Faculty Bibliography

Bone tissue regeneration is a complex process that proceeds along the well-established wound healing pathway of hemostasis, inflammation, proliferation, and remodeling. Recently, tissue engineering efforts have focused on the application of biological and technological principles for the development of soft and hard tissue substitutes. Aim is directed towards boosting pathways of the healing process to restore form and function of tissue deficits. Continued development of synthetic scaffolds, cell therapies, and signaling biomolecules seeks to minimize the need for autografting. Despite being the current gold standard treatment, it is limited by donor sites' size and shape, as well as donor site morbidity. Since the advent of computer-aided design/computer-aided manufacturing (CAD/CAM) and additive manufacturing (AM) techniques (3D printing), bioengineering has expanded markedly while continuing to present innovative approaches to oral and craniofacial skeletal reconstruction. Prime examples include customizable, high-strength, load bearing, bioactive ceramic scaffolds. Porous macro- and micro-architecture along with the surface topography of 3D printed scaffolds favors osteoconduction and vascular in-growth, as well as the incorporation of stem and/or other osteoprogenitor cells and growth factors. This includes platelet concentrates (PCs), bone morphogenetic proteins (BMPs), and some pharmacological agents, such as dipyridamole (DIPY), an adenosine A 2A receptor indirect agonist that enhances osteogenic and osteoinductive capacity, thus improving bone formation. This two-part review commences by presenting current biological and engineering principles of bone regeneration utilized to produce 3D-printed ceramic scaffolds with the goal to create a viable alternative to autografts for craniofacial skeleton reconstruction. Part II comprehensively examines recent preclinical data to elucidate the potential clinical translation of such 3D-printed ceramic scaffolds.

Hydrogels with long-term storage stability, controllable sustained-release properties, and biocompatibility have been garnering attention as carriers for drug/growth factor delivery in tissue engineering applications. Chitosan (CS)/Graphene Oxide (GO)/Hydroxyethyl cellulose (HEC)/β-glycerol phosphate (β-GP) hydrogel is capable of forming a 3D gel network at physiological temperature (37 °C), rendering it an excellent candidate for use as an injectable biomaterial. This work focused on an injectable thermo-responsive CS/GO/HEC/β-GP hydrogel, which was designed to deliver Atsttrin, an engineered derivative of a known chondrogenic and anti-inflammatory growth factor-like molecule progranulin. The combination of the CS/GO/HEC/β-GP hydrogel and Atsttrin provides a unique biochemical and biomechanical environment to enhance fracture healing. CS/GO/HEC/β-GP hydrogels with increased amounts of GO exhibited rapid sol-gel transition, higher viscosity, and sustained release of Atsttrin. In addition, these hydrogels exhibited a porous interconnected structure. The combination of Atsttrin and hydrogel successfully promoted chondrogenesis and osteogenesis of bone marrow mesenchymal stem cells (bmMSCs) in vitro. Furthermore, the work also presented in vivo evidence that injection of Atsttrin-loaded CS/GO/HEC/β-GP hydrogel stimulated diabetic fracture healing by simultaneously inhibiting inflammatory and stimulating cartilage regeneration and endochondral bone formation signaling pathways. Collectively, the developed injectable thermo-responsive CS/GO/HEC/βG-P hydrogel yielded to be minimally invasive, as well as capable of prolonged and sustained delivery of Atsttrin, for therapeutic application in impaired fracture healing, particularly diabetic fracture healing.

