Osteoarthritis is a debilitating disease characterized by progressive deterioration of the osteochondral unit.1 Derangements in the subchondral bone (SCB) play a critical role in the initiation of osteochondral insufficiency, osteoarthritis progression, and the presence of pain.2–7 Bone marrow lesions (BMLs) have been increasingly diagnosed in osteoarthritic patients on magnetic resonance imaging (MRI), corresponding to areas of microtrabecular damage, necrosis, and fibrosis.2,4 BMLs have also been strongly associated with the presence and severity of knee pain in humans8 and are predictive of subchondral attrition,9 loss of cartilage volume,10 and disease progression.5 As a result, treatment strategies supporting the SCB and articular cartilage have become increasingly important.
Numerous surgical techniques are available to address cartilage defects associated with joint disease6,11–20; however, lesion repair is hampered by the inferior molecular composition of the repair tissue, lack of tissue integration, and failure to reform normal SCB and calcified cartilage layers.1 A current mainstay for treating full-thickness cartilage defects is microfracture,13 a marrow stimulation technique involving multifocal penetration of the subchondral plate to allow infiltration of marrow contents for improved cartilage repair. Despite yielding positive short-term results in some populations,18,21 it ultimately results in progressive joint deterioration and long-term failure secondary to its adverse effects on the SCB.6,12,18,22–28
Subchondroplasty, a treatment for osteoarthritis-related BMLs in humans, has resulted in improved pain and function in patients with severe knee osteoarthritis.3,29–33 The injection of a flowable, osteoconductive, calcium phosphate bone substitute material (BSM) into the BML may help stabilize mechanical SCB insufficiency and stimulate repair by providing a scaffold for the ingrowth of endogenous osteocytes.3 It is possible that this technique could augment current surgical therapies, including microfracture, to allow better tissue integration and subchondral stability.
An intervention with the capacity to relieve symptoms, repair SCB, and alter the long-term progression of joint deterioration is currently lacking. Using a validated equine model of articular cartilage loss, the objective of this pilot study was to assess the feasibility of a modified subchondroplasty (mSCP) technique as a potential treatment strategy in horses to target all affected osteochondral tissues, improve the integration of implanted materials, and reduce the negative side effects of microfracture. We aimed to evaluate (1) the administration of 2 injectable materials into experimentally created, full-thickness cartilage defects treated with microfracture using the mSCP technique, and (2) the initial integration of and short-term host tissue response to the injected materials. We hypothesized that subjectively, the mSCP technique would be a simple, reproducible technique for the filling of full-thickness cartilage defects in the medial trochlear ridge of the femur in horses and that this technique would result in the perfusion of the injected material into the targeted defect with minimal adverse effects on the host tissues.
Materials and Methods
Study design
This was a randomized, controlled pilot study using 3 normal horses of mixed breeds (all mares; age, 4 to 8 years; weight, 323 to 517 kg). All procedures were approved by the Institutional Animal Care and Use Committee at Colorado State University (No. 19-9232A). Horses without perceptible lameness at the walk or trot (grade 0 according to the American Association of Equine Practitioners [AAEP] Lameness Scale), palpable femoropatellar joint effusion, or radiographic evidence of stifle disease were used.
Two full-thickness cartilage defects were created on the medial trochlear ridge of each femur using a previously-validated model,16,34,35 resulting in 4 defects per horse. Defects were randomly assigned to receive 1 of 4 treatments in addition to microfracture (Figure 1): (1) autologous fibrin graft via subchondral injection of a fibrin glue matrix (FG), (2) autologous fibrin graft via direct injection of FG, (3) subchondral injection of a BSM combined with an autologous fibrin graft (direct injection), and (4) negative control to assess the effects of the defect and mSCP drill tract creation alone. Treatments were assigned using a random number generator (Supplementary Table S1). It was not the goal of this study to compare the efficacy of mSCP to other techniques; thus, treatments 2 and 4 were a point of reference for assessing treatments 1 and 3, which were performed to assess the use of 2 different injectable materials for this technique. Horses were euthanized 2 weeks postoperatively with an overdose of barbiturate for advanced diagnostic imaging, microcomputed tomography (microCT), and gross and histologic examination of the SCB and cartilage defects.
