategy cite superior outcomes in lip and nose symmetry following reconstruction.⁵⁵ However, others promote the staged approach, citing significantly less adverse effects on the growing maxilla.⁵⁶ Importantly, a recent randomized controlled trial did not find any significant difference in speech outcomes or fistula formation between the two approaches.⁵⁷ Velopharyngeal competence that supports normal speech production is critical in palatoplasty. Formation of a water-tight barrier between oral and nasal cavities should limit complications, such as hypernasality, speech distortion, and oronasal fistulae. A universal requirement essential to all these goals is a tension-free, water-tight repair of the palate to minimize risk of complications at the site of closure.⁵⁸ This is a key consideration in any tissue engineering approach to such repair.

Osteogenic Scaffolds for Surgical Correction of Cleft Defects

While numerous innovations in bone substitute materials have been published for decades, treatment of bone defects with autologous bone grafting remains the “gold standard” against which other approaches are compared.⁵⁹ Autologous bone in the adult skeleton is a natural scaffold that is both osteoconductive and osteoinductive as well as favorable for promoting osteoblast functions. Nonetheless, autologous pediatric bone has reduced cortical thickness and lower osteoinductive potential.⁶⁰ Infants and children who receive autologous bone hence suffer restrictions in maxillofacial growth that require revision surgeries to achieve facial symmetry and full palatal closure. Often harvested from the iliac crest of the pediatric patient, the morbidity associated with the procedure is substantial, requiring postoperative pain management and rehabilitation.^61,62 Additionally, all harvested autografts must be customized to fit each patient's hard or soft tissue cleft, often with the expectations for biomechanical durability and hydrostatic seal with host tissue. Such precision fit is not simple or reliable with autograft harvests. Clearly, autologous pediatric bone as a gold standard for CLP repair requires improvements to address diverse patient demands and clinical unmet needs.⁶³

Tailored bone tissue engineering strategies to overcome these physiologic and biomechanical hurdles must employ osteogenic scaffolds that are customizable, biocompatible, biodegradable, and capable of integration with both surrounding hard and soft tissues to restore form and function without impeding craniofacial development.^64,65 Furthermore, the ideal scaffold should provide mechanical support and stability at the surgical site until sufficient new bone is fully matured and able to withstand normal mechanical loading.⁶⁶ As a general rule, any scaffold material should be sufficiently robust to resist compressive and tensile forces that arise from movements of the tongue, soft palate, and overlying sinuses and mid-face structures. Additionally, standard surgical repair and tissue bed reconstruction is no longer the simple, direct outcome desired. Rapid, reliable, and functional tissue restoration and regeneration to accommodate full-defect reconstruction through patient-specific, individualized implants endowed with biological adjuvants (growth factors, cells, small molecules) now dominate most new approaches.

Controlled spatiotemporal orchestration of bone-specific signals, cues, and growth factors in vivo is critical to successful bone tissue engineering strategies. A scaffold designed for cleft palate correction must provide a careful balance of degradation kinetics and biomechanical properties imparting both stability and flexibility while encouraging de novo bone to infiltrate and fully restore the defect spatially and mechanically, prompted by exogenous or endogenous biological cues.⁶⁷

Customized implant fabrication for CLP

Donor-site morbidity associated with autograft harvesting for CLP can be limited by careful preoperative planning that considers the individual aspects of each CLP repair and volumes of autograft harvest required. In such cases, 3D CLP site imaging using computerized tomography (CT) or magnetic resonance imaging (MRI) facilitates 3D construction of a virtual image of the CLP site for anatomical considerations and modeling of various repair strategies. Recently, the use of an intraoral scanner has been reported in the 3D modeling of a bilateral cleft defect in a 3-month-old patient.⁶⁸ Translation of this technology into the cleft clinical care model would allow for real-time 3D mapping of patient-specific defects for preoperative virtual surgical planning, individualized scaffold design, and rapid prototyping of custom implants best suited to repair bone defects^69,70 and, specifically, CLP sites. Finite element analysis can be applied to various implant models to predict and optimize the implant structural strength, including any fixation devices required for implant stabilization during integration.⁷¹ More significantly, the virtual image of the CLP site can also be used to avoid autografting altogether through integration with 3D printing capabilities to build custom individualized scaffolds from accepted, diverse biocompatible scaffolding components to address CLP requirements.^72,73

A 3D scaffold plays a critical role in biomechanical support, defect filling, host tissue integration through host cell migration and tissue neogenesis, and metabolic/catabolic transport features. Using patient-specific imaging data, 3D printing with additive manufacturing allows fabrication of implants tailored to specific, complex geometries, mechanical properties, and morphologies, with specific porous fractions and morphologies also as variables.⁷⁴ Such a 3D customized fabrication capability expedites CLP surgical procedures with implants created peri- or intraoperatively, minimizing additional requirements for patient-specific modifications or protracted fit issues in real time during repair surgery. Additionally, 3D scaffolds can be rapidly prototyped with variable bulk properties, biological additives, and materials to facilitate screening and testing for optimization. Use of coprinted radio-opaque agents can facilitate their monitoring in vivo. Host-deposited mineralized tissue within an implanted scaffold can also be followed using micro-CT both in vitro and in vivo.^75,76

Three-dimensional printed patient-specific implants using additive manufacturing are increasingly approved for bone repair applications.⁷⁷ Nonetheless, 3D implant design requirements span an enormous variable space, and few are shown to be optimal for any or all patients in regenerating bone or soft tissue. Biomaterial selection, added growth factor cues and dosing regimens, added cells, mechanical properties, porosity and transport considerations, and degradation features remain to be determined, especially for CLP 3D implants. How these variables are rationally modified in patient-specific ways is unknown. CLP sites likely have anisotropic loading over both time and space in healing, and biomaterials that reliably integrate with appropriate regenerative capacity in this context are not yet validated clinically. Hence, customized 3D scaffold printing for tissue regeneration remains largely empirical without clear design specifications for accommodating patient-specific needs in healing, tissue growth, and load bearing that promotes healing and functional restoration. Optimal cleft repair implants might also best accommodate dynamic, time-dependent anatomical, mechanical, and physiological changes occurring naturally as the cleft site evolves with each patient. Scaffolds that respond to patient-specific cleft site changes must be considered. Virtual CLP implant-site imaging produces a seductive morphological basis to manufacture implants to fit individual sites. However, beyond implant shape, specifications for biomaterial composition, load distribution, osteoconductive or inductive properties, and anisotropic structural features (e.g., density, porosity, and local modulus) remain unproven and require timely, costly in vivo experimentation to understand.

To date, myriad combinations of form, biomaterials, gradients, porosities, and biological additives have been 3D printed for bone regenerative use.^78,79 To accommodate local physiological, mechanical, and metabolic demands, in addition to biological orchestration of tissue regeneration and scaffold degradation specific to each application, the parameter set for constructing customized scaffolds using 3D rapid prototyping is daunting.^80,81 Lacking reliable design specifications for 3D-manufactured implants comprising numerous components, the field relies on trial/error and multiparametric experimental designs, seeking optimized performance in each context. Most approaches use resorbable scaffold designs to avoid multiple surgeries. Personalized scaffolds that are suitably osteoconductive or osteoinductive for hard tissue CLP repair are poorly explored and largely unvalidated in vivo. Diverse materials, composites, and blends are available to fabricate CLP scaffolds, with or without 3D printing methods, but much experimental work remains to show their clinical performance benefits over traditional autograft gold standard approaches.

