Molly S. Shoichet

Engineering three-dimensional bone tissue in vitro using biodegradable scaffolds: Investigating initial cell-seeding density and culture period

New three-dimensional (3D) scaffolds for bone tissue engineering have been developed throughout which bone cells grow, differentiate, and produce mineralized matrix. In this study, the percentage of cells anchoring to our polymer scaffolds as a function of initial cell seeding density was established; we then investigated bone tissue formation throughout our scaffolds as a function of initial cell seeding density and time in culture. Initial cell seeding densities ranging from 0.5 to 10 x 10(6) cells/cm(3) were seeded onto 3D scaffolds. After 1 h in culture, we determined that 25% of initial seeded cells had adhered to the scaffolds in static culture conditions. The cell-seeded scaffolds remained in culture for 3 and 6 weeks, to investigate the effect of initial cell seeding density on bone tissue formation in vitro. Further cultures using 1 x 10(6) cells/cm(3) were maintained for 1 h and 1, 2, 4, and 6 weeks to study bone tissue formation as a function of culture period. After 3 and 6 weeks in culture, scaffolds seeded with 1 x 10(6) cells/cm(3) showed similar tissue formation as those seeded with higher initial cell seeding densities. When initial cell seeding densities of 1 x 10(6) cells/cm(3) were used, osteocalcin immunolabeling indicative of osteoblast differentiation was seen throughout the scaffolds after only 2 weeks of culture. Von Kossa and tetracycline labeling, indicative of mineralization, occurred after 3 weeks. These results demonstrated that differentiated bone tissue was formed throughout 3D scaffolds after 2 weeks in culture using an optimized initial cell density, whereas mineralization of the tissue only occurred after 3 weeks. Furthermore, after 6 weeks in culture, newly formed bone tissue had replaced degrading polymer.

Spatially controlled simultaneous patterning of multiple growth factors in three-dimensional hydrogels

Three-dimensional (3D) protein-patterned scaffolds provide a more biomimetic environment for cell culture than traditional two-dimensional surfaces, but simultaneous 3D protein patterning has proved difficult. We developed a method to spatially control the immobilization of different growth factors in distinct volumes in 3D hydrogels, and to specifically guide differentiation of stem/progenitor cells therein. Stem-cell differentiation factors sonic hedgehog (SHH) and ciliary neurotrophic factor (CNTF) were simultaneously immobilized using orthogonal physical binding pairs, barnase-barstar and streptavidin-biotin, respectively. Barnase and streptavidin were sequentially immobilized using two-photon chemistry for subsequent concurrent complexation with fusion proteins barstar-SHH and biotin-CNTF, resulting in bioactive 3D patterned hydrogels. The technique should be broadly applicable to the patterning of a wide range of proteins.

Stability of hydrogels used in cell encapsulation : An in vitro comparison of alginate and agarose

The present studies were undertaken to evaluate the in vitro gel stability of the hydrogels alginate and agarose. Gel strength (of alginate and agarose) and protein diffusion (of alginate only) were shown to correlate with gel stability and to be useful techniques to monitor gel stability over time. The gel strengths of alginate and agarose were followed for a 90‐day period using gel strength as a measure of gel stability. The gel strength of agarose diminished in the presence of cells because the cells likely interfered with the hydrogen bond formation required for agarose gelation. In the presence of cells, the gel strength of agarose decreased by an average of 25% from time 0 to 60 days, thereafter maintaining that value to 90 days. The gel strength of calcium‐ or barium‐crosslinked alginate decreased over 90 days, with an equilibrium gel strength being achieved after 30 days. The presence of cells did not further decrease alginate gel strength. The gel strengths of calcium‐ and barium‐crosslinked alginates were similar at 60 days—350 ± 20 g and 300 ± 60 g, respectively—indicating equivalence in their stability. The stability of calcium‐crosslinked sodium alginate gels over a 60‐day time period was monitored by diffusion of proteins ranging in molecular weight from 14.5 to 155 kD. From these diffusion measurements, the average pore size of the calcium‐crosslinked alginate gels was estimated, using a semi‐empirical model, to increase from ∼176 to 289 Å over a period of 60 days.

In vitro degradation of a novel poly(lactide-co-glycolide) 75/25 foam

Macroporous poly(lactide-co-glycolide) PLGA 75/25 foams were prepared for application in bone tissue engineering. Their in vitro degradation behaviour was followed over a 30 week period at 37 degrees C and at one of three pHs: (1) pH 5.0, which mimics the acidic environment produced by activated macrophages, (2) pH 7.4, which reproduces normal physiological conditions and (3) an intermediate pH 6.4. The degradation of the PLGA 75/25 foams was studied by measuring changes in mass, molecular weight and morphology. The degradation profile of foams maintained at pH 5.0, 6.4 and 7.4 was similar until week 16, after which foams maintained at pH 6.4 and 7.4 had comparable degradation patterns whereas foams maintained at pH 5.0 degraded faster. For example, mass loss was less than 3% for foams maintained at all three pHs until week 16; however, by week 30, foams maintained at pH 6.4 and 7.4 had lost 30% of their mass whereas foams maintained at pH 5.0 had lost 90% of their mass. Foams maintained at pH 6.4 and 7.4 showed a similar constant decrease in molecular weight over the entire degradation study. Foams maintained at pH 5.0 had a similar rate of molecular weight loss as those maintained at pH 6.4 and 7.4 until week 16, after which the rate of molecular weight loss of foams maintained at pH 5.0 was accelerated. The morphology of the foams maintained at pH 6.4 and 7.4 was unchanged for 25 weeks. Foams maintained at pH 5.0 collapsed after week 18. Thus the PLGA 75/25 foams, described herein, maintained their 3-D morphology at physiological pH for over 6 months, which is an important feature for tissue engineering applications.