Schematic (transverse plane) of the 4 treatment groups, including the route of injection of the fibrin glue matrix and bone substitute material (BSM). Two 15-mm-diameter full-thickness cartilage defects were made on the medial trochlear ridge of each femur and treated with microfracture prior to treatment administration.
Citation: American Journal of Veterinary Research 84, 4; 10.2460/ajvr.22.12.0219
Injectate preparation: autologous fibrin glue matrix
Within 4 hours preoperatively, blood was aseptically collected from each horse into individual 450-mL double blood collection bags containing CPDA#1 anticoagulant solution (JorVet; Jorgensen Laboratories). The FG was aseptically prepared as a solution of 75% fibrinogen in autologous serum. The fibrinogen concentration was selected based on preliminary ex vivo evaluation comparing autologous FG containing 25%, 50%, 75%, or 100% fibrinogen for the mSCP technique in equine cadavers. Autologous FG containing 75% fibrinogen had the best injection properties without compromising the coagulation of the final graft. An equal volume of sterile 0.54 kU/mL bovine thrombin (Sigma-Aldrich) was injected simultaneously with the FG through a joined mixing tip and dual-syringe injection system (DUPLOJECT; Baxter Healthcare Inc).
Injectate preparation: bone substitute material
A commercially available calcium phosphate BSM (AccuFill; Zimmer Biomet) was acquired for this study. Using a closed, controlled mixing system (AccuMix system; Zimmer Biomet), the BSM was prepared intraoperatively immediately prior to injection according to manufacturer instructions.36
Surgical technique
Horses were administered perioperative cefazolin (11 mg/kg body weight, intravenously [IV], every 8 hours), gentamicin (6.6 mg/kg body weight, IV, every 24 hours), and phenylbutazone (4.4 mg/kg body weight, IV, once). With both stifles in full extension, the medial trochlear ridge of each femur was exposed via a cranial femoropatellar joint arthrotomy. Using a 15-mm-diameter chondral biopsy punch, 2 impressions were made in the articular cartilage, 1 proximal and 1 distal, approximately 1-cm apart. A bone curette was used to remove the articular and calcified cartilage layers within each defect.34 The site was flushed with sterile saline to remove debris. Microfracture was performed using a 40° surgical awl (Chondral Pick 40°, Ref. AR-1762; Arthrex, Inc), with ten 2-mm-diameter perforations made uniformly through the SCB plate with a bony bridge present between holes (Supplementary Figure S1).
For treatments 1, 3, and 4, the closed tip of a mosquito hemostat was placed at the caudal-most margin within the medial femoropatellar joint pouch, aligned with the respective defect. A stab incision was made over the tip of the hemostats. An aiming device, positioned with the drill sleeve through the stab incision and the pointed tip at the center of the axial defect margin, was used to guide a 2.0-mm drill bit in a caudoabaxial-cranioaxial direction toward the corresponding defect. The drill bit was advanced under radiographic guidance (Supplementary Figure S2), aiming to stop the tip of the drill bit approximately 10-mm below the subchondral plate.4,32 For treatment 4, no treatments were applied following the creation of the drill tract.
For treatment 1, a 2-inch 11-gauge Jamshidi cannula with sharp trocar and multidirectional outflow fenestrations (Wright Medical Group) was inserted into the drill tract. The defect was dried using sterile cotton-tip applicators. The FG was injected through the cannula such that it perfused into the defect through the microfracture holes. The injection was continued until the graft was slightly proud to the adjacent cartilage surface, as contraction of the FG during coagulation reduces matrix size. The trocar was replaced, and the FG was allowed to set up for 3 minutes before the cannula was removed.
Treatment 2 represented a method of treating full-thickness cartilage defects clinically; therefore, a drill tract was not made following the creation of the defect. The base of the defect was dried, and the defect was filled via direct injection of the FG using the dual-syringe injection system.
For treatment 3, the Jamshidi cannula was inserted into the drill tract as in treatment 1. Immediately after mixing, the BSM was injected through the cannula until it began filling the microfracture holes while still remaining below the superficial margin of the subchondral plate (Supplementary Figure S3). The trocar was reinserted into the cannula and left in place for 8 to 10 minutes. During this time, the base of the cartilage defect was dried and the autologous fibrin graft was applied via direct injection.