Skeletal stem and progenitor cells (SSPCs) perform bone maintenance and repair. With age, they produce fewer osteoblasts and more adipocytes leading to a loss of skeletal integrity. The molecular mechanisms that underlie this detrimental transformation are largely unknown. Single-cell RNA sequencing revealed that Notch signaling becomes elevated in SSPCs during aging. To examine the role of increased Notch activity, we deleted Nicastrin, an essential Notch pathway component, in SSPCs in vivo. Middle-aged conditional knockout mice displayed elevated SSPC osteo-lineage gene expression, increased trabecular bone mass, reduced bone marrow adiposity, and enhanced bone repair. Thus, Notch regulates SSPC cell fate decisions, and moderating Notch signaling ameliorates the skeletal aging phenotype, increasing bone mass even beyond that of young mice. Finally, we identified the transcription factor Ebf3 as a downstream mediator of Notch signaling in SSPCs that is dysregulated with aging, highlighting it as a promising therapeutic target to rejuvenate the aged skeleton.

The aim of this study was to evaluate the bone healing of tight-fit implants placed in the maxilla and mandible of subjects compromised with metabolic syndrome (MS) and type-2 Diabetes Mellitus (T2DM). Eighteen Göttingen minipigs were randomly distributed into three groups: (i) control (normal diet), (ii) MS (cafeteria diet for obesity induction), (iii) T2DM (cafeteria diet for obesity induction + Streptozotocin for T2DM induction). Maxillary and mandibular premolars and molar were extracted. After 8 weeks of healing, implants with progressive small buttress threads were placed, and allowed to integrate for 6 weeks after which the implant/bone blocks were retrieved for histological processing. Qualitative and quantitative histomorphometric analyses (percentage of bone-to-implant contact, %BIC, and bone area fraction occupancy within implant threads, %BAFO) were performed. The bone healing process around the implant occurred predominantly through interfacial remodeling with subsequent bone apposition. Data as a function of systemic condition yielded significantly higher %BIC and %BAFO values for healthy and MS relative to T2DM. Data as a function of maxilla and mandible did not yield significant differences for either %BIC and %BAFO. When considering both factors, healthy and MS subjects had %BIC and %BAFO trend towards higher values in the mandible relative to maxilla, whereas T2DM yielded higher %BIC and %BAFO in the maxilla relative to mandible. All systemic conditions presented comparable levels of %BIC and %BAFO in the maxilla; healthy and MS presented significantly higher %BIC and %BAFO relative to T2DM in the mandible. T2DM presented lower amounts of bone formation around implants relative to MS and healthy. Implants placed in the maxilla and in the mandible showed comparable amounts of bone in proximity to implants.

Collagen, an abundant extracellular matrix protein, has shown hemostatic, chemotactic, and cell adhesive characteristics, making it an attractive choice for the fabrication of tissue engineering scaffolds. The aim of this study was to synthesize a fibrillar colloidal gel from Type 1 bovine collagen, as well as three dimensionally (3D) print scaffolds with engineered pore architectures. 3D-printed scaffolds were also subjected to post-processing through chemical crosslinking (in N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide) and lyophilization. The scaffolds were physicochemically characterized through Fourier Transform Infrared Spectroscopy (FTIR), Thermogravimetric Analysis, Differential Scanning Calorimetry, and mechanical (tensile) testing. In vitro experiments using Presto Blue and Alkaline Phosphatase assays were conducted to assess cellular viability and the scaffolds' ability to promote cellular proliferation and differentiation. Rheological analysis indicated shear thinning capabilities in the collagen gels. Crosslinked and lyophilized 3D-printed scaffolds were thermally stable at 37 °C and did not show signs of denaturation, although crosslinking resulted in poor mechanical strength. PB and ALP assays showed no signs of cytotoxicity as a result of crosslinking. Fibrillar collagen was successfully formulated into a colloidal gel for extrusion through a direct inkjet writing printer. 3D-printed scaffolds promoted cellular attachment and proliferation, making them a promising material for customized, patient-specific tissue regenerative applications.

