Extracorporeal shockwave therapy in osteoporotic osteoarthritis of the knee in rats: an experiment in animals

Fifty-six 8-weeks-old female Sprague–Dawley (SD) rats (BioLASCO, Taipei, Taiwan) were used in the experiment. The experimental protocol of the animal study was approved by the Animal Care Committee of Kaohsiung Chang Gung Memorial Hospital. The animals were kept at the Laboratory Animal Center for 1 week before experiment. The rats were housed at 23°C ± 1°C with a 12-hour light-and-dark cycle and given food and water.

SD rats were randomly divided into seven groups with eight rats in each group. Group 1 was designated the sham group and received sham laparotomy without ovariectomy and sham arthrotomy of the left knee without anterior cruciate ligament transaction (ACLT) or medial meniscectomy (MM). Group 2 was designated the OA group and received ACLT and MM of the left knees [8]. Group 3 was designated the OP group and received bilateral ovariectomies [30]. Group 4 was designated the OA + OP group and received ACLT and MM of the left knee and bilateral ovariectomies. Group 5 was designated the OA + ESWT and received ACLT + MM of the left knee and ESWT. Group 6 was designated the OP + ESWT and received bilateral ovariectomies plus ESWT. Group 7 was designated the OA + OP + ESWT and received ACLT and MM of the left knee, bilateral ovariectomies, and ESWT.

Postoperatively, the animals were cared for by a veterinarian. The surgical site and the activities of the animals were observed daily. After BMD measurement, all animals were killed at 12 weeks after surgery. Left-knee specimens were harvested for examination of gross pathologic lesions, micro-CT scan, bone-strength test, histopathologic examination, and immunohistochemical analysis.

The left knee was prepared in a surgically sterile fashion. Through miniarthrotomy, the ACL was transected with a scalpel, and MM was performed by excising the entire medial meniscus. The knee joint was irrigated, and the incision was closed. Prophylactic antibiotics with ampicillin, 50 mg/kg body weight, were given for 5 days after surgery. Postoperatively, the animals were cared for by a veterinarian. The surgical site and the activities of the animals were observed daily.

The rats were anesthetized with an intramuscular injection of phenobarbital (50 mg/kg body weight). The abdomen was scrubbed and prepared in surgically sterile fashion. A midline incision was made, and laparotomy was performed. Bilateral ovariectomies were performed by excision of both ovaries. The wound was irrigated and closed in routine fashion.

ESWT was performed in half of animals (eight rats with eight knees in each group) at 1 week after surgery when the wounds healed. The animals were sedated with a 1:1 volume mixture of Rompun (containing xylazine; 5 mg/kg) with Zoletil (containing zolazepam and tiletamine; 20 mg/kg) while receiving ESWT. The source of the shockwave was an OssaTron (Saunwave, Alpharetta, GA, USA). Application of 800 impulses of shockwave at energy-flux density of 0.22 mJ/mm² was given to the proximal medial tibial condyle at 0.5 cm below the joint line and 0.5 cm from the medial skin edge in a single session [27, 31, 32].

The BMD values within the region of interest (ROI) in medial proximal tibia and distal femur condyle was measured by using dual-energy X-ray absorptiometry (DEXA) (Hologic QDR 4500 W, Hologic, Bedford, MA, USA) before the animals were killed. BMD was used to assess the changes in bone density around the knee in different conditions.

The knee joint was examined under a magnification scope. The gross pathologic lesions with arthritic changes on femoral condoyle and tibial plateau were identified and quantified separately by the semiquantitative scale [19], The severity of joint-surface damage was scored and categorized as follows: (a) normal, (b) discoloration, mild surface irregularities or pitting, (c) partial-thickness erosion or fibrillation, and (d) full-thickness erosion and/or osteophytes. The overall scores were obtained by summing the cartilage scores of the lesions in femoral condoyle and tibial plateau cartilages in eight knees of each group [19].

