Biomimetic Nanofibrous 3D Materials for Craniofacial Bone Tissue Engineering

Abstract

Repair of large bone defects using biomaterials-based strategies has been a significant challenge due to the complex characteristics required for tissue regeneration, especially in the craniofacial region. Tissue engineering strategies aimed at restoration of function face challenges in material selection, synthesis technique, and choice of bioactive factor release in combination with all aforementioned facets. Biomimetic nanofibrous (NF) scaffolds are attractive vehicles for tissue engineering due to their ability to promote endogenous bone regeneration by mimicking the shape and chemistry of natural bone extracellular matrix (ECM). To date, several techniques for generation of biomimetic NF scaffolds have been discovered, each possessing several advantages and drawbacks. This spotlight highlights two of the more popular techniques for biomimetic NF scaffold synthesis: electrospinning and thermally-induced phase separation (TIPS), covering development from inception in each technique as well as discussing the most recent innovations in each fabrication method.

Graphical Abstract

INTRODUCTION:

Repair of large bone defects using biomaterials-based strategies has been a significant challenge due to the complex characteristics required for tissue regeneration. Autologous bone grafts have provided a gold standard for repair for several decades, but are marred by availability concerns, high associated cost, and donor site morbidity^1–3. Emergence of tissue engineering strategies for defect repair has also presented challenges in material synthesis technique and utilization^{4, 5}. Additional consideration to biomaterial design must be taken in the craniofacial region due to the complex geometry of defects, proximity to vital organs, and heightened inflammatory response^{6, 7}. Graft substitute materials such as metals and ceramics can be synthesized in many shapes, seeking to restore the mechanical strength of defect regions, however are non-biodegradable and can cause complications via stress shielding^{8, 9}. Additionally, these non-biomimetic strategies fail to restore the function of bone long-term. Biomimicry has long been a guiding principle for material design for scaffold-based tissue repair, where biomaterials seek to emulate the native extracellular matrix (ECM), both morphologically and chemically, of the tissue they aim to repair. Biomimetic scaffolds for bone repair mimic the bone matrix morphology, with nanofiber-like structures paired with highly interconnected macro-porous structure for in-growth of reparative cells and release of instructive factors^{10, 11}. Additionally, biomaterials aim to emulate the function of ECM via acting as a reservoir/bioactive factor release system. To date, several techniques to produce nanofibrous (NF) scaffolds have been studied, namely thermally-induced phase separation (TIPS), electrospinning, self-assembly, and more recently the blowspinning/airbrushing-style technique^12–15. Of the techniques, the latter two have seen relatively limited use in bone tissue engineering, especially in the craniofacial region, so this article will focus on the development of the formerly listed techniques. Conversely, the electrospinning and TIPS technique have seen significant usage in bone tissue engineering strategies to date. Electrospinning produces nanofibers with finely controlled diameter and orientation, but traditionally is limited in ability to generate 3D structures necessary for bone and craniofacial engineering purposes^{16, 17}. Phase separation techniques can generate NF scaffolds with three-dimensional (3D) features including interconnected macroporous structure when utilized in combination with a porogen, however are typically limited by a lengthy and complicated manufacturing process, and may use toxic reagents during preparation¹⁸. In this short review, we will briefly discuss these current techniques used for fabrication of 3D NF polymer scaffolds and their development through the past few decades, while highlighting our recent work on providing innovative new approaches to these 3D NF scaffolds and scaffolds-based drug delivery techniques for endogenous craniofacial bone regeneration.