Natural polymer scaffolds

Protein scaffolds

Given their intrinsic biocompatibility and biomimetic properties (as well as ability to promote cell attachment), natural proteins have regulatory approval in numerous medical device and implant applications.⁸² Several matricellular proteins have recognized evaluations as bone tissue engineering scaffold materials,^83,84 with some rapid prototyping and limited CLP repair applications described. Examples include collagens,⁸⁵ gelatins,⁸⁶ silks,⁸⁷ fibrin,^88,89 and ECM,⁹⁰ some of which are available in medical grades and diverse forms (e.g., sponges, sheets, pastes, glues) and are approved by the U.S. Food and Drug Administration (FDA) for application in bone tissue engineering (e.g., Collapat^® [BioMet, Inc.], BioGide [GeistlichPharma AG, Switzerland], and Collagraft™ [Collagen Corp.]).⁹¹ Multiple preclinical studies have utilized fibrin,^92,93 collagen,^92,94–97 and gelatin⁹⁸ independently or in combination with other natural scaffold materials, including polysaccharides, proteins, and inorganic calcium solids, to successfully repair cleft palate bony defects in preclinical models. Collagen scaffolds have also been employed to manage alveolar clefts in 8- to 15-year-old human patients,⁹⁹ but secondary palatal cleft repair with natural scaffolds has not yet been reported in human infants. Despite commercial availability and clinical use, proteinaceous biomaterial performance in bone regeneration is anatomic site-specific and inconsistent in craniomaxillofacial repair. Clinical data specific to cleft repair are not available.

Demineralized bone matrix

A well-studied autologous bone graft alternative is demineralized bone matrix (DBM), widely cited in the dental and orthopedic literature as an effective conduit to repair osseous defects.¹⁰⁰ As a pooled, commercially processed product of cadaver bone donors, DBM naturally contains a multitude of endogenous osteogenic growth factors.¹⁰¹ Proposed benefits of using DBM in lieu of autologous bone include unlimited supply, lack of donor-site morbidity, reduced operative time (saving substantial cost to the patient and hospital system), numerous commercial forms, and relatively easy supplementation with drugs or additional osteogenic growth factors.¹⁰²

DBM properties and its consequent clinical performance are known to be affected by recognized differences in donor bone, and its preparation and processing methods used in producing DBM. Variations in residual calcium content from demineralization processes, DBM bone-derived particle sizes and distributions, and variable endogenous growth factor contents can be substantial.¹⁰³ Biological bioactivity assays of DBM in vitro and in vivo attempt to provide a reference “osteoinductive index” (OI).¹⁰³ Nonetheless, DBM OI variability across lots and manufacturers can be substantial. Assessing and predicting DBM potency in osteogenesis in vitro and in vivo is therefore problematic; DBM OI values are not standardized, and not controlled or consistent from tissue bank sources in marketed DMB products. Despite a substantial clinical record of DMB use and history, OI variability, methods used for bioactivity assay, lack of comparisons between in vitro and various preclinical animal healing in osteogenesis models, and inconsistent human therapeutic experiences make DBM use and efficacy still controversial.

One known way to modify existing DBM compositions is using them as drug delivery vehicles to improve osteogenic capabilities. Versatile drug delivery from DBM using custom selections of therapeutic drugs, bioactive agents, and cells to enhance DBMs' own properties and exploit its clinical use as local bone delivery matrix is widely reported and exploited.¹⁰¹ This strategy can be used to mitigate potential therapeutic insufficiencies associated with DBM variability, modifying DBM with pharmaceuticals of many classes, including osteogenic growth factors and diverse cell phenotypes, yielding a combination medical device (i.e., an implant with medical device function such as bone filling/augmentation as its primary mode of action and drug delivery as a secondary mode of action). Consequently, DBM is commercially accessible in many different physical forms (e.g., pastes, foams, fabrics, blocks), and exhibits intrinsic osteogenic scaffold properties, in addition to properties attractive and necessary for drug delivery applications.¹⁰¹

However, DBM does come with an increased price tag compared with other marketed scaffold materials, as well as potential immunostimulatory activity and risk of transmission of communicable disease from donor to recipient (although rare).¹⁰⁴ This increased cost and potential adverse events associated with DBM use in alveolar cleft reconstruction must be weighed against the cost savings and decreased morbidity of autologous graft harvest; the average cost for 5 mL of DBM is ∼1000 USD, in comparison to the average operating room cost of ∼62 USD per minute.¹⁰⁵ Thorough evaluation of surgical outcomes using DMB compared with autograft bone must be prioritized. Proponents of DBM use in alveolar cleft reconstruction report equivalent or superior outcomes compared with gold standard iliac crest bone graft.^102,106 However, larger clinical trials are required to substantiate DBM's use as a superior method to reconstruct bony defects of the palate. A very recent report for 3D printing of hydroxyapatite ceramic and DBM applied in spine fusion as a recombinant growth factor-free composite shows promise to overcome limitations of currently used bone graft substitutes for spine fusion.¹⁰⁷ That this prototyping and demonstrated osteogenic properties might be extended to palate repair appears promising.

Polysaccharide scaffolds

Natural polysaccharides include alginate, chitosan, and hyaluronic acid, also enjoy clinical use in diverse forms, and depending on their physical and chemical processing, can exhibit intrinsic biological and mechanical properties similar to natural proteins.¹⁰⁸ Several polysaccharides have been employed in scaffolds for bone tissue engineering.¹⁰⁹ Notably, cellulose-based materials, possessing enormous natural diversity, have some tissue engineering history,¹¹⁰ but are not readily biodegradable by humans, limiting their utility in tissue engineering. As the most exploited, commercialized and well-studied, hyaluronic acid (0.25 g/mL aqueous hydrogel)¹¹¹ is readily delivered as an injectable gel⁹³ with flow properties suitable for complete filling of cleft defect tunnels, with some evidence to modulate cellular responsiveness to osteogenic signaling molecules through hyaluronin/CD44 receptor interactions to stimulate intracellular Smad protein activity.^111,112 However, many polysaccharides intrinsically lack requisite cell-adhesive properties and therefore, while biocompatible in a broader sense, are often blended with matricellular proteins (e.g., collagen or gelatin), or ceramic particulates or cell-binding peptides to improve cell retention and growth.¹¹³ Despite the recent promise of alginate-based scaffolds in bone regeneration,¹¹⁴ nanocomposites with graphene oxide and gelatin¹¹⁵ have not yet been applied to cleft palate repair in vivo. Importantly, alginates alone are not sufficient to engineer bone as they require coadditives to stimulate osteoblast activity and proangiogenic signaling (e.g., growth factors, collagen, ECM, stem cells, etc.).¹¹⁶ Such composite constructs have been shown to be effective in bone tissue engineering in the maxillofacial region,¹¹⁷ and may prove useful specifically for cleft defects.

