A multipurpose brachytherapy catheter to enable intratumoral injection.

PURPOSE: To create and test a multipurpose brachytherapy catheter prototype enabling intratumoral injection and brachytherapy after a single catheter insertion. METHODS AND MATERIALS: The design of the prototype consists of an outer tube and an inner syringe tube that can be filled with injectable agent. The outer sheath and inner syringe tube were constructed using polytetrafluoroethylene tubing, and the other components were 3D printed using dental resin and polylactic acid material. To demonstrate functionality, we injected in vitro phantoms with dyed saline. For proof of concept, we demonstrated the potential for the prototype to deliver cell therapy, enhance tumor delineation, deliver tattoo ink for pathology marking, avoid toxicity through local delivery of chemotherapy, and facilitate combination brachytherapy and immunotherapy. RESULTS: The prototype enables accurate injection in vitro and in vivo without altering dosimetry. To illustrate the potential for delivery of cell therapies, we injected luciferase-expressing splenocytes and confirmed their delivery with bioluminescence imaging. To demonstrate feasibility of radiographically visualizing injected material, we delivered iohexol contrast intratumorally and confirmed tumor retention using Faxitron x-ray imaging. In addition, we show the potential of intratumoral administration to reduce toxicity associated with cyclophosphamide compared with systemic administration. To demonstrate feasibility, we treated tumor-bearing mice with brachytherapy (192Ir source, 2 Gy to 5 mm) in combination with intratumoral injection of 375,000 U of interleukin 2 and observed no increased toxicity. CONCLUSIONS: These results demonstrate that a prototype multipurpose brachytherapy catheter enables accurate intratumoral injection and support the feasibility of combining intratumoral injection with brachytherapy.

Biomineral coating increases bone formation by ex vivo BMP-7 gene therapy in rapid prototyped poly(L-lactic acid) (PLLA) and poly(ε-caprolactone) (PCL) porous scaffolds.

Porousbiodegradable polymer scaffolds are widely utilized for bone tissue engineering, but are not osteoconductive like calcium phosphate scaffolds. We combine indirect solid freeform fabrication (SFF), ex vivo gene therapy, with biomineral coating to compare the effect of biomineral coating on bone regeneration for Poly (L-lactic acid) (PLLA) and Poly (ε-caprolactone) (PCL) scaffolds with the same porous architecture. Scanning electron microscope (SEM) and micro-computed tomography (µ-CT) demonstrate PLLA and PCL scaffolds have the same porous architecture and are completely coated. All scaffolds are seeded with human gingival fibroblasts (HGF) transduced with adenovirus encoded with either bone morphogenetic protein 7 (BMP-7) or green fluorescent protein (GFP), and implanted into mice subcutaneously for 3 and 10 weeks. Only scaffolds with BMP-7 transduced HGFs show mineralized tissue formation. At 3 weeks some blood vessel-like structures are observed in coated PLLA and PCL scaffolds, but there is no significant difference in bone ingrowth between the coated and uncoated scaffolds for either PLLA or PCL. At 10 weeks, however, coated scaffolds (both PLLA and PCL) have significantly more bone ingrowth than uncoated scaffolds, which have more fibrous tissue. Coated PLLA scaffolds have improved mechanical properties compared with uncoated PLLA scaffolds due to increased bone ingrowth.

How does the pathophysiological context influence delivery of bone growth factors?

"Orthobiologics" represents an important category of therapeutics for the regeneration of bone defects caused by injuries or diseases, and bone growth factors are a particularly rapidly growing sub-category. Clinical application of bone growth factors has accelerated in the last two decades with the introduction of BMPs into clinical bone repair. Optimal use of growth factor-mediated treatments heavily relies on controlled delivery, which can substantially influence the local growth factor dose, release kinetics, and biological activity. The characteristics of the surrounding environment, or "context", during delivery can dictate growth factor loading efficiency, release and biological activity. This review discusses the influence of the surrounding environment on therapeutic delivery of bone growth factors. We specifically focus on pathophysiological components, including soluble components and cells, and how they can actively influence the therapeutic delivery and perhaps efficacy of bone growth factors.