The joint capsule and subcutaneous fascia were closed using 2-0 absorbable monofilament suture. The skin was closed using 2-0 nylon. A sterile tie-over bandage was secured over the incisions prior to recovery from general anesthesia. Intravenous antimicrobials and phenylbutazone were continued for 72 hours, with the phenylbutazone dose reduced to 2.2 mg/kg body weight, IV, every 12 hours. Horses were rested in a 3.65-m X 3.65-m box stall. The tie-over bandages were replaced every 3 days throughout the study.
Lameness examination
Complete lameness examinations were performed by the primary author the day before surgery (day −1) and on day 14. Lameness was graded (0 to 5) according to the AAEP Lameness Scale. The response to stifle flexion was graded (0 to 4) as no response, slight, mild, moderate, or severe. Range of motion through the stifle was graded (0 to 3) as normal or a mild, moderate, or severe reduction in range of motion. Femoropatellar joint effusion was graded (0 to 3) as none, mild, moderate, or severe. Details of the subjective scoring systems are provided elsewhere (Supplementary Table S2).
Digital radiography
Routine radiographic views of both stifles (lateromedial, flexed lateromedial, caudocranial, and caudolateral-craniomedial oblique) were obtained on days 1 and 14 postoperatively. Radiographs were evaluated by a board-certified radiologist blinded to defect treatment. Changes in the SCB surrounding the defects were subjectively compared between time points.
Postmortem evaluation
MRI and CT
All images were assessed by a board-certified radiologist blinded to defect treatment. The MRI of each stifle was performed using a high-field magnet (3-Tesla; Siemens MAGNETOM Skyra, Siemens Medical Solutions USA, Inc). MRI sequences are described elsewhere (Supplementary Table S3). Multiplanar T2-weighted dual echo steady state, T1 VIBE, and intermediate weighted fat-suppressed sequences (Dixon technique) were used to track the injectate and detect changes in the SCB and articular cartilage. Descriptions of the tissue signal within each defect, the percentage of the defect that was filled, changes in the surrounding bone, injectate distribution, and other relevant changes were recorded.
Dual-energy CT was performed with a multi-slice helical scanner (Siemens SOMATOM Definition AS; Siemens Medical Solutions USA, Inc) using 80 kV and 140 kV, effective 240 mAs, 3.0-mm slice thickness, and 270-mm field of view. Transverse, frontal, and sagittal slices were obtained in bone and soft tissue windows. CT imaging was performed as a complement to MRI to further characterize changes in the subchondral and trabecular bone and to better visualize the BSM distribution.
Gross evaluation
The femoropatellar joints were disarticulated to assess defect appearance and examine the joint for pathologic or iatrogenic lesions that may have occurred secondary to the procedure. Defects were photographed and graded semiobjectively for injectate retention and defect filling (1 = 0 to 25%, 2 = 26 to 50%, 3 = 51 to 75%, and 4 = 76 to 100% defect volume filled). The femoral trochleae were sectioned en bloc, wrapped in sterile gauze soaked with a proteinase inhibitor solution (2 mM Na2-EDTA, 1 mM phenylmethanesulfonyl fluoride, 5 mM benzamide-HCl, and 10 mM N-ethylmaleimide in Dulbecco’s phosphate-buffered saline), and stored at 4° C. Within 24 hours, a dissection saw was used to create bone blocks for microCT analysis.
Microcomputed tomography
For each block to fit properly within the scanner, the cut margins were 5 to 8 mm from the peripheral defect margins in the sagittal and transverse planes and 20 to 25 mm deep to the articular surface. The blocks were scanned at a resolution of 37-µm (Scanco µCT 80, Scanco USA Inc) with resultant DICOM images at an isotropic resolution of 37-µm. A cylindrical volume of interest (VOI; diameter of 15-mm and depth of 10-mm) was chosen to include the defect, microfracture holes, and some of the underlying trabecular bone. Quantitative measures of bone quality within each VOI were measured using Scanco µCT 80 analysis software: (1) bone volume fraction (BV/TV), (2) bone mineral density (BMD; mgHA/cm3), (3) mean trabecular number (Tb.N; 1/mm), (4) mean trabecular thickness (Tb.Th; mm), (5) mean trabecular spacing (Tb.Sp; mm), and (6) specific bone surface (BS/BV; 1/mm).