BACKGROUND:3D-printed bioceramic scaffolds composed of 100% beta(β)-tricalcium phosphate augmented with dipyridamole (3DPBC-DIPY) can regenerate bone across critically sized defects in skeletally mature and immature animal models. Prior to human application, safe and effective bone formation should be demonstrated in a large translational animal model. This study evaluated the ability of 3DPBC-DIPY scaffolds to restore critically sized calvarial defects in a skeletally immature, growing minipig. METHODS:Unilateral calvarial defects (~1.4cm) were created in six-week-old Göttingen minipigs (n=12). Four defects were filled with a 1000µ M 3DPBC-DIPY scaffold with a cap (a solid barrier on the ectocortical side of the scaffold to prevent soft tissue infiltration), four defects were filled with a 1000µM 3DPBC-DIPY scaffold without a cap, and four defects served as negative controls (no scaffold). Animals were euthanized 12-weeks post-operatively. Calvaria were subjected to micro-computed tomography, 3D-reconstruction with volumetric analysis, qualitative histologic analysis, and nanoindentation. RESULTS:Scaffold-induced bone growth was statistically greater than negative controls (p≤0.001) and the scaffolds with caps produced significantly more bone generation compared to the scaffolds without caps (p≤0.001). Histological analysis revealed woven and lamellar bone with the presence of haversian canals throughout the regenerated bone. Additionally, cranial sutures were observed to be patent and there was no evidence of ectopic bone formation or excess inflammatory response. Reduced elastic modulus (Er) and hardness (H) of scaffold-regenerated bone were found to be statistically equivalent to native bone (p = 0.148 for Er of scaffolds with and without caps, and p = 0.228 and p = 0.902, for H of scaffolds with and without caps, respectively). CONCLUSION/CONCLUSIONS:3DPBC-DIPY scaffolds have the capacity to regenerate bone across critically sized calvarial defects in a skeletally immature translational pig model.

OBJECTIVE:) ceramic structures fabricated using additive manufacturing (3D printing). METHODS:powder was combined with ammonium polyacrylate, hydroxypropyl methylcellulose, and polyelectrolyte to create a colloidal gel, which was then used for printing. A digital CAD model of a disc was designed, and the G-code transferred to a custom built DIW 3D printer. The control group samples were prepared using pre-crystallized ceramic blocks, which were cut to obtain discs with same dimensions as the AR group. Disc-shaped specimens from both groups were crystallized at 840 °C. Mechanical properties were evaluated using biaxial flexural strength test (BFS) and Vickers hardness test. Representative fractographic images of the specimens were acquired using scanning electron microscopy (SEM) to analyze the fracture origin and crack propagation. Energy-dispersive X-ray spectroscopy (EDS) and attenuated total reflection Fourier transform infrared spectroscopy (FTIR-ATR) were used for chemical analysis, and X-ray diffractometry (XRD) was performed to analyze the crystalline phases. RESULTS:) in the AM group. SEM micrographs showed a more porous microstructure associated with the 3D printed samples. SIGNIFICANCE/CONCLUSIONS:crystals and higher degrees of porosity.

Defects characterized as large osseous voids in bone, in certain circumstances, are difficult to treat, requiring extensive treatments which lead to an increased financial burden, pain, and prolonged hospital stays. Grafts exist to aid in bone tissue regeneration (BTR), among which ceramic-based grafts have become increasingly popular due to their biocompatibility and resorbability. BTR using bioceramic materials such as β-tricalcium phosphate has seen tremendous progress and has been extensively used in the fabrication of biomimetic scaffolds through the three-dimensional printing (3DP) workflow. 3DP has hence revolutionized BTR by offering unparalleled potential for the creation of complex, patient, and anatomic location-specific structures. More importantly, it has enabled the production of biomimetic scaffolds with porous structures that mimic the natural extracellular matrix while allowing for cell growth-a critical factor in determining the overall success of the BTR modality. While the concept of 3DP bioceramic bone tissue scaffolds for human applications is nascent, numerous studies have highlighted its potential in restoring both form and function of critically sized defects in a wide variety of translational models. In this review, we summarize these recent advancements and present a review of the engineering principles and methodologies that are vital for using 3DP technology for craniomaxillofacial reconstructive applications. Moreover, we highlight future advances in the field of dynamic 3D printed constructs via shape-memory effect, and comment on pharmacological manipulation and bioactive molecules required to treat a wider range of boney defects.