Electrospinning

The evolution of biomaterials design over last several decades has also led to an expanded knowledge of the role of the biomaterial in tissue engineering. Mimicking particular morphological features of native extracellular matrix of bone has become one primary strategy in scaffold fabrication, as we can look to nature’s design for inspiration. In particular, collagenous ECM has a fiber diameter of ~150nm, with hierarchically structured micro and macroporous features¹⁹. One particular strategy to form nanofibrous materials with similar diameter is electrospinning, a popular technique applicable to a wide range of polymers that produces finely controlled nanofiber-diameter fibers. Early applications of electrospun nanofibers began use in the 1990s where various researchers demonstrated capability of several organic polymers capability of producing nanofibers through the electrospinning process²⁰. Utilization of electrospinning in regenerative medicine and in vitro applications were quickly realized with epithelial and wound-dressing applications, where the high surface area of fibers produced a favorable environment for cellular attachment with various types of polymers^{21, 22}. Some pioneering work by Jayasinghe and coworkers in 2006 explored ‘cellular electrospinning’ to evaluate feasibility for electrospinning biological architectures in 2D and 3D²³. This demonstrated capability of deposition of cellular material in a coaxial manner with biocompatible polymers within the confines of a 2D layout. However, without an interconnected porous morphology with hierarchically structured macro- and micro-pores, electrospinning scaffolds were limited to use in 2D applications only. Ding and coworkers in 2014 reported ultralight nanofiber-assembled 3D structures with overlaid electrospun fiber mats, where 3D shaping via electrospinning could be conceptually synthesized²⁴. However, the structures lacked suitable pore sizes for cell ingrowth/penetration, and utilized toxic crosslinking agents which would be potentially harmful to cells and local healthy tissue. ‘Wet electrospun’ scaffolds reported by Kobayashi and coworkers in 2008 demonstrated concept of nanofiber collection in charged liquid solutions, generating 3D shape after freeze drying²⁵. These wet solution-based electrospun scaffolds had nanofiber diameters several orders of magnitude higher than that of native ECM, making them unsuitable as a biomimetic material. Greiner and coworkers reported self-assembled 3D shapes after electrospinning of poly(MA-coMMA-co-MABP) sponges with tunable shapes in 2015 and successfully cultured cells after coating with hydrophilic poly(vinyl alcohol)²⁶. Yet, the non-biodedgradability of the material would significantly impede its usage in tissue engineering applications.

In order to develop a biocompatible 3D electrospinning based technique, our lab in collaboration with Hao Fong’s group introduced thermally-induced self-assembled (TISA) technique with polycaprolactone (PCL) nanofibers²⁷. Two significant impediments toward 3D electrospun scaffolds in biomedical applications were the 1. lack of interconnected micro and macroporous structures allowing for cell and nutrient penetration and 2. biocompatible and biodegradable materials suitable for use in tissue engineering applications. The TISA technique introduced a convenient self-assembly method in solution that was reliable and tunable; after obtaining electrospun mats, individual nanofibers were separated via mechanical grinding, where they were placed in solution and heated to slightly under the melting point of PCL. Suspensions are then quenched in freezing conditions, and then freeze dried to obtain 3D shapes (Figure 1). Resulting scaffolds possessed very high average porosity at over 96% and low density on average of 0.041 g/cm3 and demonstrated elastic properties with a low modulus upon mechanical testing. Additionally, it was found that by altering the viscosity of the assembly solution, the overall porosity of the scaffold could be controlled. Biological studies of the resulting scaffolds showed favorable results for osteogenic and chondrogenic differentiation of stem cells both in vitro and in vivo environments. Compared to PCL thermally induced phase separated plus porogen (TIPS&P) scaffolds, TISA PCL scaffolds showed a marked increase in early osteogenic marker alkaline phosphatase (ALP) of cultured cells, and early gene marker Runx2 as well as late stage markers bone sialoprotein and osteocalcin. Interestingly, cartilage marker Sox9 showed a significant increase as well, which was likely due to the softness of the scaffolds encouraging chondrogenic differentiation over more direct osteogenic differentiation of stem cells. In vivo ectopic mouse model showed a significant increase in BMP2-induced new bone formed after 4 weeks of culture, however the new bone was still overall low in amount and concentrated only around the edges of scaffolds with limited formation in the bulk of scaffolds.

Figure 1.

Schematic for the novel approach of generating 3D electrospun scaffolds from 2D mats via the thermally-induced (nanofiber) self-agglomeration followed by freeze drying. Reproduced with permission from [27]. Copyright 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