Chitosan

Chitosan is a deacetylated form of the invertebrate exoskeleton biopolymer, chitin, which displays many promising characteristics that include its compatibility with other natural materials used for bone tissue engineering.^118–121 Chitosan-based delivery systems seeded with bone morphogenetic protein (BMP)-2 demonstrated increases in osteocalcin synthesis in cultured human embryonic palatal cells.^122,123 More recently, hydrophobically modified glycol chitosan nanocontrolled delivery of trichloroacetic acid and epidermal growth factor in vivo in critical-sized defects in palatal soft tissues in a canine model, demonstrating successful regeneration of these palatal defects.¹²⁴ While chitosan-based scaffolds have yet to be clinically assessed, many forms for tissue-engineered bone are reported. Three-dimensional-bioprinted cryogels formed from chitosan and/or gelatin-based scaffolds for patient-specific palatal cleft reconstruction have been successfully designed through CT image constructions of CLP repair sites.¹²⁵ These valuable proof-of-concept studies provide rationale for further investigation of chitosan and its composites as a viable scaffold material in palatal repair.

Poly(hydroxyalkanoate)-based Scaffolds

Poly(hydroxyalkanoate)s, or PHAs, are a family of natural biodegradable polyesters produced and stored within microorganisms (i.e., bacteria, fungi) as insoluble granules. PHAs exhibit a range of physical and mechanical properties comparable to polyalkenes and thermoplastic elastomers. PHA polymer properties are a function of their natural production conditions: microbial strain, carbon source for monomers, and fermentation conditions. PHAs can be polymerized from carbohydrates, alcohols, fats and oils, and organic acids, including lactic acid to vary polymer properties, yielding variable solid state and degradation properties. Several PHAs have been employed in scaffold-based bone tissue engineering, including 3D printing.^126,127 While PHAs have not been reported for in vivo cleft repair, their future use is promising given their efficacy in other bone regenerative applications.^{126,128–131}

Synthetic biomaterials for cleft scaffolds

Given their previous published history in other tissue engineering applications, degradable synthetic biomaterials have recently been applied in cleft animal models (alveolar and palatal clefts)¹³² and in humans (alveolar cleft).¹³³ In the context of cleft repair, clinically familiar medical grade and resorbable poly(ɛ-caprolactone) (PCL), poly(lactide-co-glycolic acid) (PLGA), poly-l-lactic acid (PLLA), poly-d-l-lactic acid, and poly(propylene fumarate) (PPF) have been investigated to date.⁸² The benefits of these materials lie in their recognized extensive clinical history, acceptable bulk mechanical properties, diverse marketed commercial medical grades, precise control of scaffold pore size, use in additive manufacturing and rapid prototyping, and ranges of biodegradation rates that can exceed 24 months for PCL, PLLA, and PPF.^82,134,135 However, only PLLA degrades to yield a completely nontoxic, weakly acidic natural byproduct.¹³⁶ In the context of palate repair, these resorbable biomedical polymers have a broad history of biomaterial use.^65,137,138 Polymer scaffolds alone generally produce less-impressive palate repair than their blends with ceramic materials (e.g., hydroxyapatite), or natural proteins (e.g., collagen, gelatin), or with osteoblast lineage-induced stem cells.^{97,129,139,142–144} More recent advancement in scaffold design has incorporated hybrid naturally derived biomaterials (e.g., collagen or chitosan) in combination with PCL and PLGA copolymer nanofibers, enabling superior osteogenic potential by bolstering the biomimetic and stimulating effects of natural polymers with the structural and mechanical stability of synthetic polymer materials.^145–147

Bioactive ceramics

Bioactive ceramics utilized in bone tissue scaffolds represent a broad class of inorganic materials, many based on calcium-based solids, and generally effective as osteoinductive agents for bone tissue engineering. Scaffolds comprising bioactive glass (Si-O), tricalcium phosphate, or hydroxyapatite are common examples.^148–150 Bioactive glass scaffolds enable controlled stimulation of the body's inherent ability to initiate and propagate bone tissue regeneration through osteoblast induction on the surface of these implant materials.¹⁴⁸ They are also 3D printed into scaffold forms.^151,152 Tricalcium phosphate bioceramic scaffolds implanted into immature rabbits during facial development demonstrate significant enhancement of osteogenic regeneration of native-like bone in calvarial and alveolar defects compared with bone repaired with autologous bone graft.^153.Biodegradation of the bioactive ceramic component was also noted and 3D morphometric facial surface analysis confirmed the preservation of normal craniofacial growth after scaffold insertion.¹⁵³

Since ceramics are notoriously hard and thus often brittle, they have also been combined into composites with natural and/or synthetic polymers during repair of cleft palates to improve properties.^154,155 Natural and synthetic polymer scaffolds are frequently augmented with bioactive inorganic materials to enhance interactions between scaffold surface, cells, and native tissue.^156–158 Importantly, calcium salts, solid particulate slurries, and their polymer composite materials can be 3D printed for ceramic scaffolds.^159,160 For instance, custom tricalcium phosphate–polyhydroxy butyrate scaffolds have been 3D printed from CT scans of CLP sites to custom fit alveolar clefts of 8- to 11-year-old patients.¹⁶¹ Importantly, analysis of safety and outcomes in tricalcium phosphate alveolar cleft repair compared with autologous bone has shown equivalent residual tissue calcification, canine eruption, and complication rates at the recipient site.¹⁶²

Preclinical and Clinical Applications of Growth Factors in Palate Tissue Engineering

Growth factors are a class of natural, soluble, secreted signaling peptides, proteins, and hormones that regulate undifferentiated cell proliferation and cell differentiation to increase or decrease specific cell populations by binding to receptors and transmitting intracellular signals. Secreted by cells to control natural signaling locally on demand, growth factors can engage cell receptors directly, but are also frequently bound by specific components of ECM for controlled presentation and even coordination of presentation with other costimulatory factors.¹⁶³ Cell–ECM interactions are therefore recognized as increasingly important for growth factor signaling¹⁶⁴; scaffolds delivering growth factors alone without ECM coordination may not be sufficient to recapitulate normal endogenous signaling.

Harnessing the natural power of growth factors to induce and accelerate healing or tissue regeneration has been a focus for tissue engineering for decades. Increasing availability of human recombinant growth factors, reduced costs, and their clinical approval as therapeutic agents has catalyzed and expanded many research studies on tissue engineering utility. Common growth factor therapeutic limitations in regenerative medicine use include the need for supraphysiological dosing, off-site adverse effects, and accommodating their extremely brief half-life of biological activity and specified durations of locally effective concentrations. Thus, scaffold technology, enabling controlled, local, or sustained growth factor release, is important for tissue regeneration strategies with growth factors. However, formulating growth factors within scaffolds is not trivial. As peptides and proteins, most growth factors have notable stability and fragility issues that are not consistent with scaffold processing and fabrication methods. More robust small molecules are being developed to emulate growth factor signaling activities but in a more practically controlled, more stable, less immunogenic, and perhaps better economical formats.¹⁶⁵

Growth factors involved in regulating bone metabolism include BMP-2, BMP-7/OP-1, FGF, PDGF, PTH, PTHrP (parathyroid hormone-related peptide), TGF-β3, VEGF, IGF, and Wnt proteins.¹⁶⁶ Nonetheless, translating osteogenic growth factor results from preclinical to clinical use has been plagued by inconsistencies in reproducing bone regeneration under growth factor control seen in animals to humans.¹⁶⁷ Enhanced bone repair has been clinically demonstrated following European Medicines Agency and FDA approval of human recombinant BMP-2, BMP-7/OP-1, PDGF, PTH, and PTHrP. A substantial record of scaffold and vehicle development accompanies these regulatory approvals for bone-specific therapeutic applications.