OBJECTIVE:To evaluate the reliability, maximum principal stress, shear stress, and crack initiation of a computer-aided design/computer-aided manufacturing (CAD/CAM) resin composite (RC) incorporating surface pre-reacted glass (S-PRG) filler for primary molar teeth. METHODS:Mandibular primary molar crowns fabricated by experimental (EB) or commercially available CAD/CAM RCs (HC) were prepared and cemented to a resinous abutment tooth using an adhesive resin cement (Cem) or a conventional glass-ionomer cement (CX). These specimens were subjected to a single compressive test (n = 5/each) and the step-stress accelerated life testing (SSALT) (n = 12/each). Data was evaluated using Weibull analyses and reliability was calculated. Afterwards, the maximum principal stress and crack initiation point of each crown was analyzed by finite element analysis. To evaluate bonding of EB and HC to dentin, microtensile bond strength (μTBS) testing was conducted using primary molar teeth (n = 10/each). RESULTS:There was no significant difference between the fracture loads of EB and HC for either cement (p > 0.05). The fracture loads of EB-CX and HC-CX were significantly lower than EB-Cem and HC-Cem (p < 0.05). The reliability at 600 N for EB-Cem was greater than that for EB-CX, HC-Cem, and HC-CX. The maximum principal stress concentrated on EB was lower than that on HC. The shear stress concentrated in the cement layer for EB-CX was higher than that for HC-CX. There was no significant difference among the μTBSs of EB-Cem, EB-CX, HC-Cem, and HC-CX (p > 0.05). SIGNIFICANCE/CONCLUSIONS:The crowns fabricated with the experimental CAD/CAM RC incorporating S-PRG filler yielded greater fracture loads and reliability than the crowns manufactured with commercially available CAD/CAM RC regardless of the luting materials. These findings suggest that the experimental CAD/CAM RC crown may be clinically useful for the restoration of primary molars.

The present study aimed to evaluate the effect of dipyridamole, an indirect adenosine 2A receptors (A_2AR), on the osseointegration of titanium implants in a large, translational pre-clinical model. Sixty tapered, acid-etched titanium implants, treated with four different coatings ((i) Type I Bovine Collagen (control), (ii) 10 μM dipyridamole (DIPY), (iii) 100 μM DIPY, and (iv) 1000 μM DIPY), were inserted in the vertebral bodies of 15 female sheep (weight ~65 kg). Qualitative and quantitative analysis were performed after 3, 6, and 12 weeks in vivo to assess histological features, and percentages of bone-to-implant contact (%BIC) and bone area fraction occupancy (%BAFO). Data was analyzed using a general linear mixed model analysis with time in vivo and coating as fixed factors. Histomorphometric analysis after 3 weeks in vivo revealed higher BIC for DIPY coated implant groups (10 μM (30.42% ± 10.62), 100 μM (36.41% ± 10.62), and 1000 μM (32.46% ± 10.62)) in comparison to the control group (17.99% ± 5.82). Further, significantly higher BAFO was observed for implants augmented with 1000 μM of DIPY (43.84% ± 9.97) compared to the control group (31.89% ± 5.46). At 6 and 12 weeks, no significant differences were observed among groups. Histological analysis evidenced similar osseointegration features and an intramembranous-type healing pattern for all groups. Qualitative observation corroborated the increased presence of woven bone formation in intimate contact with the surface of the implant and within the threads at 3 weeks with increased concentrations of DIPY. Coating the implant surface with dipyridamole yielded a favorable effect with regard to BIC and BAFO at 3 weeks in vivo. These findings suggest a positive effect of DIPY on the early stages of osseointegration.