Early loss of subchondral bone following microfracture is counteracted by bone marrow aspirate in a translational model of osteochondral repair

Abstract

Microfracture of cartilage defects may induce alterations of the subchondral bone in the mid- and long-term, yet very little is known about their onset. Possibly, these changes may be avoided by an enhanced microfracture technique with additional application of bone marrow aspirate. In this study, full-thickness chondral defects in the knee joints of minipigs were either treated with (1) debridement down to the subchondral bone plate alone, (2) debridement with microfracture, or (3) microfracture with additional application of bone marrow aspirate. At 4 weeks after microfracture, the loss of subchondral bone below the defects largely exceeded the original microfracture holes. Of note, a significant increase of osteoclast density was identified in defects treated with microfracture alone compared with debridement only. Both changes were significantly counteracted by the adjunct treatment with bone marrow. Debridement and microfracture without or with bone marrow were equivalent regarding the early cartilage repair. These data suggest that microfracture induced a substantial early resorption of the subchondral bone and also highlight the potential value of bone marrow aspirate as an adjunct to counteract these alterations. Clinical studies are warranted to further elucidate early events of osteochondral repair and the effect of enhanced microfracture techniques.

Figure 1. Schematic drawing depicting the study design.

Standardized circular (diameter 4 mm), full thickness chondral defects were created in the trochlear facets of the hind legs of minipigs. (a) Three treatments were applied including debridement alone (debridement group), debridement and microfracture (microfracture group), and debridement and bone marrow aspirate enhanced microfracture (enhanced microfracture group). (b) Top view of defects following the three treatments. (c) The trocar-shaped microfracture awl tip consists of a distal trihedral head (length 2.6 mm), a middle cylindrical body (length 2.4 mm, diameter 1.2 mm), and a proximal penetration stop, allowing a standardized penetration depth to 5.0 mm. Articular cartilage: light blue; calcified cartilage: dark blue; subchondral bone: grey; bone marrow aspirate: red.

Figure 2. Macroscopic, histological and immunohistochemical analyses of the osteochondral unit.

Standardized circular full-thickness chondral defects in the trochlear groove of minipigs were treated by (1) debridement (debridement group), (2) debridement and microfracture (microfracture group), and (3) debridement, microfracture, and application of bone marrow aspirate (enhanced microfracture group). By macroscopic grading, no significant differences in articular cartilage repair were detected between debridement group. (a), microfracture group (b), and enhanced microfracture group (c). According to Sellers et al., histological analysis of the cartilaginous repair tissue stained with safranin O (d–i) and hematoxylin and eosin (j–o) also yielded no significant differences between debridement (d,g,j,m), microfracture (e,h,k,n), and enhanced microfracture group (f,i,l,o), except for a significantly higher percentage of subchondral bone within defects treated by debridement alone (v). Immunoreactivity to type-II collagen⁶⁴ was similar between defects treated with debridement (p,s), microfracture (q,t), and enhanced microfracture (r,u). (g–i,m–o and s–u) show the corresponding higher magnification images of the area within the dotted lines in the upper images. Black triangles denote defect borders (d–u). Scale bars: 2.0 mm (a–c), 0.5 mm (d–f,j–l,p–r), and 1.0 mm (g–i,m–o,s–u).

Figure 3. Histomorphometric analyses of subchondral bone changes below the cartilage defects treated with the three experimental strategies.

(a) Representative images showing the pattern of subchondral bone changes in sections through the central region of defects stained with Masson-Goldner trichrome staining. The yellow dotted boxes indicate the defined region of interest (ROI) (3.0 mm × 4.0 mm) selected for the histomorphometric analyses. The ROI was divided into superficial, middle and deep zone (each zone 1.0 mm × 4.0 mm). (b) No significant differences were seen between the three groups for BV/TV, Tb.Th, Tb.Sp, and Tb.N. (c) Typical higher-magnification image of the interface between the repair tissue and the subchondral bone, showing an accumulation of osteoclasts (black arrowheads) and osteoblasts (black arrows) as well as newly formed bone (black stars). The asterisks indicate the marrow cavity and the white arrowheads indicate blood vessels. (d) Density of osteoclasts and osteoblasts within the defined ROI. Compared with enhanced microfracture and debridement only, a significant higher density of osteoclasts was detected in the microfracture group (without bone marrow aspirate). Microfracture without and with bone marrow aspirate recruited more osteoblasts than debridement alone to the treated subchondral bone. (e) Percentage of areas occupied by marrow cavity (white), original (mature) bone (dark red), new bone (light red), and repair tissue (green) within the defined ROI of defects from the three individual groups. Compared with microfracture alone, a significant larger area of marrow cavity and higher preservation rate of original (mature) bone were observed in the enhanced microfracture group. *Indicates a significant difference between debridement and microfracture group, ^#indicates a significant difference between debridement and enhanced microfracture, and ^§indicates difference between microfracture and enhanced microfracture. Scale bars: 1.0 mm (a); 200 μm (c).