Bone morphogenetic proteins

BMPs comprise a family of over 15 structurally related multifunctional growth factors belonging to the transforming growth factor-beta (TGF-β) super family.¹⁶⁸ Among the many growth factors studied for craniofacial applications, recombinant human BMPs (rhBMPs) have demonstrated the most research activity and highest potential in human clinical trials.¹⁶⁹ Specific to bone formation, BMPs are generally considered to be highly potent stimulators of de novo osteogenesis, driving many processes in the bone formation continuum from mesenchymal stem cell (MSC) migration to osteoblast differentiation.^170–174 Inclusion of BMPs in palatal scaffolds is proposed to provide a platform for localized growth factor release to improve the rate and reliability of clinical palatal closure. Delivery of these potent osteogenic growth factors must be highly targeted, however, as BMPs are not bone tissue specific and exhibit off-target and often adverse effects when their delivery solely intends bone regeneration. BMP receptors exist on all mammalian cells, so their unmitigated release may lead to ectopic bone formation and other adverse effects.^{170,175–177}

rhBMP-2, -4, and -7 have been evaluated for use in cleft palate, with rhBMP-2 and rhBMP-7 available as commercially available human products indicated to improve local bone regeneration. Clinical studies have demonstrated that BMPs are, at least, equivalent in terms of bone quantity produced as autologous bone grafts for the repair of alveolar cleft.^{106,171,178,179} However, controversy exists over the specific quality of bone produced through BMP-driven intervention.^175,180

Bone morphogenetic protein-2

Preclinical models of palatal repair with adjunctive BMP-2 have been reported to demonstrate successful in vitro osteogenic differentiation of palate connective tissue cells derived from a TGF-β3 murine cleft model.¹⁸¹ In a canine cleft palate model, the cleft margins of 6-week-old pups were treated subperiosteally with an injectable hyaluronic acid-based hydrogel scaffold composite containing hydroxyapatite and BMP-2 4 weeks before medial mucosal edge removal and subsequent adhesion. Compared with conventional palatoplasty, this minimally invasive canine approach resulted in improved postoperative healing and radiographic indications of palatal bone growth.¹¹¹

The commercially available human spine repair Infuse™ system comprises rhBMP-2 on an absorbable collagen sponge (ACS) scaffold. Infuse is currently FDA-approved for sinus augmentation and extraction socket-associated localized alveolar ridge augmentations, but has been employed in a range of off-label maxillofacial procedures, including alveolar cleft repairs in children.¹⁸² Recent meta-analyses of alveolar ridge donation have found autologous bone and rhBMP-2 grafts to be similarly effective in maxillary alveolar reconstruction.^62,183 There was no statistically significant difference in bone formation in patients with unilateral cleft lip and palate after a 1-year follow-up, neither was the difference in bone height between the two treatment groups with 6- or 12-month follow-up.⁶² However, rhBMP-2 grafting was associated with an average of 1-day shorter hospital stays, presumably due to reduced morbidity through obviation of bone harvesting.

Although several cases of adverse events at the site of implant are reported,¹⁸⁴ intraoral infection has been reported to occur less frequently with the Infuse system than with iliac crest bone grafting, as it is presumed that particulate autologous bone serves as an environment more conducive to microbial colonization and growth on exposure to pathogens during intraoral transit.¹⁷³ Moreover, despite the considerable scaffold cost, cost savings associated with a procedural shortening by 102 min offsets the implant product expense.¹⁷³ One main drawback to the Infuse system is that the efficacy of rhBMPs/ACS scaffold is reduced by the rhBMP-2 burst release mechanism, yielding short-term supraphysiological doses, rapid agent clearance, and inefficient long-term growth factor dissemination into tissues.¹⁸⁴ Delivery through degradable polymeric systems and other scaffolds has been proposed to overcome this burst delivery and allow for more controlled release.¹⁸³ An alternative hydrogel scaffold system was subsequently proposed to reduce the dose of rhBMP-2 required to achieve therapeutic effects, and this system was found to increase bone formation relative to the ACS scaffold.¹⁸⁵ In vitro and in vivo studies using rhBMP-2 delivered in bone grafts have shown the production of mature, vascularized bone.¹⁸⁶

Bone morphogenetic protein-7

A recent 10-year follow-up study evidenced the safe use of BMP-7 for the reconstruction of alveolar clefts (nine unilateral and two bilateral) through the commercial OP-1 system, consisting of 3.5 mg of rhBMP-7 on a type I bovine collagen carrier.¹⁸⁷ In both unilateral and bilateral clefting cases in this study, rhBMP-7 was shown radiographically and clinically to be successful in regenerating bone at the alveolar cleft. Thus, operation time, donor-site morbidity, and hospital stays were reduced. That rhBMP-7 will continue to be used in bone grafts for reconstructing alveolar cleft defects remains to be seen, but studies continue.

Platelet-derived growth factors

Recombinant human platelet-derived growth factors (rhPDGFs) include four isoforms with dimeric structure primarily secreted from alpha granules of circulating platelets. The developmental roles of PDGF isoforms -AA, -AB, and -BB have been well delineated as key potentiators in the morphogenesis of cranial and cardiac neural crest, lung, intestine, skeleton, and blood vessels.¹⁸⁸ Furthermore, of these isoforms, rhPDGF-BB has been the most highly investigated in its role as a propagator of wound healing.¹⁸⁹ The initial evidence establishing PDGF as a key potentiator of bone repair came in 1989, and again in 1991, from Lynch et al. through their investigation of PDGFs role in periodontal defect regeneration in a canine animal model.^190,191 There is substantial preclinical and clinical supporting evidence for rhPDGF use in the treatment of periodontal bony defects, largely supported by the mitogenic and chemotactic activity of rhPDGF to stimulate periodontal and periosseous implant bone regeneration through direct receptor activation of fibroblasts, osteoblasts, bone marrow-derived stem cells (BMSCs), and pericytes.^192,193 Approved as a biological agent by the FDA in 2005 specifically for use in treating osseous periodontal defects and associated gingival recession,¹⁹⁴ clinical trials have demonstrated significant improvement in periodontal ligament attachment gain as well as alveolar bone fill following purified rhPDGF therapy with a bone substitute osteoconductive matrix carrier (β-tricalcium phosphate).^195,196

Larger randomized clinical trials investigating use of rhPDGF in a variety of osteoinductive and osteoconductive matrix carriers (e.g., mineralized or demineralized freeze-dried bone allograft, an organic bone xenograft) have added evidence for both safety and efficacy of rhPDGF to improve oral wound healing, osteogenesis, and bony defect resolution.^196–198 Clinical outcomes have demonstrated long-term follow-up of treated patients who consistently retain the observed structural and functional improvements in bone defects following rhPDGF therapy.¹⁹⁹

A recent meta-analysis of 63 clinical studies utilizing rhPDGF in oral regeneration was performed by Tavelli et al.¹⁸⁷ The authors assessed use of rhPDGF in combination with a wide range of osteogenic matrices, such as allografts, xenografts, and alloplastic biomaterials for periodontal regeneration, gingival bone recession, and alveolar bone regeneration as safe and efficacious, stating strong evidence in support of its intrabony regenerative outcomes in these tissues. Collective clinical outcomes now being increasingly generated for rhPDGF in oral bone regeneration show promise for the applicability of this biological therapy in cleft palate surgical correction as a potential augmentative intervention to promote bone growth and wound healing.