While new 3D NF PCL scaffolds formed via the TISA technique could act as a synthetic ECM that encouraged new bone formation for bone tissue engineering, several new challenges were identified in the research. One of the limiting factors of PCL-based scaffolds is the innate hydrophobicity of PCL, a factor commonly linked to weaker effects on cell adhesion and penetration into scaffolds. Additionally, the bio-inertness of PCL and slow degradation rate may have also impacted and slowed growth of new bone in vivo. And while chondrogenic differentiation was significantly improved in scaffolds, the softness/low mechanical properties likely did not provide adequate support for cells to further improve osteogenic differentiation. In order to address these limitations, polylactic acid (PLA), another biocompatible FDA-approved biopolymer was used in blends with PCL and adapted to the TISA process to make new TISA blend scaffolds, where various blend ratios were generated and evaluated for their effectiveness in improving osteogenic response of stem cells^{28, 29}. Compared to neat PCL-3D scaffolds, PCL/PLA-3D blend scaffolds had higher mechanical properties in addition to lowering the hydrophobicity of nanofibers, leading to significant increase in cellular penetration in scaffolds and viability in vitro. They not only promoted the osteogenic differentiation of hMSCs but also significantly improved new bone formation in a large cranial bone defect mouse model (Figure 2). Synthetic hydroxyapatite (HA) was another addition utilized to more closely mimic the natural bone niche. HA demonstrated a positive net effect on osteogenic differentiation and new bone formation in animal models, where it could be coated evenly and uniformly throughout the 3D construct, also without compromising porous integrity³⁰ (Figure 3). As the TISA process is further studied, understanding the modifications to the technique to process even more types of polymers will be critical to further the development of the technique.

Figure 2.

H&E staining of the repaired calvarias after 6 weeks of implantation in vivo. Reproduced with permission from [28]. Copyright 2016 Elsevier Ltd.

Figure 3.

TISA-PCL scaffolds morphology before (A-C low to high magnification) and after (D-F low to high magnification) even coating with HA. Reproduced with permission from [30]. Copyright 2017 Elsevier Ltd.

Another limitation to the neat PCL 3D ES scaffolds is the lack of inherent drug-binding/release capacity often critical for induction of osteogenic stem cell differentiation, especially in animal models. As the understanding of the role of the biomaterial in tissue engineering evolves, it has become further evident in recent years that the biomaterial plays a significant role in the delivery of bioactive factors, so designing materials to best accommodate release of active factors is critical^{30, 31}. BMP2, one of the most widely studied and utilized molecules in bone tissue regeneration presents significant clinical challenges, mostly notably its high cost and low half-life in solution. In order to address these shortcomings, we used a low-cost BMP2 activator Phenamil for use with PCL 3D TISA scaffolds³⁰. PCL scaffolds coated with the mineral-like HA closely mimicking the natural bone niche, showing a synergistic effect when combined with phenamil; osteogenic differentiation of stem cells was significantly improved with the addition of both HA+phenamil. HA coating of scaffolds alone showed a positive effect on the mechanical properties of the composite scaffolds, which also likely contributed to the improved osteogenic differentiation of stem cells (Figure 3). Moreover, biomimetic mineral deposition on TISA scaffold is ideal for drug delivery by encapsulation of bioactive agents (e.g., phenamil) into the HA crystalline lattice network (unpublished work by our group). The TISA technique while showing great promise for the field of bone tissue engineering does have a few limitations to discuss; firstly the bundling of nanofibers is inconsistent with the mechanical grinding process, leading to variability of some scaffolds. Also, while the overall porosity can be controlled by varying the viscosity of the self-assembly solution, the minute porous structure is uncontrollable without a porogen; a particular step that could be rectified in future experiments with an additional porogen template²⁷.

Thermally-Induced Phase Separation

In parallel to electrospinning, phase-separation based techniques have seen high popularity with significant advances in adaptations to the technology over the past several decades as well. Originally proposed by Castro in the 1980s, liquid-solid phase separated materials have seen a surge in popularity through the 90s and turn of the century³². Phase separated scaffolds present via a polymer-rich and polymer-lean phase, which are separated via cooled liquid-liquid phase separation and subsequent solvent removal, leaving a polymer-rich phase with a nanofibrous morphology. Early applications for biomedical uses were proposed by P. Ma and coworkers, who utilized the technique using PLLA as a base material for a thermally-induced phase separated (TIPS) hydroxyapatite scaffold as a biomimetic composite material in 1999³³. Resulting scaffolds had uniform nanofibrous matrices with high degree of porosity but were insufficient for allowing movement of cells/bioactive factors due to lack of a macroporous structure. In order to form a porous microstructure, Ma’s group later utilized a pre-made porogen template, synthesizing phase separated scaffolds around the porogen before it was leached via appropriate solvent, leaving nanofibrous scaffolds with a predesigned interconnected porous network^{34, 35}. Nanofibrous TIPS&P scaffolds allowed for mobility of cellular material and growth factors, however typically possessed weak mechanical properties. Additionally, TIPS&P technique of monopolymeric synthesis often had limited/poor osteoconductivity and lacked inherent drug binding/delivery capacity for bioactive factors, which are key toward successful integration into host for effective new bone growth. Further improvements to functionalize scaffolds with apatites or other functional groups helped improve mechanical strength and osteogenic capabilities of scaffolds³⁶.

