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Porous tissue-engineered 3D-printed scaffolds are a compelling alternative to autografts for the treatment of large periorbital bone defects. Matching the defect-specific geometry has long been considered an optimal strategy to restore pre-injury anatomy. However, studies in large animal models have revealed that biomaterial-induced bone formation largely occurs around the scaffold periphery. Such ectopic bone formation in the periorbital region can affect vision and cause disfigurement. To enhance anatomic reconstruction, geometric mismatches are introduced in the scaffolds used to treat full thickness zygomatic defects created bilaterally in adult Yucatan minipigs. 3D-printed, anatomically-mirrored scaffolds are used in combination with autologous stromal vascular fraction of cells (SVF) for treatment. An advanced image-registration workflow is developed to quantify the post-surgical geometric mismatch and correlate it with the spatial pattern of the regenerating bone. Osteoconductive bone growth on the dorsal and ventral aspect of the defect enhances scaffold integration with the native bone while medio-lateral bone growth leads to failure of the scaffolds to integrate. A strong positive correlation is found between geometric mismatch and orthotopic bone deposition at the defect site. The data suggest that strategic mismatch >20% could improve bone scaffold design to promote enhanced regeneration, osseointegration, and long-term scaffold survivability.

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Critical-sized midfacial bone defects present a unique clinical challenge due to their complex three-dimensional shapes and intimate associations with sensory organs. To address this challenge, a point-of-care treatment strategy for functional, long-term regeneration of 2 cm full-thickness segmental defects in the zygomatic arches of Yucatan minipigs is evaluated. A digital workflow is used to 3D-print anatomically precise, porous, biodegradable scaffolds from clinical-grade poly-ε-caprolactone and decellularized bone composites. The autologous stromal vascular fraction of cells (SVF) is isolated from adipose tissue extracts and infused into the scaffolds that are implanted into the zygomatic ostectomies. Bone regeneration is assessed up to 52 weeks post-operatively in acellular (AC) and SVF groups (BV/DV = 0.64 ± 0.10 and 0.65 ± 0.10 respectively). In both treated groups, bone grows from the adjacent tissues and restores the native anatomy. Significantly higher torque is required to fracture the bone-scaffold interface in the SVF (7.11 ± 2.31 N m) compared to AC groups (2.83 ± 0.23 N m). Three-dimensional microcomputed tomography analysis reveals two distinct regenerative patterns: osteoconduction along the periphery of scaffolds to form dense lamellar bone and small islands of woven bone deposits growing along the struts in the scaffold interior. Overall, this study validates the efficacy of using 3D-printed bioactive scaffolds with autologous SVF to restore geometrically complex midfacial bone defects of clinically relevant sizes while also highlighting remaining challenges to be addressed prior to clinical translation.

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Low oxygen (O2) diffusion into large tissue engineered scaffolds hinders the therapeutic efficacy of transplanted cells. To overcome this, we previously studied hollow, hyperbarically-loaded microtanks (µtanks) to serve as O2 reservoirs. To adapt these for bone regeneration, we fabricated biodegradable µtanks from polyvinyl alcohol and poly (lactic-co-glycolic acid) and embedded them to form 3D-printed, porous poly-ε-caprolactone (PCL)-µtank scaffolds. PCL-µtank scaffolds were loaded with pure O2 at 300-500 psi. When placed at atmospheric pressures, the scaffolds released O2 over a period of up to 8 h. We confirmed the inhibitory effects of hypoxia on the osteogenic differentiation of human adipose-derived stem cells (hASCs and we validated that µtank-mediated transient hyperoxia had no toxic impacts on hASCs, possibly due to upregulation of endogenous antioxidant regulator genes. We assessed bone regeneration in vivo by implanting O2-loaded, hASC-seeded, PCL-µtank scaffolds into murine calvarial defects (4 mm diameters × 0.6 mm height) and subcutaneously (4 mm diameter × 8 mm height). In both cases we observed increased deposition of extracellular matrix in the O2 delivery group along with greater osteopontin coverages and higher mineral deposition. This study provides evidence that even short-term O2 delivery from PCL-µtank scaffolds may enhance hASC-mediated bone tissue regeneration.

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3D-printing is a powerful manufacturing tool that can create precise microscale architectures across macroscale geometries. Within biomedical research, 3D-printing of various materials has been used to fabricate rigid scaffolds for cell and tissue engineering constructs with precise microarchitecture to direct cell behavior and macroscale geometry provides patient specificity. While 3D-printing hardware has become low-cost due to modeling and rapid prototyping applications, there is no common paradigm or platform for the controlled design and manufacture of 3D-printed constructs for tissue engineering. Specifically, controlling the tissue engineering features of pore size, porosity, and pore arrangement is difficult using currently available software. We have developed a MATLAB approach termed scafSLICR to design and manufacture tissue-engineered scaffolds with precise microarchitecture and with simple options to enable spatially patterned pore properties. Using scafSLICR, we designed, manufactured, and characterized porous scaffolds in acrylonitrile butadiene styrene with a variety of pore sizes, porosities, and gradients. We found that transitions between different porous regions maintained an open, connected porous network without compromising mechanical integrity. Further, we demonstrated the usefulness of scafSLICR in patterning different porous designs throughout large anatomic shapes and in preparing craniofacial tissue engineering bone scaffolds. Finally, scafSLICR is distributed as open-source MATLAB scripts and as a stand-alone graphical interface.

