Biomaterials-aided mandibular reconstruction using in vivo bioreactors.

Large mandibular defects are clinically challenging to reconstruct due to the complex anatomy of the jaw and the limited availability of appropriate tissue for repair. We envision leveraging current advances in fabrication and biomaterials to create implantable devices that generate bone within the patients themselves suitable for their own specific anatomical pathology. The in vivo bioreactor strategy facilitates the generation of large autologous vascularized bony tissue of customized geometry without the addition of exogenous growth factors or cells. To translate this technology, we investigated its success in reconstructing a mandibular defect of physiologically relevant size in sheep. We fabricated and implanted 3D-printed in vivo bioreactors against rib periosteum and utilized biomaterial-based space maintenance to preserve the native anatomical mandibular structure in the defect site before reconstruction. Nine weeks after bioreactor implantation, the ovine mandibles were repaired with the autologous bony tissue generated from the in vivo bioreactors. We evaluated tissues generated in bioreactors by radiographic, histological, mechanical, and biomolecular assays and repaired mandibles by radiographic and histological assays. Biomaterial-aided mandibular reconstruction was successful in a large superior marginal defect in five of six (83%) sheep. Given that these studies utilized clinically available biomaterials, such as bone cement and ceramic particles, this strategy is designed for rapid human translation to improve outcomes in patients with large mandibular defects.

Econazole-releasing porous space maintainers for fungal periprosthetic joint infection.

While antibiotic-eluting polymethylmethacrylate space maintainers have shown efficacy in the treatment of bacterial periprosthetic joint infection and osteomyelitis, antifungal-eluting space maintainers are associated with greater limitations for treatment of fungal musculoskeletal infections including limited elution concentration and duration. In this study, we have designed a porous econazole-eluting space maintainer capable of greater inhibition of fungal growth than traditional solid space maintainers. The eluted econazole demonstrated bioactivity in a concentration-dependent manner against the most common species responsible for fungal periprosthetic joint infection as well as staphylococci. Lastly, these porous space maintainers retain compressive mechanical properties appropriate to maintain space before definitive repair of the joint or bony defect.

Synthesis and Characterization of Diol-Based Unsaturated Polyesters: Poly(diol fumarate) and Poly(diol fumarate-co-succinate).

In this work, we describe the synthesis and characterization of variants of poly(diol fumarate) and poly(diol fumarate-co-succinate). Through a Fischer esterification, α,ω-diols and dicarboxylic acids were polymerized to form aliphatic polyester comacromers. Because of the carbon-carbon double bond of fumaric acid, incorporating it into the macromer backbone structure resulted in unsaturated chains. By choosing α,ω-diols of different lengths (1,6-hexanediol, 1,8-octanediol, and 1,10-decanediol) and controlling the amount of fumaric acid in the dicarboxylic acid monomer feed (33, 50, and 100 mol %), nine diol-based macromer variants were synthesized and characterized for molecular weight, number of unsaturated bonds per chain, and thermal properties. Degradation and in vitro cytotoxicity were also measured in a subset of macromers. As proof-of-principle, macromer networks were photo-cross-linked to demonstrate the ability to perform free radical addition using the unsaturated macromer backbone. Cross-linked macromer networks were also characterized for physicochemical properties (swelling, sol fraction, compressive modulus) based on diol length and amount of unsaturated bonds. A statistical model was built using data generated from these diol-based macromers and macromer networks to evaluate the impact of monomer inputs on final macromer and macromer network properties. With the ability to be modified by free radical addition, biodegradable unsaturated polyesters serve as important macromers in the design of devices such as drug delivery vehicles and tissue scaffolds. Given the ability to extensively control final macromer properties based on monomer input parameters, poly(diol fumarate) and poly(diol fumarate-co-succinate) represent an exciting new class of macromers.

Repair of complex ovine segmental mandibulectomy utilizing customized tissue engineered bony flaps.