Figure 4. Qualitative and quantitative analysis of subchondral bone changes underlying defects treated with the three strategies.

(a) Representative micro-CT images of subchondral bone changes. The top row shows a 3D reconstruction of characteristic defects. The subchondral bone plate and subarticular spongiosa are colored in blue and red, respectively. The yellow dashed ellipse shows the margin of the defect. The middle and bottom rows represent two different sections of the defects of the top row. Removal of the calcified cartilage layer in the debridement group induced subchondral bone loss (red arrows). In defects from the microfracture and enhanced microfracture group, residual microfracture holes and peri-hole bone resorption were present. The yellow dashed lines indicate the projected cement lines, and the red asterisks indicate the preserved bone bridge between two residual microfracture holes. Scale bar: 2 mm. (b) Comparison of the micro-CT parameters of SBP-defect between the three treatment groups. ^*P < 0.05. (c) Comparison of the micro-CT parameters of SAS-defect between the three treatment groups. ^*P < 0.05.

Figure 5. Mathematical modeling of subchondral bone volume changes within defect region following the three treatments.

(a) Comparison of the calculated BV/TV of SBP-defect [SBP-defect(calculated)] and the measured BV/TV of SBP-defect [SBP-defect(measured)]. (b) Comparison of BV/TV of SAS-defect(calculated) and BV/TV of SAS-defect(measured). (c) Comparison of preservation rate of BV/TV of SBP-defect(calculated) and SAS-defect(calculated) of the three groups. No apparent differences were found in preservation rates of BV/TV of SBP-defect(calculated) between the three groups, but additional application of aspirates yielded the highest preservation rate of BV/TV of SAS-defect(calculated). *P < 0.05.

Figure 6. Standardized regions of interest (ROI) for the quantitative micro-CT evaluation of the subchondral bone.

(a) Four standardized ROIs were defined on micro-CT images. SBP-defect involved exclusively the subchondral bone plate within the defect, and the underlying subarticular spongiosa (SAS-defect). SBP-adjacent and SAS-adjacent were located laterally neighboring SBP-defect and SAS-defect correspondingly. The maximum total vertical depth of SBP and SAS was 3.0 mm. Overlapping of individual ROIs was strictly avoided. (b) Schematic of the ROIs for subchondral bone evaluation. Note the ~10-fold larger ROI of the subarticular spongiosa compared with the subchondral bone plate.

Figure 7. Mathematical modeling for calculating the change of subchondral bone volume following the microfracture procedure.

(a) Schematic of the microfracture awl tip of Fig. 4c.(b) Volume of interest (VOI) of the entire subchondral bone underlying the full-thickness chondral defect is composed of two cylinders. The top cylinder is VOI of SBP-defect with height 0.1 mm (average value of 20 measurements) and diameter 4.0 mm, and the bottom one is VOI of SAS-defect with height 2.9 mm and diameter 4.0 mm. (c) In this model, the three microfracture impactions excise triple volume of the microfracture awl tip. Of note, when interacting with the subchondral bone, most of the trihedral head of the awl tip penetrates beyond the defined region of VOI of the subchondral bone (SBP-defect and SAS-defect). Therefore, the excised subchondral bone within the VOI of the subchondral bone is approximately equal to two cylindrical sections: one is within SBP-defect (height 0.1 mm; diameter 1.2 mm), and the other is within SAS-defect (height 2.9 mm; diameter 1.2 mm).