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Large craniofacial bone defects remain a clinical challenge due to their complex shapes and large volumes. Stem cell-based technologies that deliver osteogenic stem cells have shown remarkable regenerative potential but are hampered by the need for extensive in vitro manipulation before implantation. To address this, we explored the bone forming potential of the clinically relevant stromal vascular fraction (SVF) cells obtained from human lipoaspirate. SVF cells can be isolated for acute use in the operating room and contain a subpopulation of adipose-derived stromal/stem cells (ASCs) that can develop mineralized tissue. ASCs can be purified from the more heterogeneous population of SVF cells via secondary and tertiary culture on tissue culture plastic. In this study, the relative potential for using SVF cells or passaged ASCs to induce robust bone regeneration was compared. Isogenic SVF and ASCs were suspended in fibrin hydrogels and seeded in three-dimensional-printed osteoinductive scaffolds of decellularized bone matrix and polycaprolactone. In vitro, both cell populations successfully mineralized the scaffold, demonstrating the robust bone formation properties of SVF. In murine critical-sized cranial defects, ASC-loaded scaffolds had greater (but not statistically significant) bone volume and bone coverage area than SVF-loaded scaffolds. However, both cell-laden interventions resulted in significantly greater bone healing than contralateral acellular controls. In conclusion, we observed substantial in vitro mineralization and robust in vivo bone regeneration in tissue-engineered bone grafts using both SVF and passaged ASCs. Impact Statement The inability to effectively regenerate bone within critical-sized craniofacial defects is a present clinical challenge and overcoming this limitation using tissue engineering strategies would significantly advance current treatment outcomes. The present study tests the feasibility of harvesting stem cells intraoperatively, combining them with three-dimensional (3D)-printed osteoinductive scaffolds and, without culturing in vitro, implanting them into the bone defect to stimulate regeneration. The data from this study demonstrated that SVF isolated from lipoaspirate and used in vivo with minimal processing could be combined with a 3D-printed bioactive material in a point-of-care approach to promote bone regeneration.

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Millions of patients worldwide require bone grafts for treatment of large, critically sized bone defects from conditions such as trauma, cancer, and congenital defects. Tissue engineered (TE) bone grafts have the potential to provide a more effective treatment than current bone grafts since they would restore fully functional bone tissue in large defects. Most bone TE approaches involve a combination of stem cells with porous, biodegradable scaffolds that provide mechanical support and degrade gradually as bone tissue is regenerated by stem cells. 3D-printing is a key technique in bone TE that can be used to fabricate functionalized scaffolds with patient-specific geometry. Using 3D-printing, composite polycaprolactone (PCL) and decellularized bone matrix (DCB) scaffolds can be produced to have the desired mechanical properties, geometry, and osteoinductivity needed for a TE bone graft. This book chapter will describe the protocols for fabricating and characterizing 3D-printed PCL:DCB scaffolds. Moreover, procedures for culturing adipose-derived stem cells (ASCs) in these scaffolds in vitro will be described to demonstrate the osteoinductivity of the scaffolds.

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INTRODUCTION: Pre-vascularization of tissue engineered grafts is a promising strategy to facilitate their improved viability following in vivo implantation. In this process, endothelial cells (ECs) form capillary-like networks that can anastomose with host vasculature. Adipose-derived stromal cells (ASCs) are a commonly used cell population for tissue engineering and contain a subpopulation of ECs capable of assembling into robust vascular networks and anastomosing with the host. However, their initial vascular assembly is significantly impaired in hypoxic conditions (2% O2). In this study, we explored the minimum period of normoxic (20% O2) pre-treatment required to enable the formation of stable vascular networks. METHODS: ASC-derived vascular structures were allowed to preassemble in fibrin hydrogels in normoxia for 0, 2, 4, or 6 days and then transplanted into hypoxic environments for 6 days. Total vascular length, pericyte coverage, cell proliferation, apoptosis rates, and ECM production was assessed. RESULTS: Vascular assembly increased with time over the 6 days of culture. We found that 4 days was the minimum period of time required for stable vascular assembly. We compared the major differences in cell behavior and network structure at Days 2 and 4. Neither proliferation nor apoptosis differed, however, the Day 4 time-point was associated with a significant increase in pericyte coverage (46.1 ± 2.6%) compared to Day 2 (24.3 ± 5.3%). CONCLUSIONS: These data suggest oxygen tension may be a mediator of EC-pericyte interactions during vascular assembly. Pre-vascularization strategies should incorporate a normoxic period of to enable successful vascular formation and development.