Vascular endothelial growth factor

Since poorly developed vasculature hinders intramembranous ossification in maxillary and palatal tissues, the vascular endothelial growth factor (VEGF) pathway that promotes angiogenesis²⁰⁰ is of critical importance when considering improvements with local growth factor delivery from scaffolds. The absence of cranial neural crest cell-derived VEGF causes a murine cleft phenotype reminiscent of DiGeorge syndrome (e.g., submucous, occult).²⁰¹ In conditional VEGFa knockout mice, cellular proliferation and vascularization is reduced within palatal shelves, which do not elongate or elevate and fail to fuse.²⁰¹ Normal ossification can be induced in primary cell cultures of palatal mesenchyme from VEGFa-knockout mice following BMP-2 stimulation, which implicates reduced BMP-2 downstream of VEGFa signaling in defective osteogenesis.²⁰¹ Furthermore, Duan et al. in 2016 identified that VEGF stimulates intramembranous bone formation during craniofacial skeletal development, specifically modulating mechanisms upstream of BMP-dependent specification and expansion of mandibular mesenchyme.²⁰² However, it is important to note that the mandibular and maxillary blood supply differ substantially, which may impact the relative dosage requirement for effective generation of new bone. It follows that adequate healing at the site of bone grafts requires sufficient VEGF-mediated neovascularization to be orchestrated and maintained.

Consistent with this role, VEGF has been described as the “master regulator” of vascular growth, required for effective coupling of angiogenesis and osteogenesis during both skeletal development and postnatal bone repair. As with many other osteogenic factors, this is achieved through sustained release of VEGF over time,²⁰⁰ rather than through transient release of high concentrations of effector molecules demonstrated to be critically important to driving both angiogenesis and osteogenesis.²⁰³ Thus, reliably mimicking complex in vivo dynamics through local VEGF delivery within a confined orofacial cleft defect poses a unique challenge in creating vasculature that could potentially support a regenerative tissue construct known to be intrinsically deficient in neovascularization.

Some evidence shows that VEGF applied locally at the site of bone healing may be an effective localized therapeutic adjunct to promote human bone repair.^204–206 Delivery of VEGF RNA and recombinant protein has been found to increase both osteogenesis and intraosseous angiogenesis.²⁰³ VEGF RNA transfection of osteoblast-like cells increased angiogenesis of the chorioallantois membrane in ovo. Intraosseous vascular branching was also stimulated in murine calvariae in the presence of VEGF RNA. Recombinant human VEGF165 protein (rhVEGF165) delivery enhanced osteogenesis as demonstrated by increased cell proliferation and calvarial bone thickness in vitro and in vivo, respectively. As VEGF interacts with multiple signaling cascades to promote bone healing in vivo, Zhang et al. sought to investigate the effects of VEGF as a stand-alone intervention and in association with BMP-2 activity.²⁰⁷ Thus, while each agent was insufficient to promote adequate bone regeneration individually, they functioned synergistically to enhance bone density and volume. A postnatal approach to promote osteogenesis may therefore lie in the combinatorial effect of VEGF with other proangiogenic and osteogenic agents in regenerating bone tissue. As locally delivered BMP and VEGF dosing, pharmacokinetics, and local bioactivities are likely spatiotemporally distinct, and perhaps synergistic, rational designs for scaffolds that coformulate both growth factors for local delivery in a bone formation context remain undefined.

Fibroblast growth factors

The osteogenic potential of fibroblast growth factor (FGF) isoforms and associated receptors has been extensively researched in rodents and nonhuman primates. FGF-2 in particular has been found to stimulate proliferation of periosteal cells, osteoprogenitors, and chondrogenitors, resulting in bony callus formation during bone healing.^208,209 Human stem cells from exfoliated deciduous teeth (SHEDs) seeded onto dense collagen hydrogels, primed with FGF-2 and exposed to hypoxic conditions before implantation, improved intramembranous bone formation within a critical-sized calvarial defect in an immunodeficient mouse model.²¹⁰

However, some have raised concern over applications of FGFs in fracture healing and bone repair, given the lack of evidence for enhanced bone mineral density or mechanical strength of the FGF-induced callus.²⁰⁹ This is likely attributable to FGF's biphasic effect on bone formation, with inhibitory effects present at high doses.^208,209 Despite further speculation regarding the efficacy of rhFGF-2 in accelerating bony repair, however, modulating FGF signaling during the formation of the craniofacial complex is thought to be a local therapeutic option worth pursuing in future studies.²¹¹ No FGF delivery specific to palate repair is reported.

Insulin-like growth factors

Insulin-like growth factors (IGFs) are important in the function of almost every organ in the body.^212,213 IGF-I and IGF-II are needed at various phases of development to complete embryonic formation. Interestingly, the overproduction of IGF-II is associated with perinatal lethality and frequently also with cleft palate.²¹⁴ Prenatal IGF-IIR agonist administration to IGF-II-null mice demonstrated a reduction of cleft palate and improved survival, invoking a key role in palatal closure.²¹⁵ In humans, IGF-II has been administered in concentrated growth factor (CGF) trials as an endogenous component of human platelets (e.g., platelet-rich plasma [PRP]; platelet-rich fibrin [PRF]) for the treatment of alveolar cleft.²¹⁶ Centrifuge-mediated collection of autologous CGFs preceded the growth factor layer incorporation into a biomimetic cell sheet applied topically after alveolar bone grafting. Results showed similar spatial formation of bone within the cleft, with the addition of harvested CGFs driving a higher bone density compared with a cellular dermal matrix. This limited work highlights the potential role of IGF-II in regulating palatal closure.

Platelet-rich plasma and PRF

PRP is the plasma fraction of autologous blood having a platelet concentration above blood baseline values (by FDA regulations PRP must contain at least 250,000 platelets per microliter). Platelet granules contain a broad portfolio of growth factors that are released during wound hemostasis to attract cells to wound sites (e.g., platelet alpha granule PDGF, IGF-I, EGF, and TGF-β). These PRP granule-stored components have powerful bioactivities to limit inflammation, improve tissue healing and regeneration, promote new capillary growth, and improve host defense through the immediate recruitment of neutrophils and macrophages, fibroblasts, and endothelial cells.²¹⁷ Also, PRP preparations contain a small number of leukocytes that synthesize interleukins (e.g., IL-10) as part of a nonspecific immune response and have demonstrated antimicrobial activity against several Gram-negative and Gram-positive bacterial and fungal pathogens.²¹⁸

PRP naturally contains many endogenous growth factors described in this review, including PDGF, VEGF, and TGF-β.²¹⁹ Nonetheless, no potency standard exists for PRP and, similar to DMB, it is recognized that PRP donor sources vary widely in levels of the myriad bioactive molecules present in clinical PRP preparations for therapeutic use.^220,221 Final concentration of platelets, leukocytes, and growth factors in the >40 commercial PRP preparation systems now available varies between and within those different techniques, as well as between and within patients.²²¹ Hence, the different PRP preparation protocols and human donor sources yield PRP products with varying compositions, components, potencies, and properties. Additionally, FDA approval of PRP as a unique biologic allows PRP to be used for diversely different orthopedic indications. However, this regulatory clearance is not regarded as approval for any specific indication. Therefore, and strangely, most PRP treatments offered for musculoskeletal indications are considered “off-label” use.²²²

PRP use exposes developing tissues to a potent array of different endogenous growth factors, possibly driving healing responses that are closer to those observed during normal palatal fusion in vivo. In the context of soft tissue healing, growth factors from PRP influence wound repair through different mechanisms: (1) rPDGF-BB accelerates deposition of provisional wound matrix; (2) rTGF-β1 accelerates deposition and maturation of collagen; and (3) rbFGF induces a profound monocellular angiogenic response, which may lead to a marked delay in wound maturation, and the possible arrest of the normal signals required to stop repair.²²³ Hence PRP's therapeutic effects in the palate repair context might be different in soft and hard tissue repair sites.