Histopathology
Following microCT, each defect was sectioned and faced through the center in the transverse plane. Bone sections were placed in 10% neutral buffered formalin for 48 hours. For decalcified bone processing, formalin-fixed tissues were placed in 10% EDTA for 3 to 6 weeks until softened. Bone sections were processed, embedded in paraffin, microtome sectioned (5-um), and stained (hematoxylin and eosin [H&E] and safranin O and fast green [SOFG] stains).
The host response was determined microscopically via blinded examination by a board-certified pathologist. Each section was semiobjectively graded based on the nature and integration of the repair tissue present, structural characteristics of the defect tissue and SCB, and degenerative changes in the adjacent normal cartilage. The grading system (Supplementary Table S4) was modified from a previously reported histological grading scale.16,37 Immunohistochemistry was used to better characterize cellular infiltrates present in select sections (Supplementary Appendix).
Data analysis
Due to the exploratory nature of this pilot study, a limited sample size was used while still allowing for variance between groups. Detection of statistically significant differences between treatments was not the primary objective; therefore, descriptive data, including surgical technique, short-term clinical side effects, gross examination, imaging findings, histologic characteristics, and subjective assessment of each treatment, were recorded. Measures of central tendency and variance (median and range) were reported for continuous data.
Results
Defects were successfully filled for all 3 treated groups without compromising coagulation of the FG in treatment 1 when injected through the SCB (Supplementary Figure S4). The size of the medial thigh musculature created variable interference with the positioning of the drill; however, the approach was completed successfully for all stifles. In horse 1, there was difficulty inserting the cannula into the drill tract for injection of the BSM (treatment 3), with multiple injection attempts made. The BSM was successfully injected 10 minutes after reconstitution without affecting the injectability of the material. All other defects were treated without complication.
Increased subcutaneous fluid accumulation was palpable on the cranial aspect of all stifles secondary to dehiscence of the joint capsule layer of the incision. Skin incisions remained intact with no evidence of drainage or infection. Horses 1 and 3 exhibited a moderate increase in left hind lameness at the walk 10 days postoperatively, characterized by a shortened cranial phase of the stride. A single dose of phenylbutazone (4.4 mg/kg body weight, IV) was administered, and the lameness improved by the end of the study. No other complications were encountered.
Lameness examination
A reduction in range of motion and bilateral increases in femoropatellar joint effusion and response to flexion was observed on day 14 compared to day −1 (grade 0). The median (range) grade on day 14 for each of these parameters was as follows: effusion 2.5 (2 to 4), stifle range of motion 1.5 (0 to 2), and response to stifle flexion 2.5 (1 to 4). On day 14, horses 1 and 3 exhibited a predominant grade 3/5 left hind limb lameness, while horse 2 exhibited a predominant grade 3/5 right hind limb lameness. Subjectively, there did not appear to be a correlation between treatments and changes in lameness parameters.
Digital radiography
Radiopaque material was visible within the medial trochlear ridge of the femurs that received treatment 3, surrounding and extending from the drill tract to the respective defect in all horses (Figure 2). This radiopacity was present at both time points, though more obvious on day 1. By day 14, there was mild to moderate widening of the microfracture holes with a defined rim of sclerosis for all defects. These changes were subjectively more apparent for defects not treated with the BSM. No other radiographic abnormalities were identified.
Lateromedial radiographs of each medial trochlear ridge of the femur on days 1 and 14 postoperatively. Defects are delineated by white lines and labeled with the corresponding treatment (1 to 4). The site of drill bit entry, surrounded by radiopaque bone substitute material, was visible in all defects from treatment 3 (arrowheads), though less apparent by day 14. The drill tract for treatment 1 (arrows) was seen in select images.