Craniofacial defects require a treatment approach that provides both robust tissues to withstand the forces of mastication and high geometric fidelity that allows restoration of facial architecture. When the surrounding soft tissue is compromised either through lack of quantity (insufficient soft tissue to enclose a graft) or quality (insufficient vascularity or inducible cells), a vascularized construct is needed for reconstruction. Tissue engineering using customized 3D printed bioreactors enables the generation of mechanically robust, vascularized bony tissues of the desired geometry. While this approach has been shown to be effective when utilized for reconstruction of non-load bearing ovine angular defects and partial segmental defects, the two-stage approach to mandibular reconstruction requires testing in a large, load-bearing defect. In this study, 5 sheep underwent bioreactor implantation and the creation of a load-bearing mandibular defect. Two bioreactor geometries were tested: a larger complex bioreactor with a central groove, and a smaller rectangular bioreactor that were filled with a mix of xenograft and autograft (initial bone volume/total volume BV/TV of 31.8 ± 1.6%). At transfer, the tissues generated within large and small bioreactors were composed of a mix of lamellar and woven bone and had BV/TV of 55.3 ± 2.6% and 59.2 ± 6.3%, respectively. After transfer of the large bioreactors to the mandibular defect, the bioreactor tissues continued to remodel, reaching a final BV/TV of 64.5 ± 6.2%. Despite recalcitrant infections, viable osteoblasts were seen within the transferred tissues to the mandibular site at the end of the study, suggesting that a vascularized customized bony flap is a potentially effective reconstructive strategy when combined with an optimal stabilization strategy and local antibiotic delivery prior to development of a deep-seated infection.

Novel polymer-based system for intrauterine delivery of everolimus for anti-cancer applications.

Non-surgical treatment options for low-grade endometrial cancer and precancerous lesions are a critical unmet need for women who wish to preserve fertility or are unable to undergo hysterectomy. The PI3K/AKT/mTOR pathway is frequently activated in endometrial cancers and has been associated with resistance to endocrine therapy, making it a compelling target for early stage disease. Oral everolimus, an inhibitor against mTORC1, has shown clinical benefit in advanced or recurrent disease but has severe adverse effects that may lead to treatment interruption or dose reduction. To overcome this, we developed a polymer-based intrauterine delivery system to achieve persistent, local delivery of everolimus without systemic exposure. In vivo studies, using a rat model, showed that a poly(propylene fumarate)-based rod loaded with everolimus achieved everolimus delivery to the endometrium with levels similar to oral administration, but with limited systemic exposure and up to 84 days of release. Biological activity of everolimus delivered with this system was confirmed, measured by reduced lumen epithelial cell height and PI3K pathway biomarkers. This study shows a promising new delivery approach for anti-cancer drugs for non-surgical treatment of low-grade endometrial cancer.

Click functionalized, tissue-specific hydrogels for osteochondral tissue engineering.

Osteochondral repair requires the induction of both articular cartilage and subchondral bone development, necessitating the presentation of multiple tissue-specific cues for these highly distinct tissues. To provide a singular hydrogel system for the repair of either tissue type, we have developed biofunctionalized, mesenchymal stem cell-laden hydrogels that can present in situ biochemical cues for either chondrogenesis or osteogenesis by simple click modification of a crosslinker, poly(glycolic acid)-poly(ethylene glycol)-poly(glycolic acid)-di(but-2-yne-1,4-dithiol) (PdBT). After modifying PdBT with either cartilage-specific biomolecules (N-cadherin peptide, chondroitin sulfate) or bone-specific biomolecules (bone marrow homing peptide 1, glycine-histidine-lysine peptide), the biofunctionalized, PdBT-crosslinked hydrogels can selectively promote the desired bone- or cartilage-like matrix synthesis and tissue-specific gene expression, with effects dependent on both biomolecule selection and concentration. Our findings establish the versatility of this click functionalized hydrogel system as well as its ability to promote in vitro development of osteochondral tissue phenotypes.

An Ovine Model of In Vivo Bioreactor-Based Bone Generation.

