Roche C. de Guzman

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.

Mechanisms of hepatocyte attachment to keratin biomaterials

Keratin biomaterials support cellular adhesion, proliferation and migration, which have led to their exploitation in a variety of biomedical applications. The mechanism of cell adhesion to keratin biomaterials, however, is poorly understood. Therefore, the goal of this work was to investigate the mechanisms by which human hair keratin-based biomaterials facilitate cellular adhesion. Hepatocytes were used as a model cell type due to the abundance of published data on cell adhesion mechanisms and their relatively copious attachment to keratin substrates. The roles of β(1)- and β(2)-integrins and the hepatic asialoglycoprotein receptor (ASGPR) in hepatocyte adhesion to keratin substrates were studied using attachment assays with and without function blocking antibodies. Blocking of the hepatic integrin subunits did not decrease hepatocyte attachment to keratin. Furthermore, adhesion to keratin did not result in the formation of focal complexes or focal adhesions, nor did it produce an upregulation of phosphorylated-focal adhesion kinase. However, inhibition of hepatic ASGPR decreased the ability of hepatocytes to attach to keratin substrates, which is indicative of the role of this glycoprotein receptor in hepatocyte binding to keratin biomaterials.

Structure-property relationships of meta-kerateine biomaterials derived from human hair.

The structure-property relationships of kerateine materials were studied by separating crude hair extracts into two protein sub-fractions, referred to as α- and γ-kerateines, followed by their de novo recombination into meta-kerateine hydrogels, sponges and films. The kerateine fractions were characterized using electrophoresis and mass spectrometry, which revealed that the α-fraction contained complexes of type I and type II keratins and that the γ-fraction was primarily protein fragments of the α-fraction along with three proteins of the KAP-1 family. Meta-kerateine materials with increased amounts of γ-kerateines showed diminished physical, mechanical and biological characteristics. Most notably, materials with higher γ-content formed less elastic and less solid-like hydrogels and sponges that were less hydrolytically stable. In addition, a model biological assay showed that meta-kerateine films with greater amounts of γ-kerateines were less supportive of hepatocyte attachment. Investigation into the mechanism of attachment revealed that hepatocyte adhesion to meta-kerateines is not mediated by the β1 integrin subunit, despite the presence of LDV binding motifs within the type I α-keratins. This work to define the role of protein composition on biomaterial function is essential for the optimization of keratin biomaterials for biomedical applications.

Alginate-matrigel microencapsulated schwann cells for inducible secretion of glial cell line derived neurotrophic factor.

Controlled expression of glial cell line derived neurotrophic factor (Gdnf) can be integrated in the development of a system for repair of injured peripheral nerves. This delivery strategy was demonstrated via inducible Gdnf from microencapsulated cells in barium alginate. The Schwann cell line RT4-D6P2T was initially modified utilizing an ecdysone-based stable transfection system to produce RT4-Gdnf cells. During construct preparation, it was found that C6 cells (where Gdnf cDNA was isolated) make three Gdnf transcript variants. Additionally, the importance of 5′ untranslated region to drive biologically-functional Gdnf synthesis was shown. Encapsulation of RT4-Gdnf in 1% alginate was then performed. It was determined that cells were able to survive at least 1 month in vitro using starting densities of 20, 200 and 2000 cells/capsule and barium ion concentrations of 10, 50, 100 and 200 mM. Most importantly, encapsulated cells secreted exogenous Gdnf upon ponasterone A induction. Mixture of basement membrane extract Matrigel™ to alginate promoted increased proliferation, cell spreading and Gdnf release. Finally, compression tests showed that cell-loaded microcapsules fractured at 75% diameter compression with 38 kPa of stress. Regulated Gdnf release from these microcapsules in vivo may potentially aid in the regeneration of damaged nerves.

Electrospinning of matrigel to deposit a basal lamina-like nanofiber surface.

Schwann cell basal lamina is a nanometer-thin extracellular matrix layer that separates the axon-bound Schwann cells from the endoneurium of the peripheral nerve. It is implicated in the promotion of nerve regeneration after transection injury by allowing Schwann cell colonization and axonal guidance. Hence, it is desired to mimic the native basal lamina for neural tissue engineering applications. In this study, basal lamina proteins from BD Matrigel (growth factor-reduced) were extracted and electrospun to deposit nonwoven nanofiber mats. Adjustment of solute protein concentration, potential difference, air gap distance and flow rate produced a basal lamina-like construct with an average surface roughness of 23 nm and composed of 100-nm-thick irregular and relatively discontinuous fibers. Culture of embryonic chick dorsal root ganglion explants demonstrated that the fabricated nanofiber layer supported explant attachment, elongation of neurites, and migration of satellite Schwann cells in a similar fashion compared to electrospun collagen type-I fibers. Furthermore, the presence of nanorough surface features significantly increased the neurite spreading and Schwann cell growth. Sciatic nerve segment incubation also showed that the construct is promigratory to nerve Schwann cells. Results, therefore, suggest that the synthetic basal lamina fibers can be utilized as a biomaterial for induction of peripheral nerve repair.

