Jae Sung Lee

Functionalizing Calcium Phosphate Biomaterials with Antibacterial Silver Particles

Implant-associated infections are among the most serious post-surgical complications of medical device implants, including prosthetic joints (e.g., hip, knee and shoulder) and fracture fixation hardware.[1,2] Infection rates related to orthopedic implants have been reduced to below 5% owing to strict hygienic protocols and intraoperative systemic prophylactic treatment.[3] However, the overall number of such infections has been continuously increasing with growing demands for surgical implantation as a result of population aging and increasing participation in recreational activities.[4] Timely diagnosis of implant-associated infections is a significant challenge, and established infections may not be effectively treated with long-term systemic antibiotic therapy.[2] Therefore, postsurgical infections often require complex additional surgical procedures such as debridement, prosthesis removal and re-implantation. Even systemic antibiotic treatment raises several concerns, such as systemic toxicity, low efficiency and need for hospitalization. Given this context, extended delivery of antimicrobial agents at the site of implantation is highly desirable to offer high local antibiotic concentration without systemic toxicity and thereby prevent postoperative, implant-associated infections.

A Modular, Hydroxyapatite-Binding Version of Vascular Endothelial Growth Factor

208 ©2011 Society For Biomaterials

Modular Peptide Growth Factors for Substrate-Mediated Stem Cell Differentiation†

Natural proteins are often multifunctional, and therefore capable of activating cell surface receptors, and also binding with high affinity and specificity to natural extracellular matrices (ECMs). To achieve these diverse functions, a strategy commonly employed by nature involves creating modular proteins, in which distinct domains within a single protein are designed to enable either cell signaling or ECM binding. For example, modular proteins such as osteocalcin (OCN) and bone sialoprotein (BSP) contain a domain that binds to hydroxyapatite (HA) - the major biomineral component in the ECM of bony tissues - and a distinct domain that interacts with integrin receptors to mediate cell adhesion.[1] Therefore, these proteins are capable of influencing cell behavior in particular locations within an organism by virtue of their non-covalent linkage to a specific ECM material.

Coating with a Modular Bone Morphogenetic Peptide Promotes Healing of a Bone-Implant Gap in an Ovine Model

Despite the potential for growth factor delivery strategies to promote orthopedic implant healing, there is a need for growth factor delivery methods that are controllable and amenable to clinical translation. We have developed a modular bone growth factor, herein termed “modular bone morphogenetic peptide (mBMP)”, which was designed to efficiently bind to the surface of orthopedic implants and also stimulate new bone formation. The purpose of this study was to coat a hydroxyapatite-titanium implant with mBMP and evaluate bone healing across a bone-implant gap in the sheep femoral condyle. The mBMP molecules efficiently bound to a hydroxyapatite-titanium implant and 64% of the initially bound mBMP molecules were released in a sustained manner over 28 days. The results demonstrated that the mBMP-coated implant group had significantly more mineralized bone filling in the implant-bone gap than the control group in C-arm computed tomography (DynaCT) scanning (25% more), histological (35% more) and microradiographic images (50% more). Push-out stiffness of the mBMP group was nearly 40% greater than that of control group whereas peak force did not show a significant difference. The results of this study demonstrated that mBMP coated on a hydroxyapatite-titanium implant stimulates new bone formation and may be useful to improve implant fixation in total joint arthroplasty applications.

Controllable protein delivery from coated surgical sutures

In contemporary tissue engineering there is a need for drug delivery approaches that offer controllable release kinetics from common surgical devices, particularly at healing tissue interfaces. We describe a generally applicable approach to release therapeutic proteins from sutures in a controllable manner. Results demonstrated that several common, commercially available surgical sutures could be coated with nano-porous hydroxyapatite mineral layers using a biomimetic process. The nanoscale morphology, composition, and dissolution rate of these coatings were controlled by varying the degree of carbonate substitution during calcium phosphate (CaP) mineral growth. Coated sutures could efficiently bind both an acidic protein and a basic protein, and subsequent sustained protein release was controllable by varying the coating composition. Interestingly, decreases in pH could trigger enhanced coating dissolution and, in turn, increased protein release kinetics. Importantly, CaP coatings on Orthocord sutures were robust enough to endure under clinically relevant conditions, as protein-containing coatings remained intact after multiple passages through sheep tendon and meniscus tissues. The use of CaP-coated Orthocord sutures in a sheep rotator cuff repair model demonstrated that suture coatings did not negatively impact their utility at a bone–tendon interface. Considering the ubiquitous use of sutures and their proximity to damaged tissue, the approach described in this study may provide an efficient, site-specific way of regulating new tissue formation in emerging tissue engineering schemes.

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

β-Tricalcium phosphate (β-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 β-TCP has a rapid and uncontrolled rate of degradation. In the current study β-TCP granules were mineral coated with the aim of influencing the dissolution rate of β-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 β-TCP granules with mineral layers possessing different dissolution properties. The presence of a biomineral coating delays the dissolution rate of the β-TCP granules. As the carbonate (CO(3)(2-)) content in the coating was increased the dissolution rate of the coated β-TCP also increased, but remained slower than the dissolution of uncoated β-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.