Controlled multiple growth factor delivery from bone tissue engineering scaffolds via designed affinity.

It is known that angiogenesis plays an important role in bone regeneration and that release of angiogenic and osteogenic growth factors can enhance bone formation. Multiple growth factors play key roles in processes that lead to tissue formation/regeneration during natural tissue development and repair. Therefore, treatments aiming to mimic tissue regeneration can benefit from multiple growth factor release, and there remains a need for simple clinically relevant approaches for dual growth factor release. We hypothesized that mineral coatings could be used as a platform for controlled incorporation and release of multiple growth factors. Specifically, mineral-coated scaffolds were "dip coated" in multiple growth factor solutions, and growth factor binding and release were dictated by the growth factor-mineral binding affinity. Beta tricalcium phosphate (ß-TCP) scaffolds were fabricated using indirect solid-free form fabrication techniques and coated with a thin conformal mineral layer. Mineral-coated ß-TCP scaffolds were sequentially dipped in recombinant human vascular endothelial growth factor (rhVEGF) and a modular bone morphogenetic peptide, a mineral-binding version of bone morphogenetic protein 2 (BMP2), solutions to allow for the incorporation of each growth factor. The dual release profile showed sustained release of both growth factors for over more than 60 days. Scaffolds releasing either rhVEGF alone or the combination of growth factors showed an increase in blood vessel ingrowth in a dose-dependent manner in a sheep intramuscular implantation model. This approach demonstrates a "modular design" approach, in which a controllable biologics carrier is integrated into a structural scaffold as a thin surface coating.

Use of micro-computed tomography to nondestructively characterize biomineral coatings on solid freeform fabricated poly (L-lactic acid) and poly ((ε-caprolactone) scaffolds in vitro and in vivo.

Biomineral coatings have been extensively used to enhance the osteoconductivity of polymeric scaffolds. Numerous porous scaffolds have previously been coated with a bone-like apatite mineral through incubation in simulated body fluid (SBF). However, characterization of the mineral layer formed on scaffolds, including the amount of mineral within the scaffolds, often requires destructive methods. We have developed a method using micro-computed tomography (µ-CT) scanning to nondestructively quantify the amount of mineral in vitro and in vivo on biodegradable scaffolds made of poly (L-lactic acid) (PLLA) and poly (ε-caprolactone) (PCL). PLLA and PCL scaffolds were fabricated using an indirect solid freeform fabrication (SFF) technique to achieve orthogonally interconnected pore architectures. Biomineral coatings were formed on the fabricated PLLA and PCL scaffolds after incubation in modified SBF (mSBF). Scanning electron microscopy and X-ray diffraction confirmed the formation of an apatite-like mineral. The scaffolds were implanted into mouse ectopic sites for 3 and 10 weeks. The presence of a biomineral coating within the porous scaffolds was confirmed through plastic embedding and µ-CT techniques. Tissue mineral content (TMC) and volume of mineral on the scaffold surfaces detected by µ-CT had a strong correlation with the amount of calcium measured by the orthocresolphthalein complex-one (OCPC) method before and after implantation. There was a strong correlation between OCPC pre- and postimplantation and µ-CT measured TMC (R(2)=0.96 preimplant; R(2)=0.90 postimplant) and mineral volume (R(2)=0.96 preimplant; R(2)=0.89 postimplant). The µ-CT technique showed increases in mineral following implantation, suggesting that µ-CT can be used to nondestructively determine the amount of calcium on coated scaffolds.

Controllable mineral coatings on PCL scaffolds as carriers for growth factor release.