Citation: American Journal of Veterinary Research 84, 4; 10.2460/ajvr.22.12.0219
Postmortem assessment
MRI and CT
MRI revealed intermediate signal tissue within each defect, seen best on the T1 VIBE sequence (Figure 3). The median (range) percentage of each defect-containing tissue was 80% (70 to 90%), 50% (30 to 70%), 90% (25 to 100%), and 75% (0 to 100%) for treatments 1 to 4, respectively. All groups had irregular SCB margins surrounded by intermediate-signal intensity (fluid signal) on the intermediate-weighted Dixon fat-suppressed images, with a rim of sclerosis along the microfracture holes on the in-phase Dixon and PD images consistent with radiographic findings. In horse 3, focal resorption of the subchondral plate associated with the microfracture holes was seen on MRI and CT, characterized by smooth bone resorption and widening of the microfracture holes. This was present in all defects except for treatment 3.
Sagittal magnetic resonance and computed tomographic (CT) images of the medial trochlear ridge of the femur, representative of findings in all 3 horses. A—T1 VIBE sequence showing intermediate signal tissue within the 2 defects (*). B—Dixon fat-suppressed image with defects from treatments 1 (proximal) and 2 (distal). Hyperintense fluid signal surrounds and extends from the proximal drill tract to the respective defect, representing the distribution pattern of the fibrin matrix. Hyperintense fluid signal also surrounds the microfracture holes from treatment 2. C—Dixon fat-suppressed image of treatments 3 (distal) and 4 (proximal). Hypointense bone substitute material extends from around the drill tract to the subchondral plate, surrounded by a rim of hyperintense fluid signal. The drill tract for treatment 4 (arrow) is also visible. D and E—Sagittal CT images showing the distribution pattern of the bone substitute material (treatment 3) in 2 CT slices.
Citation: American Journal of Veterinary Research 84, 4; 10.2460/ajvr.22.12.0219
Signal patterns consistent with FG and BSM extended from the drill tract to the corresponding defects for treatments 1 and 3, respectively, with focal dispersion of each through the SCB. For treatment 1, FG was seen on sagittal fat-suppressed images as a hyperintense fluid signal surrounding and extending from the drill tract to the corresponding defect (Figure 3). There was no appreciable fluid signal within the trabecular bone surrounding the drill tract for treatment 4, suggesting that the creation of the drill tract alone did not induce enough trauma to create the fluid pattern seen for treatment 1. For treatment 3, sagittal fat-suppressed images revealed a circumferential hypointense signal around the drill tract that extended to the defect margin. This hypointense signal, confirmed to be BSM based on the matching dispersion pattern on CT, was surrounded by a rim of hyperintense fluid signal. No lesions were identified on CT that were not visible on MRI.
Additional MRI lesions included iatrogenic damage to the medial patellar ligament in 5 stifles, characterized by moderate to marked transverse fiber disruption at the level of the corresponding drill tract. In horse 2, surface irregularity was identified in the cranial medial meniscotibial ligament bilaterally, as well as marked fluid signal along the distal apex of both patellae. Horse 3 had similar patellar abnormalities, with moderate fluid signal in the distal apex bilaterally. Dehiscence of the joint capsule at the arthrotomy site was evident in all horses.
Gross evaluation
Gross evaluation revealed complete coverage of the base of the defects (Supplemental Figure S5), subjectively filled to an overall median (range) depth of 78% (30 to 100%). The defects with the least amount of tissue present were from treatments 3 (BSM) and 4 (control) for horse 2, both containing translucent, mildly hemorrhagic tissue filling to approximately 30% of the defect depth. Median grades (1 to 4) for defect fill were 3, 4, 3, and 4 for treatments 1 to 4, respectively. Individual defect grades are provided elsewhere (Supplemental Table S5).
Partial thickness wear lines were present along the proximal articular surface of the patella, bilaterally, in horses 1 and 2. These lesions were not identified on diagnostic imaging. The synovial lining was moderately hemorrhagic in appearance and mildly thickened. Dehiscence of the joint capsule incision and iatrogenic injury to the medial patellar ligaments were confirmed.
