Challenges Associated with Regeneration of Orbital Floor Bone

Abstract

Orbital floor fractures are a serious consequence of craniofacial trauma and account for ∼60%–70% of all orbital fractures. Unfortunately, the body's natural response to orbital floor defects generally may not restore proper function and facial aesthetics, which is complicated by the thin bone and adjacent sinuses. Current clinical treatments include alloplastic implants and autologous grafts; however, each has associated disadvantages and sequelae. This review has outlined necessary components for a successful tissue-engineered construct for orbital floor repair. In addition, current successes and progress in the literature specific to orbital floors and craniofacial research have been reviewed. Finally, challenges and future directions have been described.

Introduction

Orbital bone development and anatomy

The orbital region is central to the functional success and aesthetic features of the facial skeleton.¹ Initially, the ocular globes, as the growth center, grow rapidly, causing an increase in the orbits. The growth of this bony structure is 85% complete by 5 years and is finalized between 7 years of age and puberty.^2,3 The main purpose of the floor is to support the globe and separate the orbital contents from the maxillary sinus; it is therefore very thin, ∼0.5 mm.^2,4 It is composed of portions of three bones: the maxilla, zygomatic, and palantine.^4,5 The floor is not completely horizontal, but has a slight convex curve where the posteromedial section is higher than the flatter anterolateral area.⁵ The orbital floor is also lined on the ocular side by periosteum.^2,6 In addition, when compared to the other orbital walls, the orbital floor demonstrates the highest degree of deformation with static loading, possibly explaining the high number of orbital fractures associated with blunt trauma.⁴

Orbital floor injuries and mechanisms

Orbital floor injuries, most commonly caused by assault and traffic accidents, can be a devastating form of craniofacial trauma.^7,8 Studies have shown that the floor is the wall most frequently involved in orbital trauma, accounting for ∼60%–70% of all orbital fractures (see Fig. 1).^9,10 As the floor is continuous with the thin medial wall, it may also act as a natural crumple zone and be involved in significant orbital traumas. If left untreated, a fractured orbital floor will likely not provide adequate support to the globe, despite standard primary and secondary bone healing mechanisms. The thin bone fragments often paired with injured periosteum, and disrupted blood supply provides a poor conduit for bone healing. Further, there is generally insufficient contact with surrounding bony edges to conduct bone formation, which leads to fibrous scar formation. This change in orbital architecture provides inadequate globe support and increased orbital volume and, as a result, altered globe function. There are instances where orbital floor fracture treatment can be delayed or possibly avoided, such as blowout fractures, to allow improvement of motility and diplopia due to absorption of blood, edema, new connective tissue septa formation, and the adjustment of the binocular fusion mechanism.¹¹ In addition, assessment of whether a surgical approach is necessary can be determined using computed tomography to evaluate scar tissue and surrounding muscle entrapment.¹² However, in general, the endogenous response to orbital fractures, in contrast to many other bone fractures, is not sufficient for proper healing.

FIG. 1.

Computed tomography scan of an orbital fracture. Both orbital floor and orbital roof fractures are present. Notice the relative thinness of the orbital floor (∼0.5 mm), even when compared to other craniofacial bones. Also, notice the lack of bony tissue surrounding the orbit and, in particular, the sinus inferior to the orbital floor.

The majority of orbital floor injuries occur from trauma, and there are two main biomechanical mechanisms proposed to explain how orbital floor defects occur. The hydraulic theory suggests that force applied to the globe results in increased intraorbital hydraulic pressure and transmission of this pressure to the walls of the orbit results in the fracture at the weakest point, which is generally the thin orbital floor.^4,13,14 The buckling theory offers that trauma to the infraorbital rim transmits force directly to the orbital floor, causing disruption of the bone without fracture of the rim and displacement of orbital contents.^4,13,14

Current clinical solutions for orbital bone defects

To treat a clinically significant orbital floor fracture, it is critical to restore the orbit to its original volume to ensure proper globe function.⁴ During surgery the herniated orbital tissues are repositioned into the orbit and an implant is used to span the defect in the floor to prevent reherniation.¹⁵ There are a wide variety of implants available, each associated with very specific advantages and disadvantages; however, literature suggests that ophthalmologists tend toward using alloplastic implants, whereas plastic and craniofacial surgeons tend toward using autologous materials.^8,15 The decision for which type of implant should take into consideration the size and shape of the defect, the presence or absence of peripheral bony ledges, the age of the patient, donor site issues, and, to a degree, patient preferences.

