Regenerating articular tissue by converging technologies

Abstract

Scaffolds for osteochondral tissue engineering should provide mechanical stability, while offering specific signals for chondral and bone regeneration with a completely interconnected porous network for cell migration, attachment, and proliferation. Composites of polymers and ceramics are often considered to satisfy these requirements. As such methods largely rely on interfacial bonding between the ceramic and polymer phase, they may often compromise the use of the interface as an instrument to direct cell fate. Alternatively, here, we have designed hybrid 3D scaffolds using a novel concept based on biomaterial assembly, thereby omitting the drawbacks of interfacial bonding. Rapid prototyped ceramic particles were integrated into the pores of polymeric 3D fiber-deposited (3DF) matrices and infused with demineralized bone matrix (DBM) to obtain constructs that display the mechanical robustness of ceramics and the flexibility of polymers, mimicking bone tissue properties. Ostechondral scaffolds were then fabricated by directly depositing a 3DF structure optimized for cartilage regeneration adjacent to the bone scaffold. Stem cell seeded scaffolds regenerated both cartilage and bone in vivo.

Figure 1. Schematic draw of the scaffold process fabrication for (a) design A and (b) design B.

(a) P = Pillar; TC = Truncated Cone; S = Spherical. (b) an optical micrograph of the porous structure of the 3DF hollow cylinder is shown; scale bar: 400 µm. (c) X-ray diffraction pattern and (d) FTIR spectrum of rapid prototyped BCP particles where characteristic peaks are highlighted. Shrinkage following sintering of BCP particles varied from 7±0.7% to 18±1.9%.

Figure 2. SEM micrographs of designs A (a, b) and B (c, d) integrated 3D hybrid scaffolds.

(a) BCP particles inserted in the pores of a 3DF 1000PEOT70PBT30 matrix. (b) microstructure of ceramic particles sintered at T = 1150°C. (c) BCP cylinder infused with DBM after freeze drying. (d) Particular of the whole construct after insertion of the two DBM foamy discs, the infused BCP cylinder in the hollow 3DFM scaffold, and final lyophilization shows the integration of all the components. Scale bar: (a) 500 µm; (b) 10 µm; (c, d) 1 mm. Pillar particles (arrows) are shown here as an exemplification. P = polymer.

Figure 3. Optical microscopy images of the osteochondral 3D scaffolds.

Without the interlocking concentric fiber system (a) both compartments could be separated easily. With the intertwined fibers (c) the osteochondral construct maintained its integrity (b) under mechanical stress. Scale bar: 1 mm. Influence of the scaffold design on the bending (d) and compressive (e, f) storage modulus in designs A (d, e) and B (f). The dynamic stiffness of the single components comprising the hybrid scaffold was also measured for comparison. (g) Stress and strain at break for design A (influence of particle design), design B, and BCP. (*) Strength values for bone are taken from Athanasiou et al. . All groups were significantly different from each other (p<0.05). Particle shape legend: I = Irregular; S = Spherical; P = Pillar; TC = Truncated Cone.

Figure 4. New bone formation in (a) rats and (b) rabbits.

(a) Polymeric-DBM 3D scaffolds were implanted for 4 weeks intramuscularly in rats and new bone (arrows) was formed in direct apposition of DBM. (b) When implatend in ulna defects, these scaffolds repaired the defect in 6 weeks with re-establishment of bone marrow (thin black arrows). Polymer degradation was also visible at this time (yellow arrows). NB = new bone; PA = polymeric 3DF scaffold; DBM = demineralized bone matrix.

Figure 5. Osteochondral Scaffolds seeded with MSCs before (a, b) and after (c–f) subcutaneous implantation in nude mice.

(a) Cell aggregates in the chondral compartment maintained a rounded morphology typical of chondrocytes; insert shows stable aggregate formation after 48–72 hours in chondrogenic media. (b) Cell attached and spread on the BCP particles in the bone part; insert shows methylene blue staining of attached cells on porous pillars. (c) Bone part of the osteochondral construct.: pores were filled with de novo bone (fuchsin red staining). Note the embedded osteocytes and the osteoblasts laying at the outer edge of the mineralized matrix. (d) Occasionally, hypertrophic cells with positive stained matrix could be seen in the pores (thionine). (e) Cartilage part of the osteochondral construct: Cartilage tissue could be observed in the chondral part. Cells exhibit a round, chondrocyte-like morphology, locate din lacunae and surrounded by positive extracellular matrix (thionine staining). (f) Hypertrophic cells in the center of mineralized matrix (fuchsin red staining) and embedded osteocytes could be also occasionally found. Scale bar: (a, b) 50 µm; (c–f) 200 µm; Insert in (a): 250 µm; (b) 600 µm.