Microcomputed tomography
Quantitative analysis revealed increased bone volume ratio, trabecular number, and trabecular thickness for treatment 3 compared to other groups, as well as reduced trabecular separation (Table 1). The BSM diffusion pattern was consistent with that seen on diagnostic imaging. There were inconsistencies in drill tract placement and depth below the subchondral plate, as well as the point at which the drill tract ended relative to the defect (Figure 4). There were also shallow, focal defects in the subchondral plate in 5 defects. Since microCT could not be performed antemortem immediately following surgery, it is unknown whether these were the result of defect debridement, early resorption of the subchondral plate, or a combination of the 2. A sclerotic rim was present around each microfracture hole and, to a lesser extent, along the drill tract margins, consistent with imaging findings. Excluding bone resorption already identified on other modalities, no other significant changes were identified.
Results of the trabecular morphometry analysis performed on 15-mm-diameter by 10-mm-depth cylindrical volumes of interest for each cartilage defect.
| Treatment 1 | Treatment 2 | Treatment 3 | Treatment 4 | |
|---|---|---|---|---|
| BV/TV | 0.342 | 0.336 | 0.494 | 0.371 |
| BMD (mg HA/cm3) | 699.619 | 697.245 | 690.515 | 691.626 |
| Tb.N (1/mm) | 1.905 | 1.873 | 2.195 | 1.997 |
| Tb.Th (mm) | 0.226 | 0.237 | 0.357 | 0.253 |
| Tb.Sp (mm) | 0.505 | 0.488 | 0.405 | 0.471 |
| BS/BV | 11.056 | 10.796 | 7.237 | 10.078 |
Means are reported for each treatment group.
BS/BV = Specific bone surface. BMD = Bone mineral density. BV/TV = Bone volume fraction. Tb.N = Trabecular number. Tb.Sp = Trabecular spacing. Tb.Th = Trabecular thickness.
Transverse linear microCT images through each 15-mm-diameter defect, demonstrating the variability in drill tract placement (blue dotted outline) for treatments 1, 2, and 4, and the dispersion pattern of the bone substitute material in treatment 3. Additional changes identified on microCT included apparent thickening of the subchondral plate across all defects in horse 2, and variable degrees of both sclerosis and resorption along the microfracture holes in all horses.
Citation: American Journal of Veterinary Research 84, 4; 10.2460/ajvr.22.12.0219
Histopathology
The results of histopathologic scoring are provided (Table 2), with images of each section shown elsewhere (Supplementary Figure S6). Due to variability in drill tract placement relative to the histologic sections, not all slides contained the drill tract for analysis. Evidence of new bone formation within the drill tract could not be examined on every slide in which a drill tract should have been present, and this parameter was excluded from the final scoring. All treatment groups had total scores less than 50% of the maximum score attainable.
Results of the subjective histologic analysis of each defect, reported by treatment group.
| Treatment 1 | Treatment 2 | Treatment 3 | Treatment 4 | |
|---|---|---|---|---|
| Nature of repair tissue (out of 4) | 0 (0–1) | 0 (0–1) | 0 (0–1) | 0 (0–1) |
| Surface regularity | 1 (0–2) | 2 (0–2) | 2 (0–2) | 2 (0–2) |
| Structural integrity | 2 (0–2) | 1 (0–1) | 2 (0–2) | 2 (0–2) |
| Thickness | 1 (0–2) | 1 (0–1) | 1 (0–2) | 1 (0–2) |
| Bonding to adjacent cartilage | 3 (0–3) | 2 (0–2) | 1 (1–1) | 1 (1–3) |
| SCB plate advancement | 3 (1–3) | 3 (2–3) | 3 (2–3) | 3 (2–3) |
| New bone formation: microfracture holes | 1 (1–1) | 1 (0–1) | 1 (0–1) | 1 (0–2) |
| New bone formation: trabecular spaces | 0 (0–0) | 0 (0–0) | 0 (0–1) | 0 (0–0) |
| Inflammatory response in SCB | 2 (2–3) | 2 (2–3) | 1 (1–2) | 2 (2–2) |
| Degenerative change: adjacent cartilage* | 1 (0–2) | 0 (0–1) | 2 (1–4) | 3 (1–3) |
| Total score | 15 (6–16) | 12 (6–13) | 13 (9–15) | 13 (9–19) |
The median (range) score across the 3 horses is reported. The maximum possible score per horse for each parameter was 3 unless otherwise noted.