There are a number of alloplastic implants that are currently in clinical use. Frequently used materials include Medpor (high-density polypropylene), poly(L-lactic acid) or poly(glycolic acid) (PGA), and titanium (see Fig. 2).^4,8,16 The benefits of using alloplastic implants include their ease of availability, they can be adapted to fractures of any defect shape before use, and they are reasonably priced.^8,15 However, these implants are foreign bodies, and after implantation, these relatively inert alloplastic materials may develop a fibrous capsule.¹⁵ Other complications have been described with these implants, including infection, implant migration or extrusion, extraocular muscle entrapment, cyst formation, residual diplopia, globe elevation, and visual loss.^8,15,17 Treatment then involves additional surgery including removing or re-exploring and repositioning the implant.^8,15

FIG. 2.

Orbital repair with a titanium mesh. Alloplastic implants, such as titanium mesh, are easily implantable, provide mechanical support, and can be somewhat malleable. However, these implants are often associated with fibrous encapsulation and migration, resulting in poor globe positioning.

Autologous grafts are frequently used for the repair of orbital floor defects, and a number of donor sites have been employed such as iliac crest, ribs, calvaria, maxillary bone, and the outer cortex of the mandible, and also cartilage donor sites have been described.^8,18–21 The advantages to using autologous grafts include good graft stability, reduced implant associated costs, and limited adverse reactions.⁸ However, there are a number of associated disadvantages, including morbidity of the donor site, increase in surgical time, limited availability, unpredictable resorption, adaptability, and the modeling properties of the graft.^8,17 In addition, there may be significant risk to the donor site. It is vital that the orbit is restored to its original volume to promote proper function and aesthetics. There are a number of variables that account for volume maintenance, including position (inlay vs. onlay), membranous or endochondral, cancellous or cortical, mechanical stress, recipient site, method of fixation, graft orientation, presence or absence of the periosteum, and rate of vascularization.²² However, grafts harvested from calvarial and facial sites tend to resorb less than those from rib, tibia, or iliac crest.²²

There are other grafts that have been used in the past, or are currently in use; however, they are less common. Allogenic implants, such as lyophilized dura mater, were successfully used, until cases involving disease transmission were reported.⁸ New sterilization techniques have shown promise with dura mater implants; however, it is only appropriate for use with small to moderate-sized defects, and long-term outcome studies have not yet been completed.¹⁷ In addition, solvent-preserved cadaveric calvarial bone grafts have been used with some success.¹ These grafts showed implant vascularization and tissue ingrowth; however, studies showing the effects of solvent treatment on graft survival are limited and long-term effects of these implants still need to be studied.¹ Other graft types, including xenografts, specifically swine bone cortex, show good integration into the surrounding tissue; however, they have relatively modest modeling properties.⁸

Sequelae associated with orbital fractures

The endogenous response to bone healing is not adequate for proper regrowth of the orbital floor, resulting in a number of associated problems. In addition, current clinical solutions are not without their share of disadvantages. Therefore, there are a variety of general sequelae that are associated with orbital floor injuries, with the two most frequent complications being enophthalmos and diploplia.^5,23 Enophthalmos is defined as recession of the globe into the orbit compared to the contralateral globe, and this is readily visible at >3 mm.⁸ Enophthalmos is thought to be caused by changes in orbital volume, destruction of restraining ligaments, fat atrophy, and remodeling of the soft tissues into a more round shape.^1,5,8,23 Diplopia (double vision) is thought to be caused from extraocular muscle dysfunction—specifically, entrapment, ischemia, hemorrhage, or nerve injury.²⁴ Other common sequelae include decreased sensation on the injured side of the face (in the distribution area of the infraorbital nerve), and this is shown to be present in more than half of cases in recovery studies.^9,23,24 In addition, unsatisfactory facial aesthetics have been associated with orbital floor fractures.⁹ Proper treatment alleviates sequelae by supporting the orbital contents, preventing soft tissue fibrosis, and restoring continuity of the orbital floor.⁸