The generation of vascularized mineralized tissues of complex geometry without the use of extrinsic growth factors or exogenous cells requires a large animal model to recapitulate the challenges seen in the clinic. The proposed versatile ovine model can be utilized to investigate the use of a customized bioreactor to generate mineralized tissue, matching the size and shape of a defect before transfer to and integration within another site. The protocol results in bioreactors that can be harvested for investigation of the effects of different biomaterials for the generation of bone or to generate tissues appropriate for repair of bony defects; this protocol focuses on reconstruction of the mandible but could be modified for orthopedic applications. The bioreactor packing material can be altered, allowing for the study of various commercially available or novel graft materials. The surgical procedure requires ∼1.5 h to implant four bioreactors adjacent to rib periosteum. After 9 weeks, the harvest of the bioreactor tissue takes approximately 1 h. If creating a craniofacial defect, an additional 2 h should be taken for mandibular defect creation and 2 to 3 h for the reconstruction. Sheep that have undergone reconstruction are typically euthanized after 12 weeks to allow for evaluation of transferred tissues. In this protocol, we discuss the necessary steps to ensure the reproducibility and analytical techniques to assess bone regeneration such as microcomputed tomography, mechanical analysis, and histology. Impact statement Bone grafting is a frequent procedure in the fields of orthopedics, otolaryngology, and oral and maxillofacial surgery. Generating customized, vascularized, and mechanically robust bony tissues while eliminating common complications such as donor site morbidity with autograft harvest or lack of suitable mechanical properties with commercially available synthetic graft would greatly improve the lives of patients. A large animal model is necessary to generate tissues of clinically relevant geometries. In this article, a reproducible ovine model of in vivo bioreactor technology toward customized bone generation is presented with broad application to tissue engineering and regenerative medicine.

Localized mandibular infection affects remote in vivo bioreactor bone generation.

Mandibular reconstruction requires functional and aesthetic repair and is further complicated by contamination from oral and skin flora. Antibiotic-releasing porous space maintainers have been developed for the local release of vancomycin and to promote soft tissue attachment. In this study, mandibular defects in six sheep were inoculated with 106 colony forming units of Staphylococcus aureus; three sheep were implanted with unloaded porous space maintainers and three sheep were implanted with vancomycin-loaded space maintainers within the defect site. During the same surgery, 3D-printed in vivo bioreactors containing autograft or xenograft were implanted adjacent to rib periosteum. After 9 weeks, animals were euthanized, and tissues were analyzed. Antibiotic-loaded space maintainers were able to prevent dehiscence of soft tissue overlying the space maintainer, reduce local inflammatory cells, eliminate the persistence of pathogens, and prevent the increase in mandibular size compared to unloaded space maintainers in this sheep model. Animals with an untreated mandibular infection formed bony tissues with greater density and maturity within the distal bioreactors. Additionally, tissues grown in autograft-filled bioreactors had higher compressive moduli and higher maximum screw pull-out forces than xenograft-filled bioreactors. In summary, we demonstrated that antibiotic-releasing space maintainers are an innovative approach to preserve a robust soft tissue pocket while clearing infection, and that local infections can increase local and remote bone growth.

A murine model of cutaneous aspergillosis for evaluation of biomaterials-based local delivery therapies.

Cutaneous fungal infection is a challenging condition to treat that primarily afflicts immunocompromised patients. Local antifungal therapy may permit the delivery of high concentrations of antifungals directly to wounds while minimizing systemic toxicities. However, the field currently lacks suitable in vivo models. Therefore, a large cutaneous wound was created in immunosuppressed mice and inoculated with Aspergillus fumigatus. We fabricated biodegradable polymer microparticles (MPs) that were capable of locally delivering antifungal and characterized in vitro release kinetics. We compared wound bed size, fungal burden, and histological presence of fungi in mice treated with antifungal-loaded MPs. Mice with a cutaneous defect but no infection, mice with infected cutaneous defect but no treatment, and infected mice treated with blank MPs were used as controls. Infection of large wounds inhibited healing and resulted in tissue invasion in an inoculum-dependent manner. MPs were capable of releasing antifungals at concentrations above A. fumigatus Minimum Inhibitory Concentration (MIC) for at least 6 days. Wounds treated with MPs had significantly decreased size compared with no treatment (64.2% vs. 19.4% wound reduction, p = 0.002) and were not significantly different from uninfected controls (64.2% vs. 58.1%, p = 0.497). This murine model may serve to better understand cutaneous fungal infection and evaluate local biomaterials-based therapies. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1867-1874, 2019.

Incorporation of fast dissolving glucose porogens and poly(lactic-co-glycolic acid) microparticles within calcium phosphate cements for bone tissue regeneration.