Scores for cellularity and quality of safranin-O and fast green staining as described in (Supplementary Table S4) were combined to represent degenerative changes in the cartilage adjacent to the defects (maximum possible score of 6 for normal cartilage).
Defects primarily contained fibrous tissue with variability in tissue volume between horses. The tissue thickness measured within each defect was significantly lower than that estimated on MRI and gross evaluation. It was noted that failure to remove the entire calcified cartilage layer in 7 defects resulted in less defect filling and poorer adherence to the underlying surface (Supplementary Figure S7). Early evidence of subchondral plate advancement was identified in multiple defects, characterized by the presence of osteoid above the level of the adjacent normal subchondral plate. No defect in which the calcified cartilage layer was incompletely removed showed evidence of subchondral plate advancement.
The BSM deposited within the trabecular space was removed during the decalcification process and could not be examined histologically. Sections previously containing BSM were identified by areas of marrow fat with a pale, washed-out appearance in H&E-stained sections suggesting devitalization. This fat was bordered by increased new woven bone formation and an increased presence of large mononuclear cells (Supplementary Figure S8), confirmed as macrophages by positive immunohistochemical staining for the markers CD18 and CD204. Neither new woven bone nor increased cellular reactivity was seen with other treatments.
Discussion
This study provided proof of concept that full-thickness cartilage defects can be filled with common injectable materials via subchondral injection, satisfying the authors’ primary objective. Postmortem diagnostic imaging demonstrated signal patterns consistent with the FG and BSM, extending from the drill tract to the targeted defects for treatments 1 and 3, respectively, without interfering with adjacent defects. Fibrous tissue was present in all 12 defects 2 weeks postoperatively with no obvious treatment effect on tissue volume or quality. No significant negative effects were noted as a direct consequence of the mSCP technique or the presence of an injectable material within the SCB.
There was variability in drill tract location including the angle of insertion and the point of entry. Anatomical variations between horses could affect the position of the aiming device, as could differences in medial thigh musculature, which caused variable interference with the positioning of the drill. In horses with more prominent musculature, the angle of drill bit insertion may need to be shallower or the point of entry positioned further cranially to maintain a consistent drill angle. There was also variability in the distance between the end of the drill tract and the subchondral plate. Minor differences in the orientation of the primary x-ray beam relative to the stifle would alter the magnification and distortion of the radiographic image,38,39 affecting the perceived depth of the drill bit below the subchondral plate. When performing subchondroplasty in humans, a targeted approach is used based on BML location, with preoperative MRI and intraoperative fluoroscopy used for guidance.36 The authors expect that a similar procedure would be followed in clinical equine cases; therefore, variability in drill tract placement may be of less significance.
The most significant complication of the described approach was iatrogenic damage to the medial patellar ligament in 5 stifles, occurring near the level of drill bit entry. Patellar lesions were identified in all horses grossly and on MRI, similar to those reported following medial patellar desmotomy.40 In future studies, intraoperative ultrasound could be used to guide the placement of the stab incision. A more direct approach, cranial to the medial patellar ligament, could also be considered.
A common short-term complaint in humans following subchondroplasty is a transient, disproportionate increase in knee pain, typically resolving within 72 hours.29,32,41,42 Compared to baseline, improvement in visual analog scale scores may not be seen until 6 weeks postoperatively.43 All horses exhibited bilateral increases in the measured lameness parameters compared to baseline, similar to previous findings for the established defect model used in this study.16 A subchondral drill tract was associated with at least 1 defect per stifle, and treatments 1 and 3 were never administered in the same stifle. No pair of treatments appeared to result in a more significant increase in lameness compared to another. As these horses did not have preexisting BMLs, the presence of injectate within a normal SCB space cannot be ruled out as a source of discomfort. A disadvantage of a bilateral design is the potential to underestimate lameness severity between limbs. Intra-articular anesthesia of the femoropatellar joints would have allowed for a more accurate assessment of lameness. The authors elected to forego this procedure to avoid disrupting defect tissue and negatively affecting the postmortem assessment. Additionally, with 2 treatments per limb, it was impossible to determine how each treatment contributed to the observed lameness. Judgement of lameness and effusion were also confounded by the medial patellar ligament damage and joint capsule dehiscence, both of which can be avoided in future studies using a revised technique under arthroscopic guidance.