Engineering Orbital Bone

Tissue engineering and regenerative medicine have been recently begun to be explored for the treatment of orbital bone defects. In bone tissue engineering in general, cells act as the osteogenic stimulate for the formation of new bone.²² In contrast, specific growth factors and cytokines can act as the osteoinductive stimulation, recruiting and inducing osteoprogenitor cells to grow into mature bone tissue through chemotaxis, mitosis, and differentiation.²² Finally, a scaffold acts as an osteoconductive medium where the scaffold serves as a surface on which the cells can attach, migrate, grow, and divide, and new blood vessels can invade.²²

The key feature in any orbital bone regeneration strategy should be the support of the globe. This support must be immediate, beginning at the time of implantation, and constant. The implant should return the orbit to the original orbital volume to decrease the likelihood of enophthalmos.⁴ However, following a traditional tissue engineering approach, the implant should degrade as the tissue regenerates. This balance of degradation and regeneration is particularly critical, as any significant loss of mechanical support can significantly affect globe position. At this time it is difficult to speculate on specific optimal rate of degradation. An implant should only elicit a moderate immune response and not induce fibrous encapsulation, which can be a common problem with alloplastic implants. Lastly, the implant should be easily handled, molded, and positioned in the floor defect. While orbital floor regeneration encompasses many aspects of bone tissue engineering, this review aims to focus on the areas specific to orbital bone engineering. The following sections briefly discuss the fundamentals of creating a tissue-engineered construct for bone, while areas specific to the orbital floor are discussed in greater depth.

Cell sources for orbital bone regeneration

To regenerate bone tissue an appropriate cell population needs to be delivered or recruited to the injured area. A number of cell types have been used in craniofacial tissue engineering with success. The periosteum is easily harvested and known to contain osteoprogenitor and chondroprogenitor cells.²⁵ Further, these periosteal cells have been shown to express known osteogenic markers in vitro and aid in regeneration of calvarial defects in vivo.²⁶ Calvarial osteoblasts have also been investigated for use in craniofacial regeneration. Studies have demonstrated the expression of osteogenic markers in vitro and the increase of bone formation in vivo when compared to groups without additional cells.^27,28 While some progress has been made with periosteal cells and calvarial osteoblasts, the most widely investigated cell type in craniofacial tissue engineering is mesenchymal stem cells (MSCs). At this time, for orbital bone regeneration the use of bone-marrow-derived MSCs may be among the most reasonable option, as it requires the harvest of tissue that is commonly obtained by the treating clinician.

In the natural healing response to bone fractures, MSCs are recruited to the area and differentiate into osteoblasts through a number of environmental cues.²⁹ MSCs have the ability to replicate as undifferentiated cells and also the potential to differentiate into a number of lineages such as bone, cartilage, adipose, tendon, ligament, and marrow stroma.^30,31 However, to be induced down the osteogenic differentiation pathway, a sufficient and appropriate amount of extracellular signals must be available.²⁹ MSCs are capable of proliferating in vitro, allowing large numbers of cells to be cultured from small harvest amounts.³² Further, studies have demonstrated that transplanted allogenic MSCs did not elicit a significant immune response.³² Lastly, stem cells have been shown to withstand low-oxygen conditions, which may be present after transplantation.³² The ease of isolation and capacity to be induced down the osteogenic differentiation pathway make MSCs an ideal cell type for bone tissue engineering.^33–35 For orbital bone regeneration, a limited number of MSCs may be expected to migrate to the defect site due to the facial anatomy: the orbit is composed of thin bones and is adjacent to the sinuses, which do not have a large cell supply to aid in regeneration. Therefore, orbital bone engineering solutions will likely require the harvesting of cells from the patient or a donor, seeding these cells in an engineered construct, and conducting in vitro culture for cell proliferation before implantation. Guided bone tissue engineering strategies will likely have limited applications in the regeneration of orbital bone.

Key signaling molecules in orbital bone regeneration

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-β (TGF-β) superfamily and are known to be secreted signaling molecules.^36,37 BMPs are present at different periods of growth: the early stages of embryogenesis, during the organogenesis phase and growth period, and in adults during fracture repair.^36,38 Currently, 16 BMPs have been identified, and the most widely investigated for bone tissue engineering are BMP-2, -4, and -7 (also known as osteogenic protein-1).^39–44 BMP-2 and BMP-7 are currently the only BMPs with recombinant human products developed for clinical applications.^32,41,45