Delivering neuroactive molecules from biodegradable microspheres for application in central nervous system disorders

Nerve growth factor (NGF) may enhance axonal regeneration following injury to the central nervous system (CNS), such as after spinal cord injury. The release profile of NGF, co-encapsulated with ovalbumin, was tailored from biodegradable polymeric microspheres using both polymer degradation and protein loading. Biodegradable polymeric microspheres were prepared from PLGA 50/50, PLGA 85/15, PCL and a blend of PCL/PLGA 50/50 (1:1, w/w), where the latter was used to further tailor the degradation rate. The amount of protein loaded in the microspheres was varied, with PCL encapsulating the greatest amount of protein and PLGA 50/50 encapsulating the least. A two-phase release profile was observed for all polymers where the first phase resulted from release of surface proteins and the second phase resulted predominantly from polymer degradation. Polymer degradation influenced the release profile most notably from PLGA 50/50 and PLGA 85/15 microspheres. The amount and bioactivity of released NGF was followed over a 91 d period using a NGF-ELISA and PC12 cells, respectively. NGF was found to be bioactive for 91 d, which is longer than previously reported.

A hydrogel-based stem cell delivery system to treat retinal degenerative diseases

Regenerative strategies for retinal degenerative diseases are limited by poor cellular survival, distribution and integration after transplantation to the sub-retinal space. To overcome this limitations a stem cell delivery system was developed, taking advantage of the minimally-invasive, injectable and biodegradable properties of a blend of hyaluronan and methylcellulose (HAMC). The physical and biological properties of this unique HAMC formulation were studied. HAMC supported retinal stem-progenitor cell (RSPC) survival and proliferation in vitro. The blend was a viscous solution, exhibiting properties ideal for delivery to the sub-retinal space. In vivo transplantation studies in mice were carried out to investigate both the biodegradability of HAMC in the sub-retinal space over 7 days and the potential of HAMC as a cell delivery vehicle. RSPCs delivered in HAMC were more evenly distributed in the sub-retinal space than those delivered in traditional saline solutions, suggesting that HAMC is a promising vehicle for cellular delivery to the degenerating retina overcoming previously reported barriers to tissue integration in the retina such as cellular aggregation and non-contiguous distribution.

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.

Patterned glass surfaces direct cell adhesion and process outgrowth of primary neurons of the central nervous system

Glass surfaces were patterned with cell-adhesive regions of laminin adhesive peptides YIGSR, RGD, and IKVAV, and cell-repulsive regions of poly(ethylene glycol) (PEG). The patterns were created by sputter-coating titanium and then gold onto glass coverslips through electron microscope grids. Gold surfaces were modified with cysteine-terminated peptides to have approximately 450 fmol/ cm2 of peptide incorporated on the glass coverslips as determined with radiolabeled CGYIGSR. Amine-functionalized glass coverslips were prepared using an amine-functionalized silane and then further modified with PEG-aldehyde by a Schiff base reduction. All surfaces were characterized by X-ray photoelectron spectroscopy and water contact angles. Hippocampal neurons, plated from a serum-free medium, adhered preferentially to peptide-functionalized surfaces over PEG-modified surfaces. Cell adhesion and neurite outgrowth were limited to the peptide region, demonstrating that neurite outgrowth could be directed by a combination of cell-adhesive and cell-repulsive cues.

Intrathecal delivery of a polymeric nanocomposite hydrogel after spinal cord injury

Major traumatic spinal cord injury (SCI) results in permanent paralysis below the site of injury. The effectiveness of systemically delivered pharmacological therapies against SCI can be limited by the blood-spinal cord barrier and side effects. Local drug delivery to the injured spinal cord can be achieved using a minimally invasive biopolymer matrix of hyaluronan and methylcellulose injected into the intrathecal space, bypassing the blood-spinal cord barrier and overcoming limitations of existing strategies. Composite hydrogels of drug-loaded poly(lactide-co-glycolide) (PLGA) nanoparticles dispersed in this biopolymer matrix meet the in vitro design criteria for prolonged local release. Using a blank (without drug) composite designed for 28-day sustained release, we presently explore the mechanism of particle-mediated hydrogel stabilization in vitro and aspects of biocompatibility and safety in vivo. The composite hydrogel is well tolerated in the intrathecal space of spinal cord injured rats, showing no increase in inflammation, scarring, or cavity volume relative to controls, and no significant effect on locomotor function up to 28 days. Furthermore, there was no effect on locomotor function in healthy animals which received the composite hydrogel, although a qualitative increase in ED-1 staining was apparent. These data support the further development of composite hydrogels of hyaluronan and methylcellulose containing PLGA nanoparticles for sustained local delivery to the injured spinal cord, an application for which there are no approved alternatives.

Tunable Growth Factor Delivery from Injectable Hydrogels for Tissue Engineering

Current sustained delivery strategies of protein therapeutics are limited by the fragility of the protein, resulting in minimal quantities of bioactive protein delivered. In order to achieve prolonged release of bioactive protein, an affinity-based approach was designed which exploits the specific binding of the Src homology 3 (SH3) domain with short proline-rich peptides. Specifically, methyl cellulose was modified with SH3-binding peptides (MC-peptide) with either a weak affinity or strong affinity for SH3. The release profile of SH3-rhFGF2 fusion protein from hyaluronan MC-SH3 peptide (HAMC-peptide) hydrogels was investigated and compared to unmodified controls. SH3-rhFGF2 release from HAMC-peptide was extended to 10 days using peptides with different binding affinities compared to the 48 h release from unmodified HAMC. This system is capable of delivering additional proteins with tunable rates of release, while maintaining bioactivity, and thus is broadly applicable.