Multiphasic construct studied in an ectopic osteochondral defect model

Abstract

In vivo osteochondral defect models predominantly consist of small animals, such as rabbits. Although they have an advantage of low cost and manageability, their joints are smaller and more easily healed compared with larger animals or humans. We hypothesized that osteochondral cores from large animals can be implanted subcutaneously in rats to create an ectopic osteochondral defect model for routine and high-throughput screening of multiphasic scaffold designs and/or tissue-engineered constructs (TECs). Bovine osteochondral plugs with 4 mm diameter osteochondral defect were fitted with novel multiphasic osteochondral grafts composed of chondrocyte-seeded alginate gels and osteoblast-seeded polycaprolactone scaffolds, prior to being implanted in rats subcutaneously with bone morphogenic protein-7. After 12 weeks of in vivo implantation, histological and micro-computed tomography analyses demonstrated that TECs are susceptible to mineralization. Additionally, there was limited bone formation in the scaffold. These results suggest that the current model requires optimization to facilitate robust bone regeneration and vascular infiltration into the defect site. Taken together, this study provides a proof-of-concept for a high-throughput osteochondral defect model. With further optimization, the presented hybrid in vivo model may address the growing need for a cost-effective way to screen osteochondral repair strategies before moving to large animal preclinical trials.

Keywords: biomaterials; in vivo model; multiphasic scaffold; osteochondral repair; tissue engineering.

Figure 1.

(a) Overview of the experimental design. Triphasic constructs were composed of chondrocyte-seeded bilayered (S, MD) 2% alginate gels and osteoblast-seeded PCL biphasic scaffolds. Alginate gels were cultured in chondrogenic media for 12 weeks and subjected to one week of compressive stimulation prior to in vivo implantation. Triphasic constructs seeded with chondrocytes and osteoblasts were imaged using stereo microscopy, confocal microscopy (rhodamine–phalloidin (red), DAPI (blue)) and scanning electron microscopy (b) (scale bar, 1 mm). Prior to the rat subcutaneous implantation, osteochondral cores prepared from bovine knees were filled with triphasic constructs and the cartilaginous compartment was covered with electrospun PCL mesh (c) (scale bar, 6 mm). Osteochondral constructs were implanted in rat subcutaneously in the dorsal pouches underneath the skin (d) and allowed to mature for 12 weeks.

Figure 2.

Zonal construct analysis after 12 weeks of pre-culture and one week of compressive loading (Comp) or free-swelling (NC) culture. (a) S and MD constructs expressed both collagen types I and II during the long-term culture. (b) DNA and (c) GAG content in constructs did not vary significantly regardless of zones or loading conditions. MD constructs were significantly stiffer compared with the S constructs under free-swelling conditions, and one-week compression decreased the stiffness in MD zone constructs (d). **p < 0.01. Scale bars, 100 µm.

Figure 3.

EPIC-µCT images and histograms of attenuation in the cartilaginous compartments (dotted area) following in vivo implantation. Lower attenuation levels indicate higher GAG content and high attenuation levels indicate bone or tissues with low GAG content. (a) Cell-free alginate gel, (b) alginate gel subjected to one week of compression and (c) non-loaded gel implanted within the cartilaginous compartment had similar attenuation levels between the different groups, and depth-dependent zonal variations were not observed. Bone-like attenuation patterns were visible in the outer rims of the bovine cartilage in all explants.

Figure 4.

µCT images showing mineralized tissues in the scaffold. Explants of the triphasic scaffold inserted in bovine core, which had cell-free (osteoblast) (a,e) and osteoblast-seeded bone compartments (patient 1: b,f; patient 2: c,g; patient 3: d,h), had little bone formation in the osseous compartment. Explants that contained alginate gels subjected to a one-week compressive loading prior implantation are shown in (e–h), and those that had non-loaded alginates are shown in (a–d). Mineralization was visible on the outer rims of the cartilage in all groups including the cell-free controls. (i) Bone volume within the bovine osteochondral defect as measured by µCT. There was no statistical difference among the three groups (cell-free n = 4, Comp and NC n = 14). (Online version in colour.)

Figure 5.

(a,b) Safranin-O and (c,d) collagen type II-stained images of bovine cartilage tissue following in vivo implantation. Areas of the cartilage corresponding to the mineralized regions are indicated by the open arrow. (b,d) Scale bars, 200 µm. (Online version in colour.)

Figure 6.

Histological and immunohistochemical images of explant cartilaginous compartment. While explants implanted with both NC (a,d,g,j,m,p,s) and Comp (b,e,h,k,n,q,t) alginate gels had retained much of the alginate constructs during the 12-week in vivo culture, those in the cell-free group had little alginate left (c,f,i,l,o,r,u). While S and MD alginate gels remained intact in the cell-seeded groups, fibrous tissue infiltration was visible in the interface between the two gels (arrows). Cells found within the alginate compartment in all groups formed dense aggregates but did not stain strongly for either collagen type I (j–o) or II (p–u). Scale bars: (a–c), 1 mm; (d–f, j–l, p–r), 200 µm; (g–j, m–o, s–u), 50 µm. (Online version in colour.)

Figure 7.

Representative imaging of the bovine bone across the different groups. vWF staining shows well-developed blood vessels in and around the bovine bone (black arrowhead) (a), while little to no defined vWF staining is visible within the osseous scaffold within the defect (b) from a NC sample. Osteocyte etching reveals well-preserved cells in the bone that are in close proximity to the infiltrated fibrous tissues (black arrowhead) (c) and those that are well within the bone (d) from a NC sample. Representative histological images from a NC sample, which indicate areas within the bone staining intensely with Safranin-O (top (e), bottom (g), white arrowhead). Sections of a compressed sample stained for ALP show depth-dependent staining patterns with more cells staining for ALP near the top portion of the bone (f) compared with those in the bottom (h) (black arrowheads). Scale bars: (a,b,e–h), 200 µm; (c,d), 50 µm. (Online version in colour.)

Figure 8.

Haematoxylin-/eosin-stained sections of osteochondral explants. (a–d) NC, (e–h) Comp and (i–l) cell-free. Fibrous tissue infiltration was seen in both the inner and outer edges near the lower portion of the bovine cartilage in all explants (a,e,i, and arrowheads in b,f,j). Fibrous tissue infiltration was also seen in the upper part of the cartilage and they were seen primarily from the inner edges of the cartilage (arrowheads in c,g,k). Fibrous tissues also fully infiltrated the bovine bone, PCL-FDM scaffolds and the PCL mesh (a,e,i,d,h,l). Scale bars: (a,e,i), 1 mm; (b–d,f–h,j–l), 200 µm. (Online version in colour.)