The family of BMPs is known to induce formation of cartilage, bone, and other like tissues of the skeleton through recruitment, commitment, and differentiation of osteoprogenitor cells.^36,44 Specifically, BMP-2 through -7 and BMP-9 have demonstrated the ability to induce the differentiation of MSCs into osteoblasts.⁴¹ Further, BMP-2 and -7 have both been shown to have chemotactic effects on osteoblasts and osteoprogenitor cells.⁴⁶ BMP-2 is also known to induce MSC chemotaxis, proliferation, and differentiation.⁴⁷

As related to orbital defects, many studies have demonstrated the use of BMPs in increasing bone formation in maxillofacial defects.³⁹ In addition, when implanted, BMP-2 has demonstrated induction throughout defects and bony healing.³⁶ It has been shown that the amount of BMP required is small relative to the volume of bone it is capable of producing.³⁶ However, while a kilogram of bone contains only a few micrograms of BMPs, milligram doses of BMPs have been shown to be required for efficacy in human models.⁴⁸

Other growth factors are being investigated for use in bone tissue engineering, including TGF-β, insulin-like growth factor I (IGF-I), fibroblast growth factor (FGF), and platelet-derived growth factor.^{29,42,43,49,50} Together, these growth factors aid at the target site by increasing MSC and osteoprogenitor cell number, promoting bone-specific genes responsible for maintaining the osteoblastic phenotype and mineralization, and thus enhancing bone formation.^43,50 Further, they can induce increased expression of the osteoblast-related extracellular matrix molecules.⁴³ Specifically, TGF-β1 is thought to increase differentiation and proliferation in osteoblasts.²⁹ TGF-β1 has also been shown to play a role in bone graft incorporation.⁵¹ IGF-I has demonstrated a chemotactic effect, while increasing proliferation and differentiation of osteoblasts.^29,51 IGF-I is produced by osteoblasts and retained in the extracellular matrix.⁵⁰ Here IGF-1 has been shown to promote cell proliferation and synthesis of type I collagen, and decrease collagenase synthesis to maintain collagen in bone microenvironments.⁵⁰ Further, IGF-I is known to activate osteocalcin expression, a marker for mature bone.⁵⁰ FGF has shown the ability to induce mesenchymal cell mitogenesis, and increase proliferation of osteoblasts.^29,42 However, it is thought to slow the differentiation of osteoprogenitors.²⁹ Additional studies have shown that FGF levels are increased during early stages of fracture healing, and that FGF upregulates osteocalcin expression.⁵⁰ Platelet-derived growth factor is known to increase proliferation of osteoblasts.^29,51 In addition, it may also aid in recruiting bone cells during remodeling and repair.⁵¹

While there has been significant progress made in the field of bone tissue engineering with the use of growth factors, there are a number of concerns to working with these proteins. Specifically, many growth factors have a very short biological half-life, which may be as short as 2 min.⁴⁹ This causes increased concern for the tissue engineer and the ability to deliver the appropriate dose to the target area. Further, BMPs are known to be osteoconductive with a dose–response ratio and act locally.⁴¹ Therefore, it is important to supply above a threshold level of BMP at the target site to induce bone formation. Thus far, in clinical settings recombinant human BMPs have been used at concentrations 10- to 1000-fold higher than those of native BMPs.⁴¹ To deliver BMPs, and ideally reduce the amount of BMP needed, it should be combined with a matrix to allow for slow release and area retention. When combined with a matrix, the BMP-matrix system allows for cell infiltration, retention of BMP at the site, and a substrate for cell growth and differentiation.³⁶ All of these concerns highlight the importance of an adequate delivery system for these growth factors to work.

Scaffolds for engineering orbital bone

Scaffold design is critical to the success of an engineered construct. In orbital bone tissue engineering, scaffolds act as a temporary framework for cells to grow and produce new matrix and functional tissue. The scaffold should be easily modified to fit the defect, whether molded or cut to the defect after fabrication or fabricated in situ. Also, while designing a scaffold, it is necessary to consider how the implant will be secured. Currently, many implants require rigid fixation such as titanium screws; however, implants may also be secured by wire or suture material.^4,15 In addition, as the target tissue is regenerated, the scaffold should degrade to allow space for the new tissue to grow. There are many parameters involved in scaffold design, including polymer composition, biodegradation, biocompatibility, and mechanical strength.

