Justin M. Saul

Controlled targeting of liposomal doxorubicin via the folate receptor in vitro

Differential expression of folate receptor has been exploited to target liposomes to tumors. Astrogliomas express low folate receptor levels and are typically surrounded by normal cells expressing little or no folate receptors. While targeting cells with high over-expression of folate receptor (KB and HeLa) has been demonstrated, it is unclear whether targeting tumors expressing low levels of folate receptor is possible. In this study, it was demonstrated that optimizing the number of targeting ligands (folic acid) enables differential liposomal doxorubicin uptake in C6 glioma while sparing healthy cortical cells. By micellization of folate conjugates and their controlled insertion into pre-formed liposomes, tight control over the number of targeting ligands per liposome was demonstrated. Doxorubicin uptake in KB and C6 cells was dependent on the number of targeting ligands, while cortical cells showed increasing non-specific uptake with ligand number. Co-culture of C6 glioma with cortical cells confirmed preferential uptake in C6 glioma relative to cortical cells. A cell kill experiment showed that folate-targeted liposomal doxorubicin is cytotoxic and slows proliferation of KB and C6 cells with minimal effect on cortical cells. Therefore modulation of targeting ligand number enables significant differential uptake of doxorubicin in cells with low levels of folate receptor.

Regenerative Medicine as Applied to General Surgery

The present review illustrates the state of the art of regenerative medicine (RM) as applied to surgical diseases and demonstrates that this field has the potential to address some of the unmet needs in surgery. RM is a multidisciplinary field whose purpose is to regenerate in vivo or ex vivo human cells, tissues, or organs to restore or establish normal function through exploitation of the potential to regenerate, which is intrinsic to human cells, tissues, and organs. RM uses cells and/or specially designed biomaterials to reach its goals and RM-based therapies are already in use in several clinical trials in most fields of surgery. The main challenges for investigators are threefold: Creation of an appropriate microenvironment ex vivo that is able to sustain cell physiology and function in order to generate the desired cells or body parts; identification and appropriate manipulation of cells that have the potential to generate parenchymal, stromal and vascular components on demand, both in vivo and ex vivo; and production of smart materials that are able to drive cell fate.

Bone regeneration with BMP-2 delivered from keratose scaffolds

Infuse(®) is used clinically to promote bone repair. Its efficacy is dependent on a crosslinked collagen carrier/scaffold system that has come under scrutiny due to an inability to control BMP-2 release, which may result in unwanted outcomes such as heterotopic ossification. In this study, keratose biomaterial was evaluated as a new carrier/scaffold. Keratose was mixed with BMP-2, fabricated into a scaffold, and implanted into a critical-size rat femoral defect. This construct showed bridging as early as 4 weeks and induced trabecular morphology characteristic of a remodeling hard fracture callus at 16 weeks. Compared to the normal cortical bone, the regenerated tissue had greater volume and mineral content but less density and ultimate shear stress values. Moreover, μ-CT, biomechanics, FTIR-ATR spectroscopy, and polarized light microscopy data showed regeneration using keratose was similar to an Infuse control. However, unlike Infuses collagen carrier system, in vitro analysis showed that BMP-2 release correlated with keratose scaffold degradation. Surprisingly, treatment with keratose only led to deposition of more bone outgrowth than the untreated negative control at the 8-week time point. The application of keratose also demonstrated a notable reduction of adipose tissues within the gap. While not able to induce osteogenesis on its own, keratose may be the first biomaterial capable of suppressing adipose tissue formation, thereby indirectly enhancing bone regeneration.

Keratin hydrogels support the sustained release of bioactive ciprofloxacin

Keratins are naturally derived proteins that can be fabricated into several biomaterial forms including hydrogels. These materials are a potential polymeric system for several tissue engineering and regenerative medicine applications due to their ability to support cell attachment, proliferation, and migration. However, little is known regarding their ability to support sustained release of therapeutic agents. This report describes the use of keratin hydrogels for sustained release of the antibiotic ciprofloxacin, which may prove useful to traumatic injury applications that would benefit from materials promoting tissue regeneration while also preventing acute infection. Hydrogels were formed from keratins obtained by oxidative extraction and known as keratose. We found that keratose hydrogels released ~60% of loaded ciprofloxacin over the first 10 days and that continued release was detectable over the course of 3 weeks. Released ciprofloxacin was bioactive, inhibiting growth of Staphylococcus aureus for 23 days in vitro and for 2 weeks in a mouse subcutaneous model. The rate of ciprofloxacin release was highly correlated with degradation of the keratin hydrogel and not consistent with simple diffusion. Further experiments indicated that ciprofloxacin binds to keratose through electrostatic interactions. These studies demonstrate the specific use of keratose hydrogels for the release of antibiotic and the potential for the more general use of this material in tissue engineering and regenerative medicine applications.