PRP-derived growth factors have also been used in combination with other regenerative media, such as human stem cells and regenerative scaffolds. For example, rhPDGF was used in conjunction with human MSCs on four human alveolar defects.¹⁴⁴ Cell-enriched scaffolds were infused with PDGF and placed in human anterior maxillary cleft defects and closed with lateral advancement gingival flaps. Postoperative cleft bone volume was assessed 3 months postoperatively using cone beam CT scans. These scans demonstrated that this technique is comparable to autologous bone repair with an average defect fill of 51.3%.

As a highly heterogeneous, complex biological mixture with a complex regulatory control based on commercial preparation methods, PRP currently has no standard compositional definition or potency, with primarily off-label clinical use. Attempts to understand its equivocal clinical efficacy in human bone formation are hampered by this lack of standardization and adequately powered clinical trials.²²⁴ Standardization and definition of PRP biological potency and more consistent focus on clinical trial designs may improve outcomes assessment of its efficacy in tissue repair strategies.

Distinct from PRP, PRF is a second-generation autologous platelet preparation²²⁵ and an emerging potentiator of bone formation in the secondary palate. PRF applications have moved toward simultaneous hard and soft tissue engineering in conjunction with MSCs and other alloplastic tissue constructs in the maxillofacial region (e.g., alveolar bone, mandible, calvarium).^99,226 PRF is composed of endogenous procoagulant agents (e.g., thrombin) in addition to calcium chloride (also present in PRP), leukocytes, and glycoproteins. Interestingly, PRF has not been shown to affect the expression of the RUNX2 (runt-related transcription factor 2), BMP-2, or RANKL (receptor activator of nuclear factor kappa-Β ligand), but rather, induces the expression of osteoprotegrin (OPG), leading to the increase of OPG/RANKL, and suggesting that PRF could boost osteoblastic differentiation.²²⁷

As a different platelet concentrate, PRF shows distinct advantages in the clinical arena by way of its ease of preparation, application, and chemical stability. Furthermore, time-release studies of PRF's effect on tissue healing have identified a time-dependent biological effect prolonging its active phase in vivo, particularly when PRF is coupled with scaffold-based delivery systems in periodontal regeneration.²²⁸ Endothelial cell migration is enhanced in the presence of PRF, allowing for formation of cellular networks essential for neoangiogenesis, neurogenesis, vascularization, and graft retention in bone healing to occur at the site of repair.²²⁹ The relative efficacy of a bioceramic bone substitute combined with PRF is presently being investigated in alveolar cleft repair in comparison to outcomes with conventional autogenous iliac grafts.²²⁹

Transforming growth factor-β

Of the three TGF-β isoforms, TGF-β3 plays the greatest role in the developing palate. TGF-β3 alone affects palatal epithelial/mesenchymal transformation and its action cannot be replaced by other TGF-β isoforms.²³⁰ TGF-β3 (10 ng/mL) induced complete palatal fusion, showing near 100% recovery within 72 h in culture. In addition to cleft palate, TGF-β3 supplementation has induced human cleft lip fusion and has been used in surgical repair to promote scarless cleft lip repair,^231,232 and enhanced recovery postsuturectomy in craniosynostotic rabbit models.²³³

Other Bone-Generating Adjuvants

Maternal folic acid deficiencies during the embryonic period can be a significant cause of clefting.²³⁴ Salt derivatives of folic acid using bioactive cations (i.e., Sr²⁺ or Zn²⁺) show potential to accelerate bone growth, as well as reduce inflammatory cascades that can impede wound healing.²³⁴ Present studies are underway to determine the viability of applying strontium folate derivatives in combination with biomimetic scaffolds for bone regeneration of palatal defects.¹⁴⁰ Bisphosphonates (BPs) and other clinically used antiresorptive drugs have been applied to treat skeletal disorders and other conditions associated with osteoclast-mediated bone resorption (i.e., osteoporosis and fibrous dysplasia).²³⁵ Although challenges exist with consistent effects of BPs in adults (e.g., suboptimal quality of induced bone formation), the pediatric patient population has benefited from their use. Greater BP efficacy in the pediatric population has been associated with greater degree of bone turnover in the pediatric alveolar environment compared with adults.²³⁶ With respect to bone grafting and BP use, there appears to be a greater clinical efficacy of bone retention. Hong et al. evaluated volume of bone in a rat cleft palate model using both local and systemic administration of zoledronic acid.²³⁶ Local delivery of zoledronic acid, clnically used in osteoporosis treatement, demonstrated heightened efficacy in reduced graft resorption with greater bony integration. The authors' caution, however, was that zoledronic acid has known adverse systemic effects in the adult human database, thus dose-dependent toxicology studies must first validate its use in humans in cleft repair. In addition, dental concerns exist from studies in the pediatric population with respect to tooth eruption patterns and orthodontic tooth movement.^237,238 Future studies must examine these risk factors in pediatric applications for palate repair.

Stem Cell Therapy

Many cell-based therapies to regenerate bone utilizing MSCs and MSC-like populations have been evaluated over the past several decades as potential regenerative interventions,²³⁹ particularly in bone tissue regeneration, as MSCs are known to differentiate into osteoblasts under certain conditions.²⁴⁰ In contrast to the invasive nature of autologous bone harvesting for grafting, autologous stem cells can be harvested by minimally invasive procedures through needle aspiration with a reduced risk of complications at the donor site. MSCs maintain the potential to differentiate into osteocytes, chondrocytes, as well as adipocytes depending on the growth factor selection in the microenvironment, and have been found to modulate immune response and to promote tissue regeneration.²⁴¹ Furthermore, MSCs can be isolated from many tissues, with bone marrow and adipose tissue being the most common adult sources in clinical practice. Other MSC/MSC-like cell populations can be extracted from a variety of organ types, including skin, umbilical cord, pancreas, heart, brain, lung, kidney, cartilage, tendon, and teeth.^242,243

Bone marrow-derived stem cells

BMSCs are considered a “gold standard” for bone tissue engineering.²³⁹ Compared with other MSCs, BMSCs demonstrate superior osteogenic and chondrogenic differentiation potential.²⁴¹ As effective contributors to bone repair processes, BMSC activities and commercially available DBM (e.g., Osteoset™ DBM) have been compared.²⁴⁴ Recently, a study using a collagen scaffold seeded with autologous BMSCs, PRF, and nanohydroxyapatite showed enhanced healing and reduced postoperative pain compared with the traditional method of cleft palate repair using iliac bone autografting.⁹⁹ A current randomized clinical trial is investigating bone formation arising from unilateral alveolar clefts with BMSCs seeded on collagen matrix (Osteovit™) compared with iliac crest autograft.