This study investigated the effects of incorporating glucose microparticles (GMPs) and poly(lactic-co-glycolic acid) microparticles (PLGA MPs) within a calcium phosphate cement on the cement's handling, physicochemical properties, and the respective pore formation. Composites were fabricated with two different weight fractions of GMPs (10 and 20 wt%) and two different weight fractions of PLGA MPs (10 and 20 wt%). Samples were assayed for porosity, pore morphology, and compressive mechanical properties. An in vitro degradation study was also conducted. Samples were exposed to a physiological solution for 3 days, 4 wks, and 8 wks in order to understand how the inclusion of GMPs and PLGA MPs affects the composite's porosity and mass loss over time. GMPs and PLGA MPs were both successfully incorporated within the composites and all formulations showed an initial setting time that is appropriate for clinical applications. Through a main effects analysis, we observed that the incorporation of GMPs had a significant effect on the overall porosity, mean pore size, mode pore size, and in vitro degradation rate of PLGA MPs as early as after 3 days (p < 0.05). After 4 wks and 8 wks, these same properties were affected by the inclusion of both types of MPs (p < 0.05). Advanced polymer chromatography confirmed that the degradation of PLGA MPs coincided with an increase in composite porosity, mean pore size, and mode pore size. Finally, it was observed that the inclusion of GMPs slowed the degradation of PLGA MPs in vitro and reduced the solution acidity due to PLGA degradation products. Our results suggest that the dual inclusion of GMPs and PLGA MPs is a valuable approach for the generation of early macropores, while also mitigating the effect of acidic degradation products from PLGA MPs on their degradation kinetics. STATEMENT OF SIGNIFICANCE: A multitude of strategies and techniques have been investigated for the introduction of macropores with calcium phosphate cements (CPC). However, many of these strategies take several weeks to months to generate a maximal porosity or the degradation products of the porogen can trigger a localized inflammatory response in vivo. As such, it was hypothesized that the fast dissolution of glucose microparticles (GMPs) in a CPC composite also incorporating poly(lactic-co-glycolic acid) (PLGA) microparticles (MPs) will create an initial macroporosity and increase the surface area within the CPC, thus enhancing the diffusion of PLGA degradation products and preventing a significant decrease in pH. Furthermore, as PLGA degradation occurs over several weeks to months, additional macroporosity will be generated at later time points within CPCs. The results offer a new method for generating macroporosity in a multimodal fashion that also mitigates the effects of acidic degradation products.

3D printing for the design and fabrication of polymer-based gradient scaffolds.

To accurately mimic the native tissue environment, tissue engineered scaffolds often need to have a highly controlled and varied display of three-dimensional (3D) architecture and geometrical cues. Additive manufacturing in tissue engineering has made possible the development of complex scaffolds that mimic the native tissue architectures. As such, architectural details that were previously unattainable or irreproducible can now be incorporated in an ordered and organized approach, further advancing the structural and chemical cues delivered to cells interacting with the scaffold. This control over the environment has given engineers the ability to unlock cellular machinery that is highly dependent upon the intricate heterogeneous environment of native tissue. Recent research into the incorporation of physical and chemical gradients within scaffolds indicates that integrating these features improves the function of a tissue engineered construct. This review covers recent advances on techniques to incorporate gradients into polymer scaffolds through additive manufacturing and evaluate the success of these techniques. As covered here, to best replicate different tissue types, one must be cognizant of the vastly different types of manufacturing techniques available to create these gradient scaffolds. We review the various types of additive manufacturing techniques that can be leveraged to fabricate scaffolds with heterogeneous properties and discuss methods to successfully characterize them. STATEMENT OF SIGNIFICANCE: Additive manufacturing techniques have given tissue engineers the ability to precisely recapitulate the native architecture present within tissue. In addition, these techniques can be leveraged to create scaffolds with both physical and chemical gradients. This work offers insight into several techniques that can be used to generate graded scaffolds, depending on the desired gradient. Furthermore, it outlines methods to determine if the designed gradient was achieved. This review will help to condense the abundance of information that has been published on the creation and characterization of gradient scaffolds and to provide a single review discussing both methods for manufacturing gradient scaffolds and evaluating the establishment of a gradient.