Howard Kim

An injectable drug delivery platform for sustained combination therapy

We report the development of a series of physical hydrogel blends composed of hyaluronan (HA) and methyl cellulose (MC) designed for independent delivery of one or more drugs, from 1 to 28 days, for ultimate application in spinal cord injury repair strategies. To achieve a diversity of release profiles we exploit the combination of fast diffusion-controlled release of dissolved solutes from the HAMC itself and slow drug release from poly(lactide-co-glycolide) particles dispersed within the gel. Delivery from the composite hydrogels was demonstrated using the neuroprotective molecules NBQX and FGF-2, which were released for 1 and 4 days, respectively; the neuroregenerative molecules dbcAMP and EGF, and proteins alpha-chymotrypsin and IgG, which were released for 28 days. alpha-chymotrypsin and IgG were selected as model proteins for the clinically relevant neurotrophin-3 and anti-NogoA. Particle loaded hydrogels were significantly more stable than HAMC alone and drug release was longer and more linear than from particles alone. The composite hydrogels are minimally swelling and injectable through a 30 gauge/200 microm inner diameter needle at particle loads up to 15 wt.% and particle diameters up to 15 microm.

Differentiation of neural stem cells in three-dimensional growth factor-immobilized chitosan hydrogel scaffolds

The adult central nervous system (CNS) contains adult neural stem/progenitor cells (NSPCs) that possess the ability to differentiate into the primary cell types found in the CNS and to regenerate lost or damaged tissue. The ability to specifically and spatially control differentiation is vital to enable cell-based CNS regenerative strategies. Here we describe the development of a protein-biomaterial system that allows rapid, stable and homogenous linking of a growth factor to a photocrosslinkable material. A bioactive recombinant fusion protein incorporating pro-neural rat interferon-γ (rIFN-γ) and the AviTag for biotinylation was successfully expressed in Escherichia coli and purified. The photocrosslinkable biopolymer, methacrylamide chitosan (MAC), was thiolated, allowing conjugation of maleimide-strepatavidin via Michael-type addition. We demonstrated that biotin-rIFN-γ binds specifically to MAC-streptavidin in stoichiometric yields at 100 and 200 ng/mL in photocrosslinked hydrogels. For cell studies, NSPCs were photo-encapsulated in 100 ng/mL biotin-rIFN-γ immobilized MAC based scaffolds and compared to similar NSPC-seeded scaffolds combining 100 ng/mL soluble biotin-rIFN-γ vs. no growth factor. Cells were cultured for 8 days after which differentiation was assayed using immunohistochemistry for lineage specific markers. Quantification showed that immobilized biotin-rIFN-γ promoted neuronal differentiation (72.8 ± 16.0%) similar to soluble biotin-rIFN-γ (71.8 ± 13.2%). The percentage of nestin-positive (stem/progenitor) cells as well as RIP-positive (oligodendrocyte) cells were significantly higher in scaffolds with soluble vs. immobilized biotin-rIFN-γ suggesting that 3-D immobilization results in a more committed lineage specification.

Extramedullary Chitosan Channels Promote Survival of Transplanted Neural Stem and Progenitor Cells and Create a Tissue Bridge After Complete Spinal Cord Transection

Transplantation of neural stem and progenitor cells (NSPCs) is a promising strategy for repair after spinal cord injury. However, the epicenter of the severely damaged spinal cord is a hostile environment that results in poor survival of the transplanted NSPCs. We examined implantation of extramedullary chitosan channels seeded with NSPCs derived from transgenic green fluorescent protein (GFP) rats after spinal cord transection (SCT). At 14 weeks, we assessed the survival, maturation, and functional results using NSPCs harvested from the brain (brain group) or spinal cord (SC group) and seeded into chitosan channels implanted between the cord stumps after complete SCT. Control SCT animals had empty chitosan channels or no channels implanted. Channels seeded with brain or spinal cord-derived NSPCs showed a tissue bridge, although the bridges were thicker in the brain group. Both cell types showed long-term survival, but the number of surviving cells in the brain group was approximately five times as great as in the SC group. In both the brain and SC groups at 14 weeks after transplantation, many host axons were present in the center of the bridge in association with the transplanted cells. At 14 weeks astrocytic and oligodendrocytic differentiation in the channels was 24.8% and 17.3%, respectively, in the brain group, and 31.8% and 9.7%, respectively, in the SC group. The channels caused minimal tissue reaction in the adjacent spinal cord. There was no improvement in locomotor function. Thus, implantation of chitosan channels seeded with NSPCs after SCT created a tissue bridge containing many surviving transplanted cells and host axons, although there was no functional improvement.