Adipose-derived stem cells

Adipose-derived stem cells (ADSCs) are similar to BMSCs in their surface marker profiles, differentiation potential, and growth properties, but have several qualities that make them a promising progenitor cell alternative to BMSCs and/or traditional bone grafts. Compared with other mesenchymal sources, ADSCs have a high stem cell-to-volume ratio, stem cell frequency is far less sensitive to aging, harvesting can easily be up scaled according to need, and can be processed within a short time frame to obtain highly enriched populations (residing in the stromal vascular fraction).²⁴⁵

Growing evidence supports ADSCs as optimal candidates for CL/P repair, given their sourcing accessibility, broad ability to differentiate into various tissue types and their added capacity to expand in vivo.²⁴⁶ In a preclinical in vivo investigation, Pourebrahim et al. applied undifferentiated ADSCs to maxillary alveolar cleft bony defects through vehicular delivery with biphasic bone substitutes, including hydroxyapatite–tricalcium phosphate scaffolds.²⁴⁶ The authors concluded that this delivery system offered comparable bone regeneration at the site of repair to autologous tissue transfer, and may be a preferable alternative to autografts given the lack of patient donor-site morbidity and operative time. Furthermore, Conejero et al. found substantial bone formation with PLLA scaffolds seeded with osteogenically differentiated ADSCs implanted into rats.¹⁴³ Blanco-Elices et al. generated bilaminar fibrin–agarose hydrogels immersed in tranexamic acid-supplemented human plasma containing cultured ADSCs and palate-derived oral mucosa fibroblasts in an attempt to reproduce palatal hard and soft tissues in rabbits.²⁴⁷ In vivo cell and tissue differentiation were sufficient to regenerate the palatal tissues, however, not to equivalent structural thickness as wild-type controls, following 4 weeks of observation.

Novel culture methods have also been examined for delivery packaging and differentiating of human stem cells for palatal bone regeneration, including spheroid delivery systems to optimize cell colonization in scaffold materials in vivo.²⁴⁸ Major limitations in clinical MSC translation include their lack of standard potency markers and consensus on stem cell potency, required in vitro cell expansion for therapeutic dosing with risk of phenotypic loss, donor-related heterogeneity in MSC quality, and lack of standardized procedures for cell culture, handling, and storage. An additional ethical consideration in the context of neonatal or pediatric cleft repair is the autologous cell sourcing as a necessity. Nonautologous stem cell therapy comes with similar risks as solid organ transplantation, such as graft-versus-host disease, viral contamination, graft rejection, and graft failure.²⁴⁹ Thus, increasing focus on exploiting allogeneic and banked human MSCs in cell therapy applications produces numerous advantages to cell-based clinical products, but may not be readily accepted as a clinical solution for children without substantial evidence for safety and efficacy. Nonetheless, while still in the initial stages of investigation, these MSC data are promising for the implementation of MSC adjunctive therapies to induce osteogenesis in cleft defects.

Tooth-derived stem cells

Within dental tissues, five main classes of MSC populations exist, including: (1) dental follicle progenitor stem cells; (2) stem cells from apical papilla; (3) periodontal ligament stem cells; (4) dental pulp stem cells (DPSCs); and (5) SHEDs.²⁵⁰ Among these different stem cell populations, SHEDs (known to have exfoliative properties) are the most easily isolated and extracted from the odontogenic tissues through minimally invasive techniques. Dental pulp tissues used for pediatric regenerative interventions are typically obtained during childhood between years 5 and 12 of age.^251,252 SHEDs in particular demonstrate high levels of proliferation, as well as multilineage differentiation capacity and immune-modulating cytokine signaling propagation.²⁵³ Similarly, DPSCs (typically more easily isolated during teenage into adulthood years) can be harvested from third molar tissues during routine tooth extraction.²⁴⁴ Additionally, both SHEDs and DPSCs exhibit potential for generating cell sheets as well as 3D spheroid cell delivery systems for local applications to palatal tissues.^141,255 Both cell types are highly concentrated with secretomes (e.g., soluble paracrine signaling molecules) to allow for their immunomodulatory, angiogenic, and neurogenic activities in vivo.²⁵⁶ The secretome profile of SHEDs specifically shows modulatory activity during differentiation toward osteogenic lineages, in turn leading to an increase in vasculogenic activity.²⁵⁷ Furthermore, regarding osteogenic potential, SHEDs have been shown to effectively form new calvarial bone in critical-sized defects with greater capacity compared with other dental tissue-derived stem cells. When seeded within dense collagen hydrogels primed with FGF-2 in hypoxic environments before implantation, the immunodeficient calvarium demonstrated increased intramembranous bone generation.²⁵⁸

Much interest is shown currently in deciphering the clinical applicability of dental tissue-derived stem cells in bone regeneration of the craniomaxillofacial skeleton. A recent comparison of the bone regeneration ability between SHEDs, human-derived dental pulp stem cells (hDPSC), and human BMSCs concluded that there are no observable differences between these cell populations in regenerative capacity.¹⁴² However, the authors suggest that SHEDs are more easily isolated clinically and, thus, may represent the most valuable source of MSC for palatal bone repair. Another study, although not specifically regarding palate bone, by de Mendonca Costa et al. evaluated the efficacy of hDPSCs in reconstruction of cranial bone defects in a nonimmunosuppressed rat model.²⁵⁹ hDPSCs were characterized in vitro as mesenchymal cells and were evaluated for cell markers that showed osteogenic, adipogenic, and myogenic differentiation, then integrated within a collagen scaffold and evaluated alongside a collagen-only group. After 1 month, both groups showed signs of bone formation. However, rats that received the collagen scaffold seeded with hDPSCs demonstrated more mature bone. Another significant finding of this study was the lack of observable graft rejection despite the use of nonimmunosuppressed rats, suggesting that hDPSCs are another potential cell source for correcting cranial defects. Yet another study used hDPSCs alongside a silk fibroin scaffold and human amniotic fluid stem cells (hAFSC) to repair cranial bone defects in immunocompromised rats.²⁶⁰ This study demonstrated that the hAFSC-seeded scaffold produced higher bone formation than the fibroin scaffold alone just after 4 weeks postoperatively. Finally, Jahanbin et al. tested the effectiveness of hDPSCs on maxillary alveolar defects in rats with a collagen matrix against iliac bone graft transplantation⁹⁷; new bone formation was evaluated 1 and 2 months after surgery. Results of the study showed maximal new bone formation in the iliac bone graft group after 2 months. However, hDPSCs did show significant bone formation after 2 months compared with the collagen scaffold control group.⁹⁷ Importantly, while the results of this study show promising initial bone formation in the time frame specified, evaluation of bone formation is typically extended to at least 6 months following intervention. Thus, these results should be scrutinized with caution, and followed by longer-term analysis of postintervention outcomes.

As more preclinical success in cell-based therapy for palatal bone regeneration is established, future expansion of longitudinal human clinical trials will be paramount to the continued investigation and essential clinical validation of the tissue engineering triad: stem cells, scaffolds, and growth factors. As discussed above, a diverse spectrum of scaffolds combined with many types of stem cells and/or myriad supportive growth factors show encouraging results of bone formation and structural success with respect to regenerated biomechanics and anatomy.²⁶¹ These promising, emerging approaches to palatal bone tissue engineering will continue to change the constantly evolving platform of possible therapeutic interventions to benefit orofacial cleft repair.