Many strategies have been developed for bone tissue engineering using natural and synthetic polymers. Natural polymers may be advantageous because they are often biocompatible and easily degraded by the body. However, natural polymers tend to have a variable molecular structure. In addition, they generally do not possess adequate mechanical integrity. Synthetic materials have been widely investigated due to their reproducibility in the lab. In addition, these materials can be modified to have desired properties, including mechanical stiffness and degradation by tailoring the fabrication methods. Further, during synthesis, various bioactive molecules can be incorporated into the scaffolds through a number of techniques.

Many of the cells involved in bone tissue engineering are anchorage dependant, and therefore the scaffold should be engineered to aid in cell attachment. Scaffolds that have large accessible surface areas are generally more favorable as cells can attach more easily. Further, the surface has to be carefully designed as to how strongly cells attach. Studies have shown that strong cell adhesion promotes cell proliferation, while a rounded morphology demonstrates their differentiation.⁵² However, the most significant surface property of polymers is the ability to provide an environment for scaffold–host interaction. Advances in polymer synthesis allow for control of the polymer and side-chain architecture. This enables inclusion of functional groups at the surface in addition to within the material. The surface of the polymer can therefore be modified with short peptide sequences or long protein chains to promote interaction with the surrounding tissue.⁵³

The scaffold macrostructure design is important to the success of a tissue construct, as a highly porous scaffold allows cells to integrate into the porous void space. Specifically, it has been shown that human osteoblasts can penetrate pores with a diameter of 20 μm; however, a larger diameter is better.⁵⁴ Migration studies with human MSCs have demonstrated that they can pass through 5 μm pores.⁵⁵ Other studies have demonstrated that interconnected pores with diameters >50 μm are favorable to new bone formation.^54,56,57 In addition, it has been shown that the minimum pore size for osteoconduction is 80–100 μm.^54,58 Lastly, for the scaffold to support new vasculature, it has been shown that the minimum pore size is 45–100 μm; however, scaffolds with pore sizes of 100–150 μm resulted in a richer blood supply.^54,59 Therefore, from these above studies it appears that a minimum pore size of 100 μm is necessary for osteoconduction and vascularization.

As mentioned above, the ideal construct would degrade so that when the tissue is completely formed, the scaffold should be wholly degraded. For synthetic polymers, degradation occurs primarily by chemical hydrolysis of hydrolytically unstable polymer backbones.⁶⁰ Degradation can alter the mechanical properties of the construct, which subsequently can alter the effectiveness of the implant. This is critical in orbital bone engineering because if insufficient mechanical support is provided, delayed enophthalmos could occur. This concern suggests that perhaps slowly degrading scaffolds, degrading over the course of months, may be appropriate. In addition, the degradation products can modify the surrounding environment of the implant. This is dependent on the biocompatibility of the degradation products and whether they are harmful to the adjacent tissue. Both of these properties are dependent on the structure, components, and fabrication techniques of the material. In addition, degradation is dependent on the location and geometry of the implant as well as the presence of catalysts, impurities, and other additives.⁶⁰

All implanted materials elicit a reaction from the host, but there is variation in the degree of response produced based upon the material. Reactions to injury include inflammation, wound healing, and foreign body responses.⁶¹ A material may be considered to be biocompatible if it produces minimal inflammatory and immune response, and is able to function properly without significant harm to the host. The goal of designing a material for implantation is to minimize the magnitude of the response and response duration.

The immune response has a direct effect on bone tissue engineering. Specifically, degradation products are thought to be the cause of failure in many orthopedic implants.⁶² These degradation particles can be phagocytosed by macrophages when <20 μm in diameter.⁶² It is thought that these particles may indirectly affect bone cells through the secretory products of macrophages drawn to the area from the immune response.⁶² Studies suggest that degradation particles directly interact with osteoblasts and affect their proliferation.⁶³ As mentioned above, biomaterial properties can affect the magnitude and duration of the host response. Characteristics of the material that can alter the immune response include the size, shape, and chemical and physical properties.⁶¹