One of the strategies taken by our group to address the lack of drug binding and release was to use a BMP-binding peptide (BBP) in combination with gelatin nanofibrous (GF) scaffolds synthesized via the TIPS&P method³⁷. GF scaffolds were functionalized by BBP via crosslinking, which was able to significantly improve the BMP2 sequestration and release from the nanofibrous scaffolds. GF-BBP scaffolds still however demonstrated relatively low mechanical properties, so we looked to further improve this with Nanosilicate nanoclay (NS), a biodegradable synthetic smectite with previously studied use in drug delivery and regenerative medicine³⁸. NS could act as a carrier for BMP2 or other molecules, while strengthening the modulus of GF scaffolds by a significant margin when incorporated into the GF matrix. Resulting osteogenic differentiation testing revealed addition of NS alone showed increase in several gene markers including ALP and BSP, as well as calcium content of cells after 21 days of culture in osteoconductive medium, suggesting NS alone is a strong improvement to the GF scaffolds possibly by strengthening endogenous BMP2 activity in addition to providing higher mechanical properties to scaffolds. In vivo mouse ectopic bone model also demonstrated significantly new bone formed after 4 weeks of implantation. Additionally, our group has developed biomimicking-based drug delivery technique, using HA as a functionalizing agent to improve the mechanical modulus and act as a drug delivery agent for phenamil as we have previously discussed^{30, 39}. One of the concerns of phenamil is the ability to increase adipogenic activity in addition to osteogenic activity of stem cells, which limits clinical applicability of the small molecule^{40, 41}. HA can act as an encapsulator for phenamil to provide sustained and localized release of drug, where addition of HA to GF scaffolds showed a marked decrease in adipogenic staining and markers PPARγ, Fabp4, and LPL. Similar to TISA, HA could evenly coat GF scaffolds while increasing mechanical properties and leaving the porous morphology still complete (Figure 4). Moreover, GF-HA scaffolds could significantly increase osteogenic gene markers Runx2/OCN/BSP over GF alone.

Figure 4.

Morphology of GF (A), GF-HA low magnification (B) and GF-HA high magnification (C), as shown by SEM. Reproduced with permission from [39]. Copyright 2019 John Wiley & Sons, Ltd.

Another one of the significant hurdles limiting TIPS&P techniques is the complexity of fabrication, where the manufacturing process can often be unwieldy and take a significant amount of time to generate scaffolds. Additionally, many types of porogens and their respective solvents are toxic and/or difficult to remove, causing downstream problems with cell viability. To address this, our group introduced a single-step ‘one pot’ TIPS method without use of any porogen in a convenient procedure⁴². This innovative approach simplifies the TIPS method utilizing aggregated microspheres to significantly shorten manufacturing time and reduce the processing steps in a simplified manner. NF scaffold porosity and pore size could be tuned via altering of polymer concentration, which in turn produced marked changes in mechanical properties and osteogenic differentiation of stem cells, suggesting great customizability for the one pot method (Figure 5). Interestingly enough, the scaffold constructs could serve as drug delivery depots for sustained growth factor release, another positive benefit for tissue engineering concerns. TIPS strategies for tissue engineering and bone especially have seen great strides in the past several decades, yet still face some challenges continuing toward clinical usage. The one-pot method while overall porosity can be controlled is not as finely tunable without utilizing porogen still. Processing and shaping material for large defect areas remains complicated due to the synthesis process for TIPS scaffolds, which has some difficulty scaling due to its non-additive nature. And finally, some materials such as natural gelatin-based still require significant manufacturing steps to protect the matrix from premature degradation either via crosslinking or use of volatile solvents for porogen leaching, limiting their development toward clinical usage.