The proximal part of the tibia and the distal part of the femur were scanned by a micro-CT scanner (Skyscan 1076; Skyscan, Luxembourg, Belgium) with isotropic voxel size of 36 × 36 × 36 μm³, as previously described [33]. In brief, the X-ray voltage was set at 100 kV, and the current, at 100 μA. X-ray projections were obtained at 0.75-degrees angular step with a scanning angular range of 180 degrees. Reconstructions of the image slices were performed with NRecon software (Skyscan), and the process generated a series of planar transverse gray-value images. Volume of interest (VOI) of bone morphometry was selected with a semiautomatic contouring method by Skyscan CT-Analyser Software (Skyscan). Three-dimensional cross-sectional images of the femoral and tibial subchondral bone regions were generated by CTVol v2.0 software. The subchondral porosity was defined as the ratio of the volume of the pores to the total volume of the subchondral region. The subchondral plate thicknesses of the femur and tibia were measured by individual point-to-point distance from the top of the cartilage to the subchondral bone plate by averaging at least six measurements per sample.

The distal femur and proximal tibia bones were harvested for bone-strength tests, including peak load and breaking moment. A 3-cm bone from the medial proximal tibia and distal femur was obtained. The bone specimens were subjected to bone-strength tests on a Material Testing System (MTS, Synergie 200; Canton, MA, USA) machine. The peak compression strength and the breaking moment of inertia were measured by using the slow-load compression technique until fracture occurred.

The bone and cartilage of the knee were subjected to histopathologic examination. The harvested specimens are fixed in 4% PBS-buffered formaldehyde and decalcified in 10% PBS-buffered EDTA at room temperature for about 2 months. Decalcified specimens were subjected to paraffin-wax embedding and dissection into 5-μm-thick sections. The specimens were stained with hematoxylin-eosin (H&E) stain and safranin O staining. The degenerative changes of the cartilage were graded histologically by using the Mankin scoring system for the assessments of cartilage structure, cartilage cells, and tidemark integrity, with a score from 0 to 15 [7]. In addition, a safranin O score was obtained according to the Osteoarthritis Research Society International (OARSI) cartilage OA grading system [34]. The scores were obtained on a 0–to-24 scale by multiplying the index of grades with stages.

The harvested knee specimens were fixed in 4% PBS-buffered formaldehyde for 48 hours and decalcified in 10% PBS-buffered EDTA solution. Decalcified tissues were embedded in paraffin wax. The specimens were cut longitudinally into 5-μm-thick sections and transferred to polylysine-coated slides (Thermo Fisher Scientific, Waltham, MA, USA). The immunohistochemical stains were done by following the protocol provided in the immunostaining kit (Abcam, Cambridge, MA, USA). In brief, tissue sections were deparaffinized in xylene, hydrated in graded ethanol, and treated with peroxide-block and protein-block reagents. Sections of the specimens were immunostained with specific antibodies for DKK-1 (Cell Signaling Technology, Danvers, MA, USA), PCNA (Santa Cruz Biotechnology, Santa Cruz, CA, USA), VEGF (Santa Cruz Biotech), and BMP-2 (Abcam) for overnight to identify the angiogenesis and osteogenesis biomarkers according to the product immunohistochemical staining protocols. The immunoreactivity in specimens was demonstrated by using a goat anti-rabbit horseradish peroxidase (HRP)-conjugated and 3′,3′-diaminobenzendine (DAB), which were provided in the kit. The immunoactivities were quantified from five areas in three sections of the same specimen by using a Zeiss Axioskop 2 plus microscope (Carl Zeiss, Gottingen, Germany). All images of each specimen were captured by using a cool CCD camera (Media Cybernetics, Silver Spring, MD, USA). Images were analyzed by using an Image-Pro Plus image-analysis software (Media Cybernetics). The percentages of immunolabeled positive cells over the total cells were calculated from articular cartilage and subchondral bone of distal femur and proximal tibia.

SPSS ver. 17.0 (SPSS Inc., Chicago, IL, USA) was used in statistical analysis. Data were expressed as mean ± SD. One-way ANOVA and Tukey tests were used to compare sham versus OA, OP, and OA + OP with ESWT and without ESWT (designated as _*P < 0.05 and _**P < 0.001). Paired sample t tests were used to compare ESWT groups and without-ESWT groups (designated as #P < 0.05 and ##P < 0.001) and independent-sample t tests were used to compare OA versus OA + OP and OA + ESWT versus OA + OP + ESWT (designated as ※P < 0.05 and ※※P < 0.001). The P values of less than 0.05 were considered significant.