Mechanical properties are of great importance in designing a scaffold for orbital floor regeneration. As the orbital floor acts as a natural crumple zone during trauma, it is important to closely mimic the native tissue to restore this natural function. An experimental study was completed qualitatively analyzing the forces applied to the orbital floor in the two proposed traumatic defect mechanisms as a result of direct injuries to the globe or orbital rim by placing strain gauges beneath the orbital floor.^13,14 In conditions simulating the buckling mechanisms, anterior strains exceeded 3756 μɛ and minimal strains were detected posteriorly. In the hydraulic simulation, significant anterior strains were reported; however, posterior gauge readings all exceeded 3756 μɛ. In addition, the average energy required to fracture the orbital floor for each of the mechanisms was 1.54 and 1.22 J for buckling and hydraulic mechanisms, respectively. Other studies performed compared the orbital content weight and the load-resisting capabilities of common orbital reconstruction materials.⁶⁴ The investigation determined that the weight of the combined orbital contents was ∼42.97 ± 4.05 g and all materials investigated provided adequate orbital support. Specifically, one material tested was dried calvarium (1.5 mm), which exhibited a yield load of 11.93 ± 5.93 kg, a yield displacement of 1.70 ± 0.17 mm, a maximum load of 12.48 ± 6.13 kg, and a maximum displacement of 2.35 ± 0.77 mm.

Current Progress in Engineering Orbital Bone

There has been significant progress made in the field of orbital bone tissue engineering; however, a fully developed, ideal scaffold has yet to be created. This section reviews current progress achieved in orbital floor engineering and success in craniofacial engineering, which may be applied to orbital floor regeneration (see Table 1).

Table 1.

A Summary of Recent In Vivo Craniofacial Studies Applicable to Orbital Floor Regeneration

Scaffold	Cell type	Growth factor	Application	Reference
EH-PEG hydrogel	None	BMP-2	Orbital floor defects in rabbits	71
Porous PCL	Bone marrow aspirate	None	Orbital defects in pigs	72
Porous PGA	Periosteal	None	Calvarial defect in rabbit	26
Porous PCL with fibrin glue	Calvarial osteoblasts and mesenchymal progenitor cells	None	Calvarial defect in rabbit	27
Porous PPF	None	None	Cranial defect in rabbit	73
Porous PPF	None	Fibronectin, TGF-β1	Cranial defect in rabbit	74
Solid PPF with bottom porous PPF layer	Bone marrow in porous PPF	TGF- β2	Cranial defect in rabbit	75

BMP-2, bone morphogenetic protein 2; PCL, polycaprolactone; PEG, poly(ethylene glycol); PGA, poly(glycolic acid); PPF, poly(propylene fumarate); TGF-β, transforming growth factor-β; EH, 5-ethyl-5-(hydroxymethyl) β,β-dimethyl-1,3-dioxane-2-ethanol.

5-Ethyl-5-(hydroxymethyl)-β,β-dimethyl-1,3-dioxane-2-ethanol (EH)-poly(ethylene glycol) hydrogels

Investigators have developed a novel class of biomaterials based upon a cyclic acetal unit. These materials may be advantageous since the cyclic acetal unit degrades by hydrolysis into neutral primary degradation products of diols and carbonyls, and thus may not experience a change in local acidity associated with many synthetic biomaterials.⁶⁵ The acidity of hydrogel degradation products may be a concern to the stable phenotypic function of encapsulated cell populations, as acidic byproducts have been shown to increase the inflammatory response and slow wound healing.^66,67 In addition, an increase in acidity is associated with an increase in the degradation rate, which may affect the mechanical support the construct is providing.^66,68 To create a cyclic-acetal-based hydrogel for cell encapsulation, the hydrophilic polymer poly(ethylene glycol) (PEG) was incorporated. Specifically, by including PEG diacrylate within the radical polymerization of the cyclic acetal monomer 5-ethyl-5-(hydroxymethyl)-β,β-dimethyl-1,3-dioxane-2-ethanol diacrylate (EH), a water-swellable EH-PEG hydrogel has been produced.⁶⁹ This hydrogel can act as a platform for orbital floor repair by the integration of MSCs and osteoinductive signals such as BMPs. Studies with these EH-PEG hydrogels have demonstrated that components required for gel crosslinking do not affect metabolic activity, viability, or expression of osteogenic markers of bone marrow stromal cells.⁷⁰ Further, bone marrow stromal cells were shown to survive long term in EH-PEG hydrogels while maintaining viability.⁷⁰ In addition, when implanted in orbital floor defects in vivo the tissue surrounding the EH-PEG constructs showed a positive progression from 7 to 28 days, indicating that the constructs were not eliciting a chronic inflammatory response.⁷¹ During the short 28-day study, complete bony bridging across the defect did not occur. However, without the periosteum, EH-PEG gels were able to deliver BMP-2 in vivo as demonstrated by new bone in the area surrounding the constructs containing high concentrations of BMP-2 at 28 days (see Fig. 3).⁷¹ This work demonstrates that EH-PEG constructs are a viable option for use in vivo of orbital floor repair.