In this study, we have developed mineral coatings on polycaprolactone scaffolds to serve as templates for growth factor binding and release. Mineral coatings were formed using a biomimetic approach that consisted in the incubation of scaffolds in modified simulated body fluids (mSBF). To modulate the properties of the mineral coating, which we hypothesized would dictate growth factor release, we used carbonate (HCO(3)) concentration in mSBF of 4.2 mm, 25 mm, and 100 mm. Analysis of the mineral coatings formed using scanning electron microscopy indicated growth of a continuous layer of mineral with different morphologies. X-ray diffraction analysis showed peaks associated with hydroxyapatite, the major inorganic constituent of human bone tissue in coatings formed in all HCO(3) concentrations. Mineral coatings with increased HCO(3) substitution showed more rapid dissolution kinetics in an environment deficient in calcium and phosphate but showed re-precipitation in an environment with the aforementioned ions. The mineral coating provided an effective mechanism for growth factor binding and release. Peptide versions of vascular endothelial growth factor (VEGF) and bone morphogenetic protein 2 (BMP2) were bound with efficiencies up to 90% to mineral mineral-coated PCL scaffolds. We also demonstrated sustained release of all growth factors with release kinetics that were strongly dependent in the solubility of the mineral coating.

Mineral coatings modulate ß-TCP stability and enable growth factor binding and release.

ß-Tricalcium phosphate (ß-TCP) is an attractive ceramic for bone tissue repair because of its similar composition to bone mineral and its osteoconductivity. However, compared with other ceramics ß-TCP has a rapid and uncontrolled rate of degradation. In the current study ß-TCP granules were mineral coated with the aim of influencing the dissolution rate of ß-TCP, and also to use the coating as a carrier for controlled release of the growth factors recombinant human vascular endothelial growth factor (rhVEGF), modular VEGF peptide (mVEGF), and modular bone morphogenetic protein 2 peptide (mBMP2). The biomineral coatings were formed by heterogeneous nucleation in aqueous solution using simulated body fluid solutions with varying concentrations of bicarbonate (HCO(3)). Our results demonstrate that we could coat ß-TCP granules with mineral layers possessing different dissolution properties. The presence of a biomineral coating delays the dissolution rate of the ß-TCP granules. As the carbonate (CO(3)(2-)) content in the coating was increased the dissolution rate of the coated ß-TCP also increased, but remained slower than the dissolution of uncoated ß-TCP. In addition, we showed sustained release of multiple growth factors, with release kinetics that were controllable by varying the identity of the growth factor or the CO(3)(2-) content in the mineral coating. Released rhVEGF induced human umbilical vein endothelial cell (HUVEC) proliferation, and mVEGF enhanced migration of mouse embryonic endothelial cells in a scratch wound healing assay, indicating that each released growth factor was biologically active.

Controlled nucleation of hydroxyapatite on alginate scaffolds for stem cell-based bone tissue engineering.

Current bone tissue engineering strategies aim to grow a tissue similar to native bone by combining cells and biologically active molecules with a scaffold material. In this study, a macroporous scaffold made from the seaweed-derived polymer alginate was synthesized and mineralized for cell-based bone tissue engineering applications. Nucleation of a bone-like hydroxyapatite mineral was achieved by incubating the scaffold in modified simulated body fluids (mSBF) for 4 weeks. Analysis using scanning electron microscopy and energy dispersive x-ray analysis indicated growth of a continuous layer of mineral primarily composed of calcium and phosphorous. X-ray diffraction analysis showed peaks associated with hydroxyapatite, the major inorganic constituent of human bone tissue. In addition to the mineral characterization, the ability to control nucleation on the surface, into the bulk of the material, or on the inner pore surfaces of scaffolds was demonstrated. Finally, human MSCs attached and proliferated on the mineralized scaffolds and cell attachment improved when seeding cells on mineral coated alginate scaffolds. This novel alginate- HAP composite material could be used in bone tissue engineering as a scaffold material to deliver cells, and perhaps also biologically active molecules.