Embryonic mouse spinal cord motor neuron hybrid cells.

Studies of motor neurons are difficult because of limitations in their isolation and culture. One solution is to produce clonal neural hybrid cells that can express motor neuron characteristics; we fused an aminopterin-sensitive and neomycin-resistant mouse neuroblastoma cell line to isolated embryonic mouse spinal cord motor neurons. Several hybrid neuron cell lines expressing high levels of choline acetyltransferase (CHAT) enzyme activity were found. These were cloned and clones with high CHAT activity isolated. The hybrid nature of cloned cells was confirmed by karyotyping and determining glucose phosphate isomerase allozymes. The availability of these embryonic clonal hybrid cells will enable molecular, physiological, and biochemical studies to define motor neuron-specific properties.

Creating permissive microenvironments for stem cell transplantation into the central nervous system

Traumatic injury to the central nervous system (CNS) is highly debilitating, with the clinical need for regenerative therapies apparent. Neural stem/progenitor cells (NSPCs) are promising because they can repopulate lost or damaged cells and tissues. However, the adult CNS does not provide an optimal milieu for exogenous NSPCs to survive, engraft, differentiate, and integrate with host tissues. This review provides an overview of tissue engineering strategies to improve stem cell therapies by providing a defined microenvironment during transplantation. The use of biomaterials for physical support, growth factor delivery, and cellular co-transplantation are discussed. Providing the proper environment for stem cell survival and host tissue integration is crucial in realizing the full potential of these cells in CNS repair strategies.

Bioengineering Neural Stem/Progenitor Cell-Coated Tubes for Spinal Cord Injury Repair

The aim of this study was to understand the survival and differentiation of neural stem/progenitor cells (NSPCs) cultured on chitosan matrices in vivo in a complete transection model of spinal cord injury. NSPCs were isolated from the subependyma of lateral ventricles of adult GFP transgenic rat forebrains. The GFP-positive neurospheres were seeded onto the inner lumen of chitosan tubes to generate multicellular sheets ex vivo. These bioengineered neurosphere tubes were implanted into a completely transected spinal cord and assessed after 5 weeks for survival and differentiation. The in vivo study showed excellent survival of NSPCs, as well as differentiation into astrocytes and oligodendrocytes. Importantly, host neurons were identified in the tissue bridge that formed within the chitosan tubes and bridged the transected cord stumps. The excellent in vivo survival of the NSPCs coupled with their differentiation and maintenance of host neurons in the regenerated tissue bridge demonstrates the promise of the chitosan tubes for stem cell delivery and tissue regeneration.

Effects of Dibutyryl Cyclic-AMP on Survival and Neuronal Differentiation of Neural Stem/Progenitor Cells Transplanted into Spinal Cord Injured Rats

Neural stem/progenitor cells (NSPCs) have great potential as a cell replacement therapy for spinal cord injury. However, poor control over transplant cell differentiation and survival remain major obstacles. In this study, we asked whether dibutyryl cyclic-AMP (dbcAMP), which was shown to induce up to 85% in vitro differentiation of NSPCs into neurons would enhance survival of transplanted NSPCs through prolonged exposure either in vitro or in vivo through the controlled release of dbcAMP encapsulated within poly(lactic-co-glycolic acid) (PLGA) microspheres and embedded within chitosan guidance channels. NSPCs, seeded in fibrin scaffolds within the channels, differentiated in vitro to betaIII-tubulin positive neurons by immunostaining and mRNA expression, in response to dbcAMP released from PLGA microspheres. After transplantation in spinal cord injured rats, the survival and differentiation of NSPCs was evaluated. Untreated NSPCs, NSPCs transplanted with dbcAMP-releasing microspheres, and NSPCs pre-differentiated with dbcAMP for 4 days in vitro were transplanted after rat spinal cord transection and assessed 2 and 6 weeks later. Interestingly, NSPC survival was highest in the dbcAMP pre-treated group, having approximately 80% survival at both time points, which is remarkable given that stem cell transplantation often results in less than 1% survival at similar times. Importantly, dbcAMP pre-treatment also resulted in the greatest number of in vivo NSPCs differentiated into neurons (37±4%), followed by dbcAMP-microsphere treated NSPCs (27±14%) and untreated NSPCs (15±7%). The reverse trend was observed for NSPC-derived oligodendrocytes and astrocytes, with these populations being highest in untreated NSPCs. This combination strategy of stem cell-loaded chitosan channels implanted in a fully transected spinal cord resulted in extensive axonal regeneration into the injury site, with improved functional recovery after 6 weeks in animals implanted with pre-differentiated stem cells in chitosan channels.