FIG. 3.

EH-PEG hydrogel in a rabbit orbital floor defect. EH-PEG hydrogels and other similar biomaterials have been suggested as potential substrates for orbital bone regeneration. EH-PEG, 5-ethyl-5-(hydroxymethyl)-β,β-dimethyl-1,3-dioxane-2-ethanol-poly(ethylene glycol).

Polycaprolactone scaffolds

A study has been completed investigating orbital defects in pigs where they coated polycaprolactone (PCL) with bone marrow to aid in regeneration.⁷² PCL is an advantageous material as it has been approved by the U.S. Food and Drug Administration for a number of uses as a bioresorbable polymer. Further, these investigators used fused deposition modeling to create a porous and highly interconnected network that should aid in the osteoconductive capacity of the scaffold and allow for vascular ingrowth. Scaffolds were implanted in medial wall defects and analyzed after 3 months using histology; however, the presence of the periosteum during the study was not discussed. All scaffolds showed a thin layer of fibrous encapsulation, indicating a mild inflammatory response with no additional signs of infection. The defects in the control group were bridged with fibrous scar tissue. PCL scaffolds without bone marrow were able to repair the defect and demonstrated the formation of new trabecular bone at the interface and within the scaffold, ∼4.5%, whereas PCL scaffolds loaded with bone marrow aspirate reconstructed the defect and also showed significantly increased bone growth into the implant of 14.1%. In both conditions, the presence of giant cells was dismissed as not being clinically relevant, however; the researchers suggest that additional studies with more time points over an extended period of time might elucidate the foreign body reaction. This study demonstrates the importance of including a cell source as shown by the increased bone growth in PCL scaffolds with bone marrow aspirates. It is important to note that after 3 months, only 14.1% of the scaffold demonstrated new bone formation. Additional modifications to the scaffold may be necessary to improve bone regeneration; however, PCL is a viable option for orbital bone engineering.

PGA constructs

Other investigators have focused on craniofacial applications, and some of their findings can be applied to orbital bone engineering. Investigators harvested and expanded the periosteum in vitro.²⁶ The periosteum is of interest because it is easily harvested, has been shown to contain osteoprogenitor and chondroprogenitor cells, and contributes to osteogenesis in bone development and fracture healing. In this study, the periosteal cells cultured under osteogenic conditions were combined with resorbable PGA scaffolds and implanted in critical-sized calvarial defects of rabbits. Investigators demonstrated that the periosteal cells showed an osteoblast phenotype in vitro by expression of osteocalcin in osteogenic conditions.²⁶ This corresponds with the literature that the periosteum contains osteoprogenitor cells.²⁵ In addition, preimplantation analysis demonstrated adherence of the periosteal cells to the PGA matrix, which is important in tissue engineering. Further, increased bone formation was found in groups with PGA scaffolds coated with periosteal cells compared to untreated PGA implants in vivo by histology.²⁶ This study again demonstrates the importance of delivering a cell population to the target site to aid in regeneration. While additional quantification of bone formation in the scaffolds may be beneficial in future studies, PGA is a promising scaffold for craniofacial tissue engineering.

PCL-seeded scaffolds

Other studies were performed using the polymer PCL described above. However, this study focuses on calvarial defects. Here the investigators compared rabbit bone-marrow-derived mesenchymal progenitor cells (MPCs) and calvarial osteoblasts in vitro²⁸ and in vivo.²⁷ Investigators demonstrated the two-dimensional differentiation potential of MPCs and then loaded both cell types onto three-dimensional porous interconnected PCL scaffolds fabricated using fused deposition modeling where their osteogenic differentiation was measured. Finally, the PCL scaffolds with a fibrin glue suspension were loaded with each cell type and implanted in critical-sized calvarial defects in rabbits. First, in the in vitro study, when seeded on three-dimensional PCL scaffolds MPCs were shown to have slightly higher alkaline phosphatase expression than osteoblasts; however, osteocalcin expression demonstrated no statistical differences. This demonstrates that both cell types show potential for use in craniofacial tissue engineering. In addition, continuous cell proliferation and homogenous cell distribution was seen throughout the PCL scaffolds. Homogenous cell distribution is a vital property of a tissue scaffold, but more importantly, measurable cell proliferation is a positive result, showing that PCL scaffolds are a promising tissue engineering construct. Further in vivo results demonstrated increased bone formation with cell-seeded scaffolds after 3 months; however, there was no significant difference between scaffolds seeded with osteoblasts or MPCs as shown by histology and radiology. Therefore, this study demonstrates that both cell types may be successful in tissue engineering applications in vitro and in vivo, and therefore it may be best to proceed with the cell type most easily harvested.

