(496c) A Poly-L-Lactide Scaffold with Continuous Gradient Pore Size That Differentially Induce Local Chondrogenesis and Osteogenesis for Osteochondral Repair

INTRODUCTION: Cartilage and bone in articular
joints are intimately linked, and a better understanding of disease and
regenerative processes calls for the simultaneous investigation of both tissues
as part of the osteochondral (OC) unit. Furthermore, scaffold-based
regenerative approaches in the joint often target both cartilage and the
subchondral bone, taking advantage of the endogenous bone marrow stem cells
made available by breaching through the OC junction. However, the production of
scaffolds for OC regeneration is challenging, as scaffolds must provide
mechanical strength while also mimicking the local cartilage and bone
microenvironments. Thermally Induced Phase Separation (TIPS) is one of the most
adaptable techniques to produce porous scaffold for tissue engineering (1). A
wide range of morphologies in terms of pore size and distribution can be
obtained by tuning TIPS processing parameters, primarily thermal history (2).
In this work, TIPS was used to produce a poly-L-lactide (PLLA) scaffold for OC
tissue engineering with a continuous pore size gradient along the sample
thickness, starting at ~70 µm diameter on one side and increased steadily to ~200
µm diameter on the opposite surface. We expect that the smaller pore size will support
the induction of chondrogenesis while the larger pore size will induce a more
osteogenic phenotype.

A microphysiological tissue system (MPS) bioreactor
has been developed to replicate in vitro the in vivo OC physiological
conditions (3). The MPS allows separate control of the chondral and osseous
environment while permitting communication between chondrocytes and osteoblasts
across the OC junction (4), similar to the conditions of OC tissue in vivo.
We have used here our MPS system to validate the TIPS-generated pore-gradient
PLLA scaffold.

MATERIALS AND METHODS: PLLA
(Resomer TN L 209 S), 1,4-dioxane (Sigma-Aldrich) and distilled water were used
to produce the polymeric foams. The ternary solution employed has a PLLA
concentration of 4 % wt, in 87:13 wt/wt dioxane/water (solvent/non-solvent). Different
thermal histories were imposed on sample sides by an experimental apparatus made
in-house using software controlled Peltier cells. Starting from a homogeneous
system, i.e. well above the cloud point temperature (41 °C), each sample
surface was brought to the same demixing temperature (30 °C) at different
cooling rates: 25°C/min (Fast Cooling Side – FCS) and 1°C/min (Slow Cooling
Side – SCS). The residence time inside the metastable region for each side was
set at 50 and 20 min, respectively, creating a monotonously variable pore size
along sample thickness. Then, both surfaces were rapidly cooled to -20°C to
freeze the as-obtained microstructure, and the solvents were extracted by
rinsing in distilled water and drying under vacuum. Cylindrical scaffolds were
punched out and uniformly seeded with 800,000 bone marrow hMSCs derived from total
joint arthroplasty patients (IRB: University of Washington), and then placed in
the MPS with the small pore side (~70 µm) in the upper chamber (UC) and the
large pore size in the lower chamber (LC). After 7 days of growth medium (GM:
DMEM, 10%fetal bovine serum (FBS), penicillin/streptomycin/fungizone) perfusion
at 2 ml/day, chondrogenic medium (CM: DMEM, 10 ng/ml TGF-β3 (PeproTech), 1%
insulin-transferrin-selenium, 50 μM L-ascorbic acid 2-phosphate (AsA2-P),
40 μg/ml L-proline) was supplied to the UC, and osteogenic medium (OM, GM +
0.1 μM dexamethasone, 50 μg/ml AsA2-P, 10 mM β -glycerophosphate)
to the LC, both at a flow rate of 2 μL/s. After 4 weeks of
differentiation, engineered OC tissues were collected and analyzed by RT-PCR
and histology.

RESULTS: At
4 weeks, the constructs exhibited strong alcian blue staining on the side
residing in the UC (exposed to CM) and alizarin red staining in the part
residing in the LC (exposed to OM). Constructs cultured only in CM (same medium
in both chambers) exhibited stronger alcian blue staining on the small pore
side, while correspondingly stronger alizarin red staining was observed in
constructs uniformly cultured in OM. OC constructs fabricated in photocrosslinkable
methacrylated gelatin using the same hMSCs and cultured in the same conditions
within the MPS were used as 3D control. Histological staining was significantly
stronger for the PLLA OC constructs. RT-PCR analysis of PLLA constructs
cultured with CM in the UC and OM in the LC confirmed upregulation of chondral
genes (COL2, ACAN, SOX9) in the upper part of the construct (small pores) and
of osseous genes (RUNX2, BSPII, OPN) in the lower part of the construct (large
pores), compared to day 0. Similar gene expression profiles for the parts in
the UC (CM) and LC (OM) in the PLLA and methacrylated gelatin OC constructs
were found.

DISCUSSION: A
spatially defined, biphasic differentiation of hMSCs within the engineered PLLA
OC constructs is observed. Small pore size in the scaffold favored chondrogenic
differentiation, likely because of enhanced cell-cell contact inducing
mesenchymal condensation, while larger pore size favored osteogenic
differentiation, possibly due to easier nutrient movement and higher biosimilarity
to the osseous, trabecular microenvironment. The PLLA pore-gradient structure is
thus a promising scaffold for OC repair. Furthermore, even without application
of osteogenic factors, hMSCs seeded in the LC (OM) of the scaffold presented
good osteogenic differentiation, confirming the validity of the MPS design. 

Overall, a single
unit biodegradable scaffold produced with local gradient pore structure may be
tailored for chondro- and osteoinduction and applied for OC repair to improve osteochondral
tissue repair, providing mechanical stability and local cues for cell
differentiation.

REFERENCES: (1) Mannella GA. J
Polymer Sci B: Polymer Physic. 2014, 52(14): 979–983. (2) Mannella GA et
al. Mater Letts. 2015, 160: 31-33. (3) Lozito TP et al. Stem Cell Res
Ther 2013, 4(Suppl 1):S6. (4) Lin H et al. Mol Pharmaceut. 2014,
11(7): 2203-12.

ACKNOWLEDGEMENTS: Commonwealth of
Pennsylvania, NIH (1UG3 TR002136-01), Ri.MED Foundation.

Fig.1 (A) Experimental
design & MPS. (B) Histology: PLLA and gelatin constructs in MPS (dual
media). (C)
PLLA cultured in only CM (pore size effect).