Binding Interactions of Keratin-Based Hair Fiber Extract to Gold, Keratin, and BMP-2.

Hair-derived keratin biomaterials composed mostly of reduced keratin proteins (kerateines) have demonstrated their utility as carriers of biologics and drugs for tissue engineering. Electrostatic forces between negatively-charged keratins and biologic macromolecules allow for effective drug retention; attraction to positively-charged growth factors like bone morphogenetic protein 2 (BMP-2) has been used as a strategy for osteoinduction. In this study, the intermolecular surface and bulk interaction properties of kerateines were investigated. Thiol-rich kerateines were chemisorbed onto gold substrates to form an irreversible 2-nm rigid layer for surface plasmon resonance analysis. Kerateine-to-kerateine cohesion was observed in pH-neutral water with an equilibrium dissociation constant (KD) of 1.8 × 10−4 M, indicating that non-coulombic attractive forces (i.e. hydrophobic and van der Waals) were at work. The association of BMP-2 to kerateine was found to be greater (KD = 1.1 × 10−7 M), within the range of specific binding. Addition of salts (phosphate-buffered saline; PBS) shortened the Debye length or the electrostatic field influence which weakened the kerateine-BMP-2 binding (KD = 3.2 × 10−5 M). BMP-2 in bulk kerateine gels provided a limited release in PBS (~ 10% dissociation in 4 weeks), suggesting that electrostatic intermolecular attraction was significant to retain BMP-2 within the keratin matrix. Complete dissociation between kerateine and BMP-2 occurred when the PBS pH was lowered (to 4.5), below the keratin isoelectric point of 5.3. This phenomenon can be attributed to the protonation of keratin at a lower pH, leading to positive-positive repulsion. Therefore, the dynamics of kerateine-BMP-2 binding is highly dependent on pH and salt concentration, as well as on BMP-2 solubility at different pH and molarity. The study findings may contribute to our understanding of the release kinetics of drugs from keratin biomaterials and allow for the development of better, more clinically relevant BMP-2-conjugated systems for bone repair and regeneration.

PEG-Immobilized Keratin for Protein Drug Sequestration and pH-Mediated Delivery

Protein drugs like growth factors are promising therapeutics for damaged-tissue repair. Their local delivery often requires biomaterial carriers for achieving the therapeutic dose range while extending efficacy. In this study, polyethylene glycol (PEG) and keratin were crosslinked and used as sponge-like scaffolds (KTN-PEG) to absorb test proteins with different isoelectric points (pI): albumin (~5), hemoglobin (~7), and lysozyme (~11). The protein release kinetics was influenced by charge at physiological pH 7.4. The keratin network, with pI 5.3, electrostatically attracted lysozyme and repulsed albumin generating the release rate profile: albumin > hemoglobin > lysozyme. However, under acidic conditions (pH 4), all proteins including keratins were positively charged and consequently intermolecular repulsion altered the release hierarchy, now determined by size (MW) diffusion: lysozyme (14 kDa) > hemoglobin (64 kDa) > albumin (66 kDa). Vascular endothelial growth factor C (VEGF-C), with properties comparable to lysozyme, was absorbed into the KTN-PEG scaffold. Endothelial cells cultured on this substrate had significantly larger numbers than on scaffolds without VEGF-C suggesting that the ionically bound and retained growth factor at neutral pH indirectly increased acute cell attachment and viability. PEG and keratin based sequestrations of proteins with basic pIs are therefore a feasible strategy with potential applications for selective biologics delivery.

Compressive strengths of PEG gels with glycerol and bioglass particles

Poly(ethylene glycol) (PEG)-based materials can potentially be used as biomechanical matrices for regenerative medicine implants including the replacement of intervertebral (IV) discs. Glycerol and other plasticizers (low-MW PEG, propylene glycol, and sorbitol) were added to the bulk PEG matrix, gelled using chemical and photochemical methods at different temperature and pressure settings, and compression properties acquired and analyzed. Incorporation of surface bioactive glass particles shortened the blood clotting time, while alginate and laponite additives improved the gel’s mechanical properties to 645 kPa compressive modulus, 12% yield strain, and 79 kPa yield strength. This IV disc-modeled system endured the cyclic loading and unloading test indicative of an elastic response; but required improvement of its biomechanical tolerance.

PABA Release from Chitosan-PCL with InducedElectric Current

Controlled drug delivery systems such as the stimulation-based biomaterial scaffolds for sequestration and release of drugs offer safety and regulated therapeutic approach. In this study, the drug: para-aminobenzoic acid (PABA) was absorbed into a crosslinked chitosan and poly(caprolactone) (PCL) hydrogel and its release kinetics quantified under different conditions. It was experimentally-observed that the higher the pH (or the more basic the pH), the slower the PABA saturation release trended over time. At the acidic environment of pH 4, PABA was released the fastest, and enhanced by the degradation of chitosan-PCL gel. When a constant electric current of 0.6 mA as applied, PABA release was induced at pH 10. However, at pH 7, PABA was stably-bound to the chitosan-PCL matrix, with or without the external current. The selective sequestration of PABA at basic pH and its stimulated release via electric current application can be further explored for clinical translatability.