The promotion of axon extension in vitro using polymer-templated fibrin scaffolds.

Biomaterial nerve cuffs are a clinical alternative to autografts and allografts as a means to repair segmental peripheral nerve defects. However, existing clinical biomaterial constructs lack true incorporation of physical guidance cues into their design. In both two- and three-dimensional systems, it is known that substrate geometry directly affects rates of axon migration. However, the ability to incorporate these cues into biomaterial scaffolds of sufficient porosity to promote robust nerve regeneration in three-dimensional systems is a challenge. We have developed fibrin constructs fabricated by a sacrificial templating approach, yielding scaffolds with multiple 10-250 μm diameter conduits depending on the diameter of the template fibers. The resulting scaffolds contained numerous, highly aligned conduits, had porosity of ∼ 80%, and showed mechanical properties comparable to native nerve (150-300 kPa Youngs modulus). We studied the effects of the conduit diameters on the rate of axon migration through the scaffold to investigate if manipulation of this geometry could be used to ultimately promote more rapid bridging of the scaffold. All diameters studied led to axon migration, but in contrast to effects of fiber diameters in other systems, the rate of axon migration was independent of conduit diameter in these templated scaffolds. However, aligned conduits did support more rapid axon migration than non-aligned, tortuous controls.

The Pharmacology of Regenerative Medicine

Regenerative medicine is a rapidly evolving multidisciplinary, translational research enterprise whose explicit purpose is to advance technologies for the repair and replacement of damaged cells, tissues, and organs. Scientific progress in the field has been steady and expectations for its robust clinical application continue to rise. The major thesis of this review is that the pharmacological sciences will contribute critically to the accelerated translational progress and clinical utility of regenerative medicine technologies. In 2007, we coined the phrase “regenerative pharmacology” to describe the enormous possibilities that could occur at the interface between pharmacology, regenerative medicine, and tissue engineering. The operational definition of regenerative pharmacology is “the application of pharmacological sciences to accelerate, optimize, and characterize (either in vitro or in vivo) the development, maturation, and function of bioengineered and regenerating tissues.” As such, regenerative pharmacology seeks to cure disease through restoration of tissue/organ function. This strategy is distinct from standard pharmacotherapy, which is often limited to the amelioration of symptoms. Our goal here is to get pharmacologists more involved in this field of research by exposing them to the tools, opportunities, challenges, and interdisciplinary expertise that will be required to ensure awareness and galvanize involvement. To this end, we illustrate ways in which the pharmacological sciences can drive future innovations in regenerative medicine and tissue engineering and thus help to revolutionize the discovery of curative therapeutics. Hopefully, the broad foundational knowledge provided herein will spark sustained conversations among experts in diverse fields of scientific research to the benefit of all.

Engineered multilayer ovarian tissue that secretes sex steroids and peptide hormones in response to gonadotropins.

Although hormone replacement therapy is an option for the loss of ovarian function, hormone delivery through pharmacological means results in various clinical complications. The present study was designed to deliver sex steroids by a functional construct fabricated using encapsulation techniques. Theca and granulosa cells isolated from ovaries of 21-day old rats were encapsulated in multilayer alginate microcapsules to recapitulate the native follicular structure. Cells encapsulated in two other schemes were used as controls to assess the importance of the multilayer structure. The endocrine functions of the encapsulated cells were assessed in vitro for a period of 30 days. Encapsulated cells showed sustained viability during long-term in vitro culture with those encapsulated in multilayer capsules secreting significantly higher and sustained concentrations of 17 β-estradiol (E(2)) than the two other encapsulation schemes (p < 0.05, n = 6) in response to follicle-stimulating hormone (FSH) and luteinizing hormone (LH). In addition, cells in the multilayer microcapsules also secreted activin and inhibin in vitro. In contrast, when granulosa and theca cells were cultured in 2D culture, progesterone (P(4)) secretion increased while E(2) secretion decreased over a 30-day period. In summary, we have designed a multilayer engineered ovarian tissue that secretes sex steroids and peptide hormones and responds to gonadotropins, thus demonstrating the ability to recapitulate native ovarian structure ex vivo.

Keratin hydrogel carrier system for simultaneous delivery of exogenous growth factors and muscle progenitor cells.

