Regeneration of the articular surface of the rabbit synovial joint by cell homing: a proof of concept study

Abstract

Background: A common approach for tissue regeneration is cell delivery, for example by direct transplantation of stem or progenitor cells. An alternative, by recruitment of endogenous cells, needs experimental evidence. We tested the hypothesis that the articular surface of the synovial joint can regenerate with a biological cue spatially embedded in an anatomically correct bioscaffold.

Methods: In this proof of concept study, the surface morphology of a rabbit proximal humeral joint was captured with laser scanning and reconstructed by computer-aided design. We fabricated an anatomically correct bioscaffold using a composite of poly-epsilon-caprolactone and hydroxyapatite. The entire articular surface of unilateral proximal humeral condyles of skeletally mature rabbits was surgically excised and replaced with bioscaffolds spatially infused with transforming growth factor beta3 (TGFbeta3)-adsorbed or TGFbeta3-free collagen hydrogel. Locomotion and weightbearing were assessed 1-2, 3-4, and 5-8 weeks after surgery. At 4 months, regenerated cartilage samples were retrieved from in vivo and assessed for surface fissure, thickness, density, chondrocyte numbers, collagen type II and aggrecan, and mechanical properties.

Findings: Ten rabbits received TGFbeta3-infused bioscaffolds, ten received TGFbeta3-free bioscaffolds, and three rabbits underwent humeral-head excision without bioscaffold replacement. All animals in the TGFbeta3-delivery group fully resumed weightbearing and locomotion 3-4 weeks after surgery, more consistently than those in the TGFbeta3-free group. Defect-only rabbits limped at all times. 4 months after surgery, TGFbeta3-infused bioscaffolds were fully covered with hyaline cartilage in the articular surface. TGFbeta3-free bioscaffolds had only isolated cartilage formation, and no cartilage formation occurred in defect-only rabbits. TGFbeta3 delivery yielded uniformly distributed chondrocytes in a matrix with collagen type II and aggrecan and had significantly greater thickness (p=0.044) and density (p<0.0001) than did cartilage formed without TGFbeta3. Compressive and shear properties of TGFbeta3-mediated articular cartilage did not differ from those of native articular cartilage, and were significantly greater than those of cartilage formed without TGFbeta3. Regenerated cartilage was avascular and integrated with regenerated subchondral bone that had well defined blood vessels. TGFbeta3 delivery recruited roughly 130% more cells in the regenerated articular cartilage than did spontaneous cell migration without TGFbeta3.

Interpretation: Our findings suggest that the entire articular surface of the synovial joint can regenerate without cell transplantation. Regeneration of complex tissues is probable by homing of endogenous cells, as exemplified by stratified avascular cartilage and vascularised bone. Whether cell homing acts as an adjunctive or alternative approach of cell delivery for regeneration of tissues with different organisational complexity warrants further investigation.

Funding: New York State Stem Cell Science; US National Institutes of Health.

Figure 1. Surgical replacement of synovial joint

Surface morphology of a rabbit joint was reconstructed (A) to design an anatomically correct bioscaffold (B) with an intramedullary stem. A 200-μm thick shell was designed, along with internal microchannels opening to the synovium cavity (C) and bone marrow (D). PCL-HA was used to fabricate bioscaffolds following computer-aided design (E). The humeral head was excised at its metaphysis junction (F), and an orthopaedic drill used to create an intramedullary tunnel for stem fixation (G). The bioscaffold (H) was implanted by press-fitting (I). In defect-only rabbits, haematoxylin and eosin staining 4 months after surgery (J) showed that little bone had regenerated in the defect; the synovial joint cavity (sc) was visible with fibrous tissue (f) covering bone and marrow (m). Safranin O staining (K) showed scarce chondrocyte-like cells (shown by arrows) in the defect area in the synovial cavity. PCL-HA=poly-ε-caprolactone hydroxyapatite.

Figure 2. Articular cartilage regeneration

India ink staining of (A) unimplanted bioscaffold, (B) TGFβ3-free and (C) TGFβ3-infused bioscaffolds after 4 months’ implantation, and (D) native cartilage. (E) Number of chondrocytes present in TGFβ3-infused and TGFβ3-free regenerated articular cartilage samples (n=8 per group). Safranin O staining of TGFβ3-free (F, I) and TGFβ3-infused (G, J) articular cartilage. Matrix density (H) and cartilage thickness (K) of TGFβ3-infused and TGFβ3-free samples (n=8 per group for both comparisons). TGFβ3=transforming growth factor β3. PCL-HA=poly-ε-caprolactone hydroxyapatite.

Figure 3. TGFβ3 delivery and quality of articular cartilage

Immunostaining for Col-II and AGC in unimplanted bioscaffold (A–D) and native (E–H), TGFβ3-free (I–L), and TGFβ3-infused (M–P) articular cartilage samples. Immunoreactivity for Col-II (Q) and AGC (R) in native, TGFβ3-free, and TGFβ3-infused samples (n=10 per group). Col-II=collagen type II. AGC=aggrecan. TGFβ3=transforming growth factor β3.

Figure 4. Vascularisation of regenerated subchondral bone

(A) Radiolucency (shown by arrow) was present in the joint cavity on excision of the condylar head. By 8 weeks (B) and 16 weeks (C) after surgery, a convex radio-opaque structure in the shape of articular condyle was present. (D) After 16 weeks’ implantation, regenerated cartilage (c) extended to subchondral bone (b), which consisted of trabecular structures (E). (F, G) Von Kossa staining indicated mineral deposition that extended from the cartilage region (c, blue area) longitudinally in microchannels (m). (H) Bone trabeculae were populated by columnar osteoblast-like cells (which are shown by arrows). (I) Regenerated bone integrated to native humeral bone (arbitrary boundary shown by dashed blue line). (J) Multiple blood vessels (example shown by arrow) were present in regenerated bone. High magnification (K) showed the presence of erythrocytes within the lumen of an endothelium-lined blood vessel (arrow). PCL-HA=poly-ε-caprolactone hydroxyapatite.

Figure 5. Mechanical properties of regenerated cartilage

E*=complex compressive modulus. E′=storage modulus. E″=loss modulus. G*=complex shear modulus. G′=shear storage modulus. G″=shear loss modulus. TGFβ3=transforming growth factor β3.