Formation of Poly(ethylene glycol)-Poly(ε-caprolactone) Nanoparticles via Nanoprecipitation

Size control of therapeutic carriers in drug delivery systems has become important due to its relevance to biodistribution in the human body and therapeutic efficacy. To understand the dependence of particle size on the formation condition during nanoprecipitation method, we prepared nanoparticles from biodegradable, amphiphilic block copolymers and investigated the particle size and structure of the resultant nanoparticles according to various process parameters. We synthesized monomethoxy poly(ethylene glycol)-poly(ε-caprolactone) block copolymer, MPEG-PCL, with different MPEG/PCL ratios via ring opening polymerization initiated from the hydroxyl end group of MPEG. Using various formulations with systematic change of the block ratio of MPEG and PCL, solvent choice, and concentration of organic phase, MPEG-PCL nanoparticles were prepared through nanoprecipitation technique. The results indicated that (i) the nanoparticles have a dual structure with an MPEG shell and a PCL core, originating from self-assembly of MPEG-PCL copolymer in aqueous condition, and (ii) the size of nanoparticles is dependent upon two sequential processes: diffusion between the organic and aqueous phases and solidification of the polymer.

High affinity binding of an engineered, modular peptide to bone tissue.

Bone grafting procedures have become common due in part to a global trend of population aging. Native bone graft is a popular choice when compared to various synthetic bone graft substitutes, owing to superior biological activity. Nonetheless, the insufficient ability of bone allograft to induce new bone formation and the insufficient remodeling of native bone grafts call for osteoinductive factors during bone repair, exemplified by recombinant human bone morphogenetic protein 2 (rhBMP2). We previously developed a modular bone morphogenetic peptide (mBMP) to address complications associated with the clinical use of rhBMP2 as a bone graft substitute. The mBMP is designed to strongly bind to hydroxyapatite, the main inorganic component of bone and teeth, and to provide pro-osteogenic properties analogous to rhBMP2. Our previous in vivo animal studies showed that mBMP bound to hydroxyapatite-coated orthopedic implants with high affinity and stimulated new bone formation. In this study, we demonstrate specific binding of mBMP to native bone grafts. The results show that mBMP binds with high affinity to both cortical and trabecular bones, and that the binding is dependent on the mBMP concentration and incubation time. Importantly, efficient mBMP binding is also achieved in an ex vivo bone bioreactor where bone tissue is maintained viable for several weeks. In addition, mBMP binding can be localized with spatial control on native bone tissue via simple methods, such as dip-coating, spotting, and direct writing. Taken together with the pro-osteogenic activity of mBMP established in previous bone repair models, these results suggest that mBMP may promote bone healing when coated on native bone grafts in a clinically compatible manner.

Immune modulation with primed mesenchymal stem cells delivered via biodegradable scaffold to repair an Achilles tendon segmental defect

Tendon healing is a complex coordinated series of events resulting in protracted recovery, limited regeneration, and scar formation. Mesenchymal stem cell (MSC) therapy has shown promise as a new technology to enhance soft tissue and bone healing. A challenge with MSC therapy involves the ability to consistently control the inflammatory response and subsequent healing. Previous studies suggest that preconditioning MSCs with inflammatory cytokines, such as IFN‐γ, TNF‐α, and IL‐1β may accelerate cutaneous wound closure. The objective of this study was to therefore elucidate these effects in tendon. That is, the in vivo healing effects of TNF‐α primed MSCs were studied using a rat Achilles segmental defect model. Rat Achilles tendons were subjected to a unilateral 3 mm segmental defect and repaired with either a PLG scaffold alone, MSC‐seeded PLG scaffold, or TNF‐α‐primed MSC‐seeded PLG scaffold. Achilles tendons were analyzed at 2 and 4 weeks post‐injury. In vivo, MSCs, regardless of priming, increased IL‐10 production and reduced the inflammatory factor, IL‐1α. Primed MSCs reduced IL‐12 production and the number of M1 macrophages, as well as increased the percent of M2 macrophages, and synthesis of the anti‐inflammatory factor IL‐4. Primed MSC treatment also increased the concentration of type I procollagen in the healing tissue and increased failure stress of the tendon 4 weeks post‐injury. Taken together delivery of TNF‐α primed MSCs via 3D PLG scaffold modulated macrophage polarization and cytokine production to further accentuate the more regenerative MSC‐induced healing response.

Collagen scaffold arrays for combinatorial screening of biophysical and biochemical regulators of cell behavior

Arrays of 3D macroporous collagen scaffolds with orthogonal gradations of structural and biomolecular cues are described. Gradient maker technology is applied to create linear biomolecular gradients within microstructurally distinct sections of a single CG scaffold array. The array set up is used to explore cell behaviors including proliferation and regulation of stem cell fate.