Poly(propylene fumarate) scaffolds

Much progress has been made with poly(propylene fumarate) (PPF) for use in craniofacial tissue engineering. First, the soft and hard tissue responses to photocrosslinked PPF scaffolds were investigated in cranial defects using a rabbit model.⁷³ Results show an organized connective tissue at 8 weeks. Further, the study demonstrated that PPF scaffolds elicit a mild immune response in both soft and hard tissues and that scaffold porosity and pore size did not significantly affect the tissue response as examined by histology. This study indicates that PPF may be an appropriate scaffold for craniofacial tissue engineering. Next, PPF scaffolds were treated with TGF-β1 and implanted into subcritical-sized cranial defects in rabbits.⁷⁴ Results show that constructs coated with TGF-β1 had significantly higher bone growth than other groups that were not coated with TGF-β1 as demonstrated by analysis of histological images quantifying bone surface area, and bone area percentage. This indicates TGF-β1 as an important growth factor in craniofacial tissue engineering. Further studies with PPF were performed by creating a construct that combined PPF with β-tricalcium phosphate.⁷⁵ In addition, these constructs were designed to contain a porous layer that was infused with bone marrow aspirate. This study examined the inclusion of TGF-β2 to the constructs in a critical-sized cranial defect in a rabbit model. Results show more bone formation found with constructs containing TGF-β2 in addition to being the strongest bone compared to other groups as supported by mechanical testing. These above studies demonstrate that PPF scaffolds are a viable construct for use in craniofacial tissue engineering.

Proposed Tissue Engineering Strategy

There are a number of disadvantages associated with current orbital clinical treatments, and tissue engineering offers a viable alternative. From the ideas presented in this article, it can be proposed that a reasonable tissue engineering approach would include harvesting MSCs from a patient, growing them to an appropriate density, incorporating them into an engineered construct, and implanting the construct with cells into the patient when the clinician judges that the patient is ready for surgery. This is a viable strategy for a number of reasons. MSCs are one of the more accessible cell types for harvest from a patient that have demonstrated osteogenic differentiation ability. Clinically, this is a viable option for repair as many patients with orbital floor fractures do not require immediate repair and allow for better clinical evaluation after clearing of the initial edema and hemorrhage.⁷⁶ Specifically, a 2-week window of observation has been suggested in the absence of urgent surgical repair, with benefit of waiting up to 2 months allowing for natural correction of some sequelae and time for the tissue engineering construct to be prepared.^76,77 In using this method, the choice of the engineered scaffold is critical. While mentioned previously, many currently used alloplastic implants elicit fibrous encapsulation of the construct. This can be reduced in tissue-engineered constructs through the proper engineering of scaffold parameters, including mechanical strength, surface properties, and degradation products. A viable construct may be the EH-PEG hydrogels described above. These scaffolds support MSC populations with increased differentiation and osteogenic signaling.⁷⁰ In addition, initial in vivo studies have shown minimal capsule formation and favorable tissue response.⁷¹

Summary

Orbital floor fractures can be a severe form of craniofacial trauma. In addition, the floor is the orbital wall most likely involved in orbital trauma. Unfortunately, the body's natural healing response to orbital floor fractures does not always restore proper function and facial aesthetics; therefore, a clinical intervention is necessary. Current common clinical treatments include alloplastic implants and autologous grafts; however, each has associated disadvantages and sequelae. Orbital bone engineering offers solutions to the current clinical techniques and can aid in regeneration of natural bone tissue that is similar in both form and function to the native orbital floor. Tissue engineering utilizes cells, signals, and scaffolds, and this review has outlined necessary components for a successful construct for orbital floor repair. In addition, current successes and progress in the literature specific to orbital floors and craniofacial research have been reviewed, and a viable tissue engineering strategy has been discussed.

Footnotes

Acknowledgments

This work was supported by the National Science Foundation (CAREER Award to J.P.F., #0448684), and the State of Maryland, Maryland Stem Cell Research Fund.

Disclosure Statement

No competing financial interests exist.

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