The mean histologic score for each treatment group was low, as expected given the short study period. Consistent with findings from the present study, healing of experimental osteochondral defects treated with microfracture is primarily through an influx of fibrin and granulation tissue during the first 6 weeks.44 Defects from horse 2 contained the poorest histologic filling and adherence to the underlying bone, both of which have been reported to be greater when the calcified cartilage layer is removed.34 The authors suspect that failure to completely remove the calcified cartilage layer during debridement of these defects may be the reason for this difference. Histology revealed early evidence of subchondral plate advancement in at least 1 defect per group, consistent with previous reports on the healing of full-thickness cartilage defects with and without microfracture.12,24,25,27,45 This was not identified in defects in which the calcified cartilage layer was incompletely removed. Frisbie et al34 reported reformation of the calcified cartilage and a greater percentage of new bone in defects without this layer. This was suggested to be a biomechanical response to loading and shear stress at the bone-cartilage junction. The exact pathophysiology is unknown, as is the significance of the findings from the present study; however, this emphasizes the need for treatments that can improve repair tissue formation while mechanically supporting the SCB and cartilage surface.
MicroCT analysis showed increased bone volume ratio, trabecular number, trabecular thickness, and reduced trabecular separation for treatment 3 compared to other groups. Similar microCT findings were demonstrated evaluating subchondroplasty for posttraumatic BMLs in a preclinical canine model.41 Due to the similarity in attenuation values between the BSM used in the present study and native bone, it was not possible to differentiate the 2 materials during analysis, which is a limitation of this study. It must be considered that the apparent differences mentioned may result from an additive effect between the BSM and the native bone, limiting our ability to examine the true effect.
Due to the exploratory nature of this study, a primarily subjective analysis of a limited sample size was performed without control horses for comparison. Detection of statistically significant differences between treatments was not possible, and data were more susceptible to individual variation between horses. Additionally, the short duration prevented the evaluation of the effects on cartilage and SCB healing. Despite these limitations, the goal of this study was not to evaluate treatment efficacy. Further long-term studies with a larger sample size are required before any conclusions can be made regarding the safety or efficacy of this technique.
The long-term consequences of microfracture are increasingly documented. By fracturing the subchondral plate, microfracture may result in subchondral plate advancement, subchondral cyst-like lesions, and sclerosis.12,27,28 The mechanism is not fully understood. Irregular SCB margins with both sclerosis and multifocal areas of resorption along the microfracture holes were identified with all treatments in the present study. Subjectively, focal resorption of the subchondral plate and widening of the microfracture holes appeared to occur to a more dramatic degree in defects not treated with the BSM; however, further research with a larger sample size is required to test the validity of this observation. The BSM used in this study crystallizes with porosity and strength similar to that of healthy cancellous bone, providing a scaffold for local remodeling and supporting the structural integrity of diseased SCB.4,46 The use of a mSCP procedure in combination with common biologic therapies may potentially help reduce some of the negative side effects reported to occur with microfracture. The best material for injection with this procedure remains to be determined.
In conclusion, this study showed that the mSCP procedure is possible as a method of administering injectable therapeutics to both the SCB and associated cartilage defects. These data will provide the foundation for larger objective preclinical studies to better define the role of this treatment in equine joint disease, specifically, the ideal material for subchondral injection using the mSCP technique.
Supplementary Materials
Supplementary materials are posted online at the journal website: avmajournals.avma.org.
Acknowledgments
The authors acknowledge the help of Mrs. Jennifer Daniels, Ms. Natalie Lombard, Mr. Ryan Shelton, and Dr. Katie Seabaugh in caring for the horses, Mrs. Nikki Phillips for laboratory assistance, Mr. Bill Brock for postmortem imaging, and Mr. Lucas Nakamura in the Orthopaedic Bioengineering Research Laboratory for microCT.
Funding was provided by the USDA National Institute of Food and Agriculture, Animal Health and Disease grant No. NI18AHDRXXXXG010. There are no conflicts of interest to declare.
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