Figure 5.

SEM images showing morphology of ‘one pot’ porogen-free TIPS scaffolds at different magnifications. Reproduced with permission from [42]. Copyright 2020 Wiley Periodicals, Inc.

Drug Delivery in 3D NF Biomimetic Scaffolds

3D NF based drug delivery biomimetic strategies for craniofacial bone tissue engineering primarily seek to emulate the structure and chemistry of native ECM in order to provide cells with an ideal environment for growth and differentiation, but also to provide for a system to strengthen endogenous bioactive factor signaling and lessen the need for exogenous drug or molecule treatment. Synthetic scaffolds are especially positioned to provide localized release of bioactive factors, reducing the risk of systemic effects of drug on side effects and damage to healthy tissue, in addition to maintaining a sustained release profile where potentially toxic high burst release is neutered. In designing biomaterials for drug release in the craniofacial region, these complications can be compounded due to the high blood supply of local tissue and high stress generated from mastication-related muscles, making precise control of drug release especially critical^{6, 7}. Additional variety in facial anatomical features from patient to patient and desire to restore aesthetic shape can further complicate shape design for biomaterials⁴³. One strategy to deliver bioactive factors involves combination/immobilization with a nano/micro-sphere carrier such as PLGA; Doxycycline, a broad-spectrum antiobiotic was incorporated into PLGA nanospheres and delivered via PLLA scaffolds developed by TIPS&P technique⁴⁴. Similarly, rhBMP-7 encapsulated PLGA nanospheres/PLLA NF scaffolds improved ectopic bone formation in mice⁴⁵. As bone regeneration is a complex and lengthy physiological process, single-drug release strategies on NF scaffolds are often insufficient to significantly improve new bone formation in animal models⁴⁶. Therefore, multiple drug strategies with finely controlled release kinetics to better mimic the natural healing process would likely further improve the regenerative capabilities of scaffolds^47,48. To achieve this, our group used mesoporous silica nanoparticles (MSNs) /GF 3D scaffold-mediated dual-drug delivery system to carry desferoxamine (DFO) and BMP2, to modulate both angiogenesis and osteogenesis for bone tissue engineering⁴⁹.

CONCLUSION AND PERSPECTIVE

Development of bioinspired 3D NF scaffolds for bone tissue engineering is a rapidly evolving and dynamic field. Over the recent developments focused on microarchitecture and morphology of scaffolds has led to well-studied interactions with cellular behavior, the role in the sequestration and release of bioactive factors remains important for tissue engineering. Additional challenge comes with manufacturing of scaffolds to fill the irregular shaped critical-sized defects, especially in the craniofacial region, while serving as a bioactive factor releasing material, as discussed before. To this end, additive manufacturing techniques such as 3D printing show promise, although cost-reduction and effective nano-scale printing are challenges to overcome, whereas most printing efforts are focused on morphological and supportive reconstruction, rather than endogenous defect repair^52,51. Injectable and crosslinkable materials capable of delivering bioactive factors may also tackle the challenge of irregular shaped defects, where several groups have developed injectable NF microspheres using TIPS techniques capable of filling complex-shaped defects in the craniofacial region^52–55. These carriers have also demonstrated capability of capturing and delivering drugs for localized release either alone or in combination with similarly mentioned immobilizing factors, indicating significant osteoinductive strength demonstrating accelerated bone regeneration^56–59. In addition, osteoinductive materials capable of osteogenic effects can reduce the need for exogenous stem cells, proteins, and harness and help strengthen the body’s innate ability to repair defects. This unmet goal in particular urges us to further understand osteogenic mechanisms and leverage new and emerging techniques such as the natural and non-immunogenic exosome based strategies or synthetic RNA-based aptamers capable of specific, targeted delivery, endowing scaffolds with desired functional moieties for more potent bone regeneration^60–63. Yet, promising techniques such as these highly functional and specific delivery techniques still see challenges in clinical translation, and have few long-term safety studies, in addition to still un-optimized synthesis and purification techniques^64,65. However, the potential for application of these emerging techniques in combination with 3D NF scaffolds will be an exciting field of research to watch unfold in the near future.