Challenges, Perspectives, and Future Directions

Osseous tissue forms by way of two distinct developmental processes: (1) endochondral ossification and (2) intramembranous ossification. Each of these unique pathways contain independent inducing factors for the promotion of bone and supporting matrix development.²⁰⁴ For example, calvarial and craniomaxillofacial bone biology (largely cancellous, spongy bone with higher vascularity than cortical long bone) is distinct from largely dense cortical and some distal/proximal trabecular long bone. Given these innate differences in structural integrity and content, it has been proposed that clinical replacement or regeneration of bone is maximized when induced by a scaffold system (e.g., DBM) mimicking the in vivo structure.²⁶² However, this rationale directly conflicts with the current “gold standard” iliac crest bone graft for correction of bony defects in the palate. The iliac crest bone structure coincides more with trabecular (endochondral) bone, while the palate is similar to other craniomaxillofacial bone structures in its cancellous, spongy (intramembranous) nature.²⁶³

Clinical effects of BMPs and other osteogenic growth factors discussed above are distinct based on bone biology (i.e., much more successful in spongy bone of the craniomaxillofacial skeleton and spine as compared with long cortical bone in the extremities).²⁶⁴ Therefore, it is important to note that palate repair using known agents/scaffolds studied in the context of long bone regeneration may prove to be different than anticipated on the basis of fundamental anatomical and physiological differences between palate versus long bone. Bone biology in each context must be better understood to guide rational approaches to reliable regeneration.

Beyond the internal complexity of intramembranous palatal bone formation, the dynamic biomechanics of orofacial development must be addressed in the specific context of cleft repair and very young patient profiles. With an initial downward vertical trajectory of migration and growth, the developing palatal shelves are largely impacted by the simultaneous development of surrounding structures such as the tongue, maxilla, mandible, nasal septum, and facial soft tissues.²⁶⁵ Midfacial growth is often negatively impacted as a result of failure of the palatal shelves to reorient horizontally (as the tongue migrates cranially between the initial palatal shelf outgrowths) and fuse at the midline.²⁶⁶ Given this multifaceted, dynamic developmental system in the craniofacial complex, one must understand the downstream structural and functional consequences of timed intervention on concurrently growing tissues in any palate repair strategy. As discussed above, closure of palatal cleft defects is carefully timed to maximize midfacial development before imposing a coronal “barrier” to the posteroanterior development of the maxilla. Thus, future solutions proposed for orofacial cleft repair must account not only for the unique internal matrix structure of the palate bone, but also for the intricate developing anatomy surrounding the palate to optimize pan-facial treatment.

Progress in research on cleft repair protocols will likely involve fabricating biomaterials matched with 3D rapid prototyping to produce individualized solutions for CL/P scaffolds in patient-specific environments. Factors include complex hard/soft tissue integration, dynamic scaffold responses to the evolving cleft physiological and mechanical environment, and consistent rapid tissue sealing critical to this specific site to prevent complications and failures. Additionally, controlled delivery of recombinant human growth factors from these finely tuned 3D-printed scaffolds could involve transplanted and repair-primed progenitor cells that are tailored for palatal shelf closure (Fig. 4). Ultimately, a clear design hypothesis must emerge to drive CLP repair strategies based on emerging biological and genetic mechanisms for these hard and soft tissue defects. The empirical approach currently driving many repair strategies in both humans and animal cleft models has not proven to yield consistent results that point to best practices for any clinical adoption.

FIG. 4.

Postnatal tissue engineering approaches for cleft palate regeneration. Upon diagnosis of palatal cleft, either prenatally or perinatally, an array of nonautologous regenerative therapies exist to couple osteoinductive growth factors, mesenchymal stem cells, and patient-specific scaffold-based delivery vehicles to the site of implantation. Properly timed within the mixed dentition stage of craniofacial development (∼7–12 years of age), such osteogenic therapies have the capacity to mitigate the need for autologous bone harvesting, decrease operative time, and overall costs, as well as improve surgical outcomes.

Challenges with scaffolds delivering biologics to tissue sites involve little evident rationale for local dosing amounts or release kinetics; combinations delivering multiple growth factors, while seductive to target multiple tissue neogenesis pathways, are even more complicated to coformulate and design for delivery in this regard. Animal models necessary to validate performance for these systems in bone regeneration are notoriously nonpredictive for human translation. Dose–response, potency, and actual tissue regenerative effects are often distinct between preclinical and clinical experiences. Hence, simple allometric scaling of growth factor-delivering scaffolds from preclinical to clinical subjects is a risk.

Stem cells harvested for bone regeneration are heterogeneous, and lack standard preparation, validation, potency, and dosing protocols. Autologous cell harvest, ex vivo cell expansion, documentation for chain-of-custody tracking, and second implant surgeries for therapeutic application requirements (∼10⁸ cells/patient) are costly and impractical, given needs to manipulate the stromal environment and possible unintended alterations to cell source genomics in manipulations.²⁶⁷ Allogeneic, off-the-shelf banked stem cell sources for bone repair remain largely unexplored, equally challenged by potency and product validation standards, and perhaps difficult to regulate use in children. Practically, regulatory pathways for multiple growth factor or growth factor/stem cell dual-loaded scaffolds will prove challenging for clinical trial designs, especially those involving pediatric subjects, and, ultimately, for gaining regulatory approval for any product addressing this compelling need. This scenario then further limits the essential commercial enthusiasm for supporting research and clinical evidence gathering for regulatory approvals of tissue-engineered products in palate repair.

Growth factor use as adjuvants in secondary palate bone tissue engineering, coupled with increasing availability and sophistication of patient-specific implant fabrication technologies, will shift cleft and craniofacial care toward more personalized and individualized approaches, following a current trend in many other disciplines.²⁶⁸ While custom scaffolds have thus far been developed from CT or MRI scans, generating 3D models of palatal and alveolar clefts with more sophisticated optical intraoral imaging techniques²⁶⁹ may mitigate the exposure of children to radiation in designing 3D-printed scaffolds in the future. Additionally, genome-wide association studies could serve to identify prenatal development pathways, genetic risk factors, and maternal/fetal health quality signatures or biomarkers that indicate CP risk. This information could serve to guide, restore, or produce individualized surgically corrective solutions associated with defective bone signaling pathways in utero to reliably produce tissue in CPs in childhood interventions.

In summary, continued development of optimized scaffold biomaterials in conjunction with sophisticated methods for formulating and locally delivering pharmacotherapeutic and biological agents should provide improved capabilities to regenerate tissues required to repair CP defects. Ultimately, more widely accessible, reliable, cost-effective, and improved surgical outcomes are sought to streamline postnatal cleft management. This progress will, in turn, serve to alter the current paradigm of multiple invasive surgical interventions occurring over many years of early childhood, substantially improving the quality of life for many young patients affected with orofacial clefting.

Disclosure Statement

No competing financial interests exist.

Funding Information

This work was supported by the National Institute of Health/National Institute of Dental and Craniofacial Research grants DE019471 and DE027255 (Rena N. D'Souza) and supported in part by the American Cleft Palate-Craniofacial Association (Shihai Jia) and PhD degree support from the Department of Biomedical Engineering, University of Utah, and the Ole and Marty Jensen Endowment Fund to Jeremie D. Oliver.