Ideal material characteristics for tissue engineering or regenerative medicine approaches to volumetric muscle loss (VML) include the ability to deliver cells, growth factors, and molecules that support tissue formation from a system with a tunable degradation profile. Two different types of human hair-derived keratins were tested as options to fulfill these VML design requirements: (1) oxidatively extracted keratin (keratose) characterized by a lack of covalent crosslinking between cysteine residues, and (2) reductively extracted keratin (kerateine) characterized by disulfide crosslinks. Human skeletal muscle myoblasts cultured on coatings of both types of keratin had increased numbers of multinucleated cells compared to collagen or Matrigel(TM) and adhesion levels greater than collagen. Rheology showed elastic moduli from 10(2) to 10(5) Pa and viscous moduli from 10(1) to 10(4) Pa depending on gel concentration and keratin type. Kerateine and keratose showed differing rates of degradation due to the presence or absence of disulfide crosslinks, which likely contributed to observed differences in release profiles of several growth factors. In vivo testing in a subcutaneous mouse model showed that keratose hydrogels can be used to deliver mouse muscle progenitor cells and growth factors. Histological assessment showed minimal inflammatory responses and an increase in markers of muscle formation.

Surface-mediated delivery of siRNA from fibrin hydrogels for knockdown of the BMP-2 binding antagonist noggin

UNLABELLED Antagonists and inhibitory molecules responsible for maintaining tissue homeostasis can present a significant barrier to healing when tissue engineering/regenerative medicine strategies are employed. One example of this situation is the up-regulation of antagonists such as noggin in response to increasing concentrations of bone morphogenetic protein-2 (BMP-2) present from endogenous bone repair processes or delivered exogenously from biomaterials (synthetic bone grafts). While recombinant human (rh)BMP-2 delivered from synthetic bone grafts has been shown to be an effective alternative to autografts and allografts, the supraphysiological doses of rhBMP-2 have led to clinically-adverse side effects. The high rhBMP-2 dosage may be required, in part, to overcome the presence of antagonists such as noggin. Small interfering RNA (siRNA) is an appealing approach to overcome this problem because it can knock-down antagonists or inhibitory molecules in a temporary manner. Here, we conducted fundamental studies on the delivery of siRNA from material surfaces as a means to knock-down antagonists like noggin. Non-viral cationic lipid (Lipofectamine)-siRNA complexes were delivered from a fibrin hydrogel surface to MC3T3-E1 preosteoblasts that were treated with a supraphysiological dose of rhBMP-2 to achieve noggin mRNA expression levels higher than cells naïve to rhBMP-2. Confocal microscopy and flow cytometry showed intracellular uptake of siRNA in over 98% of MC3T3-E1 cells after 48 h. Doses of 0.5 and 1 μg noggin siRNA were able to significantly reduce noggin mRNA to levels equivalent to those in MC3T3-E1 cells not exposed to rhBMP-2 with no effects on cell viability. STATEMENT OF SIGNIFICANCE Small interfering RNA (siRNA) has been considered for treatment of diseases ranging from Alzheimers to cancer. However, the ability to use siRNA in conjunction with biomaterials to direct tissue regeneration processes has received relatively little attention. Using the bone morphogenetic protein 2 antagonist, noggin, as a model, this research describes an approach to knock-down molecules that are inhibitory to desired regenerative pathways at the mRNA level via siRNA delivery from a hydrogel surface. Interactions between the material (fibrin) surface and polycation-siRNA complexes, release of the siRNA from the material surface, high levels of cellular uptake/internalization of siRNA, and significant knockdown of the targeting (noggin) mRNA are demonstrated. Broader future applications include those to nerve regeneration, cardiovascular tissue engineering, directing (stem) cell behavior, and mitigating inflammatory responses to materials.

Biomaterials for the Delivery of Growth Factors and Other Therapeutic Agents in Tissue Engineering Approaches to Bone Regeneration

Bone fracture followed by delayed or non-union typically requires bone graft intervention. Autologous bone grafts remain the clinical “gold standard”. Recently, synthetic bone grafts such as Medtronics Infuse Bone Graft have opened the possibility to pharmacological and tissue engineering strategies to bone repair following fracture. This clinically-available strategy uses an absorbable collagen sponge as a carrier material for recombinant human bone morphogenetic protein 2 (rhBMP-2) and a similar strategy has been employed by Stryker with BMP-7, also known as osteogenic protein-1 (OP-1). A key advantage to this approach is its “off-the-shelf” nature, but there are clear drawbacks to these products such as edema, inflammation, and ectopic bone growth. While there are clinical challenges associated with a lack of controlled release of rhBMP-2 and OP-1, these are among the first clinical examples to wed understanding of biological principles with biochemical production of proteins and pharmacological principles to promote tissue regeneration (known as regenerative pharmacology). After considering the clinical challenges with such synthetic bone grafts, this review considers the various biomaterial carriers under investigation to promote bone regeneration. This is followed by a survey of the literature where various pharmacological approaches and molecular targets are considered as future strategies to promote more rapid and mature bone regeneration. From the review, it should be clear that pharmacological understanding is a key aspect to developing these strategies.