In vitro validation of gradient porous scaffolds for osteochondral engineering and defect repair

Introduction: Osteochondral defects affect over 60% of patients that undergo knee surgery and present a major health and mobility concern. These defects are characterized by damage to both the articular cartilage and underlying subchondral bone, which often result from either acute trauma, bone disorders, or long-term degeneration. Current treatments include microfracture and tissue graft transplant, but can lead to additional complications for patients, such as fibrocartilage formation or donor tissue complexities. Tissue engineering has emerged as a plausible alternative, however a tissue construct that promotes biphasic cartilage and bone tissue formation remains challenging—integrating scaffolds with different properties often results in delamination while other methods require non-trivial localized delivery of growth factors. To tackle the problem of osteochondral repair, we developed and validated a scaffold using a Poly-L-Lactic Acid (PLLA) gradient porous scaffold that spans both cartilage and bone compartments. Using knowledge of cell and embryonic differentiation, we hypothesize that a gradient porous tissue construct will drive tissue specific formation for a cartilage-bone interface. Specifically, small pores will create a densely packed cell region favorable for chondrogenesis via mesenchymal condensation while large pores favoring loosely spread cells will be more supportive of osteogenesis. We further leverage our biphasic bioreactor that allows us to mimic the osteochondral environment to validate our scaffold in vitro. We tested our scaffolds using human and rabbit bone marrow-derived mesenchymal stem cells (MSCs).

Materials and

Methods: PLLA Scaffold Preparation.
PLLA scaffolds were created as previously described using Thermally Induced Phase Separation (TIPS) to form a continuous gradient of pore sizes that range from ~70μm on the cartilaginous side to ~200μm on the osseous side.

Validation of rabbit MSCs for cell differentiation.
Rabbit bone marrow derived MSCs were collected from whole bone marrow aspirate via centrifugation and cells were plated in growth medium containing 5ng/mL FGF2. MSCs were tested for trilineage differentiation at 14 and 28 days.

PLLA scaffold seeding with MSCs and differentiation.
Cylindrical PLLA scaffolds were seeded with 200K MSCs suspended in 40 μL and allowed to expand for 7 days before undergoing 21 days of osteochondral differentiation in the biphasic bioreactor to mimic the cartilage-bone interface.

Cryosectioning of collected PLLA scaffolds for histological analysis.
Collected scaffolds were embedded and sectioned in the cryostat at 10 μm. Tape stabilization was used to preserve non-decalcified samples for optimal staining. Each section and tape was transferred together and mounted onto slides using a 0.75% chitosan in 0.375% acetic acid solution, tissue side upwards.

Characterization of osteochondral differentiation.
Histology was performed to probe protein and matrix deposition, including H&E, Alcian Blue, Alizarin Red, and Safranin-O staining. Top (cartilaginous) and bottom (osseous) thirds of scaffolds were separated and analyzed with RT-qPCR to quantitatively compare chondrogenic and osteogenic gene expression (Chondrogenic: SOX9, COL2, ACAN, Osteogenic: RUNX2, ALP, SPP1).

Results, Conclusions, and Discussions:

Results:
Human MSC Validation. PLLA scaffolds seeded with human MSCs show local differentiation of bone and cartilage tissue in their respective regions. Histological analysis of MSC-seeded PLLA scaffolds showed tissue specific formation of cartilage and bone within small pore and large pore regions respectively. Safranin-O staining of the scaffold showed increased Glycosaminoglycan (GAG) presence on the small pore size region of the scaffold (Figure 1). In contrast, Alizarin Red staining depicted strong calcium presence on the large pore size region of the scaffold.
Rabbit MSC Validation. Trilineage differentiation and 3D pellet chondrogenesis of the harvested rabbit MSCs validated the cells ability to differentiate into different tissue types (Figure 2). Safranin O staining of pellets revealed increased presence of proteoglycans and nuclei when comparing D14 with D28 images. Strong cartilage matrix presence was also indicated by Safranin O staining in comparison to non-cartilage or bone regions stained with Fast Green.

Discussions:
Validation of our harvested MSCs confirms their ability to differentiate into cartilage and bone when it is seeded into PLLA scaffolds and differentiated in the biphasic bioreactor. Subsequent analysis of the PLLA scaffolds revealed an increased presence of GAGs indicative of cartilaginous tissue formation on the small pore side versus a higher presence of calcium pointing to osseous tissue formation on the large pore size. Histological and gene expression analysis of the construct validates the PLLA gradient porous scaffold as a promising scaffold for simultaneous pore size driven tissue differentiation across an osteochondral junction.

Conclusions:
Our study shows simultaneous chondrogenic and osteogenic differentiation of MSCs seeded into the PLLA scaffold after differentiation in the biphasic bioreactor. Validation of the scaffold in vitro provides critical supporting data prior to in vivo implantation into animals for clinical translation. A scaffold that can drive local differentiation independent of local growth factor delivery would have a translationally significant potential for osteochondral defect repair.

Acknowledgements (Optional):