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The treatment of craniofacial defects can present many challenges due to the variety of tissue-specific requirements and the complexity of anatomical structures in that region. 3D-printing technologies provide clinicians, engineers and scientists with the ability to create patient-specific solutions for craniofacial defects. Currently, there are three key strategies that utilize these technologies to restore both appearance and function to patients: rehabilitation, reconstruction and regeneration. In rehabilitation, 3D-printing can be used to create prostheses to replace or cover damaged tissues. Reconstruction, through plastic surgery, can also leverage 3D-printing technologies to create custom cutting guides, fixation devices, practice models and implanted medical devices to improve patient outcomes. Regeneration of tissue attempts to replace defects with biological materials. 3D-printing can be used to create either scaffolds or living, cellular constructs to signal tissue-forming cells to regenerate defect regions. By integrating these three approaches, 3D-printing technologies afford the opportunity to develop personalized treatment plans and design-driven manufacturing solutions to improve aesthetic and functional outcomes for patients with craniofacial defects.

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Three-dimensional (3D)-printing facilitates rapid, custom manufacturing of bone scaffolds with a wide range of material choices. Recent studies have demonstrated the potential for 3D-printing bioactive (i.e., osteo-inductive) scaffolds for use in bone regeneration applications. In this study, we 3D-printed porous poly-ɛ-caprolactone (PCL) scaffolds using a fused deposition modeling (FDM) process and functionalized them with mineral additives that have been widely used commercially and clinically: tricalcium phosphate (TCP), hydroxyapatite (HA), Bio-Oss (BO), or decellularized bone matrix (DCB). We assessed the "print quality" of the composite scaffolds and found that the print quality of PCL-TCP, PCL-BO, and PCL-DCB measured ∼0.7 and was statistically lower than PCL and PCL-HA scaffolds (∼0.8). We found that the incorporation of mineral particles did not significantly decrease the compressive modulus of the graft, which was on the order of 260 MPa for solid blocks and ranged from 32 to 83 MPa for porous scaffolds. Raman spectroscopy revealed the surfaces of the scaffolds maintained the chemical profile of their dopants following the printing process. We evaluated the osteo-inductive properties of each scaffold composite by culturing adipose-derived stromal/stem cells in vitro and assessing their differentiation into osteoblasts. The calcium content (normalized to DNA) increased significantly in PCL-TCP (p < 0.05), PCL-BO (p < 0.001), and PCL-DCB (p < 0.0001) groups relative to PCL only. The calcium content also increased in PCL-HA but was not statistically significant (p > 0.05). Collagen 1 expression was 10-fold greater than PCL in PCL-BO and PCL-DCB (p < 0.05) and osteocalcin expression was 10-fold greater in PCL-BO and PCL-DCB (p < 0.05) as measured by quantitative-real time-polymerase chain reaction. This study suggests that PCL-BO and PCL-DCB hybrid material may be advantageous for bone healing applications over PCL-HA or PCL-TCP blends.

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Tissue-engineered approaches to regenerate bone in the craniomaxillofacial region utilize biomaterial scaffolds to provide structural and biological cues to stem cells to stimulate osteogenic differentiation. Bioactive scaffolds are typically comprised of natural components but often lack the manufacturability of synthetic materials. To circumvent this trade-off, we 3D printed materials comprised of decellularized bone (DCB) matrix particles combined with polycaprolactone (PCL) to create novel hybrid DCB:PCL scaffolds for bone regeneration. Hybrid scaffolds were readily printable at compositions of up to 70% bone by mass and displayed robust mechanical properties. Assessments of surface features revealed both collagenous and mineral components of bone were present. Qualitative and quantitative assessments showed increased surface roughness relative to that of pure PCL scaffolds. These findings correlated with enhanced cell adhesion on hybrid surfaces relative to that on pure surfaces. Human adipose-derived stem cells (hASCs) cultured in DCB:PCL scaffolds without soluble osteogenic cues exhibited significant upregulation of osteogenic genes in hybrid scaffolds relative to pure PCL scaffolds. In the presence of soluble phosphate, hybrid scaffolds resulted in increased calcification. The hASC-seeded scaffolds were implanted into critical-sized murine calvarial defects and yielded greater bone regeneration in DCB:PCL scaffolds compared to that in PCL-only at 1 and 3 months post-transplantation. Taken together, these results demonstrate that 3D printed DCB:PCL scaffolds might be effective for stimulating bone regeneration.

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Growth factors are essential orchestrators of the normal bone fracture healing response. For non-union defects, delivery of exogenous growth factors to the injured site significantly improves healing outcomes. However, current clinical methods for scaffold-based growth factor delivery are fairly rudimentary, and there is a need for greater spatial and temporal regulation to increase their in vivo efficacy. Various approaches used to provide spatiotemporal control of growth factor delivery from bone tissue engineering scaffolds include physical entrapment, chemical binding, surface modifications, biomineralization, micro- and nanoparticle encapsulation, and genetically engineered cells. Here, we provide a brief review of these technologies, describing the fundamental mechanisms used to regulate release kinetics. Examples of their use in pre-clinical studies are discussed, and their capacities to provide tunable, growth factor delivery are compared. These advanced scaffold systems have the potential to provide safer, more effective therapies for bone regeneration than the systems currently employed in the clinic.

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