Chitosan implants in the rat spinal cord: Biocompatibility and biodegradation

Biomaterials are becoming increasingly popular for use in spinal cord repair, but few studies have investigated their long-term biocompatibility in central nervous system tissue. In this study, chitosan was compared with two commercial materials, degradable polyglycolide (vicryl and polyglactin 910) and nondegradable expanded poly(tetrafluoroethylene) (Gore-Tex and ePTFE), in terms of host tissue response and biodegradation in the rat spinal cord in two different spinal cord implantation models. In an uninjured model, implants were placed in the spinal cord intrathecal space for up to 6 months. At 1 month, vicryl implants elicited an elevated macrophage/microglia response compared to chitosan and Gore-Tex, which subsided in all groups by 6 months. Fibrous encapsulation was observed for all three materials. At 6 months, the in vivo degradation of vicryl was complete, while Gore-Tex showed no signs of degradation, as assessed by mass loss and SEM. Chitosan implants showed evidence of chain degradation at 6 months as demonstrated by differential hematoxylin and eosin staining; however, this did not result in mass loss. In the second model, implants were placed directly into the spinal cord for up to 12 months. This resulted in increased immune and inflammatory responses but did not alter degradation profiles. The same trends observed for the materials in the intrathecal space were mirrored in the spinal cord tissue. These results demonstrate that chitosan is a relatively inert biomaterial that does not elicit a chronic immune response and is suitable for long-term applications for repair of the spinal cord.

Chitosan Channels Containing Spinal Cord-Derived Stem/Progenitor Cells for Repair of Subacute Spinal Cord Injury in the Rat

OBJECTIVE: We evaluated the survival and differentiation capacity of neural stem/progenitor cells (NSPCs) derived from the adult rat spinal cord and seeded on intramedullary chitosan channels that were implanted in a subacute rat spinal cord injury model. METHODS: We implanted into the injured spinal cord a chitosan channel filled with NSPCs harvested from the spinal cord of adult transgenic rats expressing green fluorescent protein 3 weeks after extradural 35g clip compression injury at T8. The NSPC-chitosan channel group was compared with 2 control groups not receiving channels: 1 receiving a direct intramedullary injection of NSPCs into the lesion cavity and 1 receiving trauma alone. The survival and differentiation of NSPCs were evaluated with immunohistochemical and histopathological techniques, and functional improvement was assessed for 6 weeks with the Basso, Beattie, and Bresnahan locomotor score. RESULTS: The NSPC-chitosan channel group showed enhanced survival of NSPCs compared with NSPCs transplanted directly into the lesion cavity, although there was no significant difference in functional recovery between the treatment and control groups. In addition, the intramedullary implantation of the chitosan channel did not worsen the functional deficit after the 35g clip injury. CONCLUSIONS: Chitosan channels enhanced the survival of transplanted NSPCs in the subacutely injured spinal cord. Functional deficits were not exacerbated by the intramedullary transplantation of chitosan channels into the site of injury.

Fibrin gels containing GDNF microspheres increase axonal regeneration after delayed peripheral nerve repair

AIM Recovery following nerve transection declines when target reconnection is delayed for prolonged periods. GDNF has previously been shown to promote motor axon regeneration following delayed nerve repair. MATERIALS & METHODS We constructed delivery systems using fibrin gels containing free GDNF or poly(lactide-co-glycolide) microspheres with GDNF. The delivery systems were implanted with fluorescent fibrinogen surrounding the common fibular (CF; peroneal) nerve in transgenic Thy-1 GFP rats (whose axons express GFP) to track degradation of the system. A delayed nerve repair model was designed by transecting the rat CF nerve, where nerve regeneration was prevented by ligating the two stumps to surrounding muscle for 2 months prior to resuture. At resuture, either a delivery system with GDNF or an additional group consisting of fibrin gels with empty microspheres were implanted surrounding the repair site. In an additional positive control, the CF was transected and repaired immediately without delay. RESULTS ELISA assays demonstrated GDNF release in vitro for 2 weeks from fibrin gels with GDNF microspheres. Implanted delivery systems, including GDNF microspheres, remained surrounding the nerve for at least 10 days compared with 3 days for free GDNF. Four weeks after repair, histomorphometry of distal nerve cross-sections taken 20 mm from the repair site demonstrated increased fiber diameter and myelin thickness due to release of GDNF from microspheres compared with empty microspheres. Additionally, the number of motoneurons that regenerated their axons to the same site increased to comparable levels as immediate repair due to the extended delivery of GDNF from microspheres. CONCLUSION These findings demonstrate that early measures of nerve regeneration after delayed nerve repair is improved by GDNF microspheres implanted at the coaptation site.