Significant difference in the sizes of gross arthritic lesions of the knee was noted between ESWT and non-ESWT. The gross pathologic lesions with and without ESWT are shown in Figure 1A. With no ESWT, the sham group showed smooth surfaces with no surface irregularity or pits. The OA group demonstrated broad areas of articular surface destruction with fibrous tissue thickening around the tibia. The OP group showed intact surfaces with decreased surface brightness and inflammation. The OA + OP group showed severe articular surface erosion, ulcerations, pitting, and osteophytes with proliferation of fibrous tissue. With ESWT, the OA group showed thickening of the articular surface with yellowish discoloration but no osteophytes. The OP group showed intact surfaces with no ulceration or discoloration. The OA + OP group demonstrated osteoarthritic changes including articular-surface erosions, ulcerations, pitting, and osteophyte formation. The statistical comparisons between ESWT and no ESWT are shown in Figure 1B; the OA and OA + OP groups showed significantly larger areas than the sham group. The OA and OA + OP group with no ESWT showed significantly larger lesions than OA and OA + OP with ESWT. The OA + OP group with ESWT showed significantly larger lesions than did those with no ESWT. It appears that ESWT is effective to ameliorate the severity of osteoporotic osteoarthritis in rat knee.

BMD values were measured with and without ESWT (Figure 2). BMD significantly decreased in groups OA, OP, and OA + OP, as compared with sham. ESWT significantly increased BMD in OA, OP, and OA + OP, as compared with sham. BMD was significantly higher with ESWT than without ESWT in OA, OP, and OA + OP groups.The bone-strength tests are shown in Figure 3. The peak load (Figure 3A,B) and the breaking moment of the femur and tibia (Figure 3C,D) significantly decreased in groups OA, OP, and OA + OP relative to sham group, with the most decrease in group OA + OP. ESWT significantly improved the peak load and breaking moment in groups OA, OP, and OA + OP as compared with the sham group. The results showed that ESWT significantly improved the BMD value and bone strength in osteoporotic osteoarthritis of the knee in rats.

The results of micro-CT scan are summarized in Figure 4. The subchondral plate thickness significantly decreased, and bone porosity increased in distal femur and proximal tibia in groups OA, OP, and OA + OP, with the most decrease noticed in group OA + OP as compared with sham. ESWT significantly improved the subchondral plate thickness and bone porosity in groups OA, OP, and OA + OP, especially in group OA + OP. ESWT seems more effective in the improvement of subchondral plate thickness and bone porosity in group OA + OP than in group OA.

The microscopic features of histopathologic examination of groups OA, OP, and OA + OP are shown in Figure 5A. The graphic illustrations with and without ESWT are shown in Figure 5B for Mankin score and Figure 5C for safranin O stain. Significant cartilage damage associated with elevations of Mankin score and safranin O stain were observed in groups OA and OA + OP, but not in group OP, as compared with sham. ESWT significantly improved the cartilage changes with decreases in Mankin score and safranin O stain in groups OA and OA + OP. (Figures 5B,C). These findings suggest that the ESWT shows chondroprotective effect in groups OA, OP, and OA + OP.

Immunohistochemical analysis of DKK-1, PCNA, VEGF, and BMP-2 in subchondral bone and articular cartilage in OA, OP, and OA + OP with and without ESWT is shown in Figure 6A-D. The representative images of microscopic features are shown in Figure 6a-d. Significant increases in DKK-1 expression (Figure 6A) and decreases in PCNA (Figure 6B), VEGF (Figure 6C), and BMP-2 (Figure 6D) expressions were noted in groups OP and OA + OP with and without ESWT, as compared with the sham group. ESWT significantly improved the changes in DKK-1, PCNA, VEGF, and BMP-2, with the most changes noticed in group OA + OP. These results demonstrated that ESWT modulates the key factors for joint repair in groups OA, OP, and OA + OP.