A total of 9 male and female dogs from 1 to 12 years old, including Maltese, Cane Corso, Shar-Pei, Labrador, Pitbull, and mixed breeds, were selected in the orthopedic clinical routine of the veterinary hospital Dr. Antônio Clemenceau. All dogs had some degree of claudication and were diagnosed with CCLR through clinical examination, drawer test, and tibial compression, as well as radiographic analysis in specific positioning for TTA according to Hoffmann et al. (2006). The selected animals had complete and unilateral CCLR of the knee joint, with three (33%) showing symptoms in the right knee and six (67%) in the left knee. Five (55%) used cage number 6 mm, three (33%) used the cage number 9 mm, and only one (11%) used the cage number 12 mm. The plates were chosen according to the radiographic pattern with the animals properly positioned (femoro-tibial-patellar joint with 135° angulation) and two plates at number 0, one plate at number 0.5, two plates at number 1, plate number 2, two plates at number 3 and a plate at number 4.
Surgery procedures
Lateral radiographic projection of the affected limb was obtained pre-operatively to assess the stifle joint. The lateral projection was centered on the stifle joint with superimposition of both femoral condyles. The stifle joint was angled at 135° in accordance to the long axis of the femur. The joint was positioned so that there was no cranial tibial translocation. A standardized TTA transparency (Focus®, Indaiatuba, Brazil) was used over the radiograph to determine the amount of TTA required to position the patellar tendon perpendicular to the tibial plateau.
The dogs were positioned in dorsal recumbency and the affected limb was aseptically prepared. All dogs received ceftriaxone (25 mg/kg) preoperative.
A medial parapatellar incision was performed 2 cm proximally to the tibial plateau and extended distally to the tibial diaphysis. The periosteum of tibial crest was reflected cranially to expose the cranial bone margin of the entire tibial crest. A previously selected TTA bone plate (Focus®, Indaiatuba, Brazil) was positioned over the tibial crest and fixed by 2 or 3 locking screws, depending on the length of the plate. After that, a bicortical osteotomy was performed in the tibial crest observing the plate contour.
The tibial crest, with attached plate, was moved cranially using a spacer and a previously selected cage (Focus®, Indaiatuba, Brazil), was placed into the osteotomy site at the proximal extent of the osteotomy and secured at its caudal margin to the tibia with a 2.0 mm screw. The cranial cage screw was secured into the tibial tuberosity and the plate was then secured distally to the tibia with the appropriately sized screws (2.0 mm; 2.7 mm or 3.5 mm). The surgical site was then sutured routinely.
Isolation and preparation of MSCs
After the TTA surgery, a subcutaneous fragment of adipose tissue (approximately 20 g) from the medial parapatellar region was collected from all animals. The specimens were stored in 199 medium (M4530, Sigma Aldrich, São Paulo, Brazil) cooled, and sent to the Biocell laboratory (Brasília, Brazil), which specializes in growth and culture of stem cells. There, the adipose tissue fragment was submitted to enzymatic treatment for extraction and expansion of the mesenchymal stem cells following a protocol described by Kang et al. (2012).
The stem cells were maintained in the culture medium (37 °C, 5% of CO2) until the eighth postoperative day, and then about 1.5 million stem cells were removed from the bottles by trypsinization and transferred with transport medium to 1.0-mL hypodermic syringes and applied to the gap formed by the osteotomy. The gap formed by the osteotomy was localized through palpation, and then the cells were injected into the gap percutaneously using a 1 ml syringe with a hypodermic needle (30 × 0.8 mm, Becton Dickinson, São Paulo, Brazil) (Fig. 1).
In the animals of the control group (GC) (n = 5), only the culture medium was applied without the MSC. In the animals of the treated group (GT) (n = 4), the culture medium was treated with MSC. Only the laboratory was aware of which animals would receive transport with MSC and which received only the transport medium. Thus, the study was characterized as double blind, since neither the surgeon nor the owner knew the composition of groups.
Qualitative evaluation of radiographic images
On the fifteenth postoperative day, the animals were evaluated for the clinical condition of the surgical wound, degree of claudication, effectiveness of the TTA technique in eliminating the shear movement in the operated knee, and the level of pain in the operated area.
The animals were X-rayed (Compact Plus 500 L, Philips Medical Systems, São Paulo, Brazil) at 15, 30, 60, 90 and 120 days after surgery. Radiographic images were evaluated by two veterinarians who classified them according to Hoffmann et al. (2006). The classification was made based on the following scores:
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zero - indicates that there was no bone healing;
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I - represents early bone production without bridging the tibial tuberosity and the axis of the tibia;
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II - significant bone bridge in one site;
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III - indicates bone bridge in two sites;
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IV - represents bone bridge at all three sites, including the region of osteotomy proximal to the cage, the region of osteotomy between the cage and the plate, and the distal region of the osteotomy to the plaque (Fig. 2).
Evaluation of the osteogenic potential of MSCs
To assess the osteogenic potential of MSC culture, some of the cells proliferated in the laboratory were induced to differentiate into bone tissue. For in vitro osteogenic differentiation, the purified MSCs were maintained in a culture medium containing ascorbic acid (50 μM), dexamethasone (0.1 μM) and beta-glycerol phosphate (10 mM) according to the protocol established by Kang et al. (2012). After cell differentiation, the detection of the bone matrix was performed by cytochemical staining with Von Kossa staining (Fig. 2).
Another part of the cells was sent to the MOFA Global laboratory (Verona, WI, USA) to perform the molecular characterization through flow cytometry (AMNIS®, Merck, Darmstadt, Germany) with quantification by image, also evaluating the degree of purity, functionality and viability of such cells, through the identification of specific molecular markers and DNA dyes. For this, 1,000,000 cells were incubated with specific antibodies, capable of identifying proteins that are found only in stem cells, called molecular markers. Such proteins, CD44, CD29, CD90, these proteins are found in the plasma membrane of stem cells with specific functions.
In addition, to analyze the cell population and to quantify the degree of functionality, antibodies were used to identify intracytoplasmic molecular markers, such as the OCT3/4 and SOX2 proteins, responsible for the maintenance of the undifferentiated state of the stem cells. Likewise, cytometry was able to identify the proportion of undifferentiated cells found in the sample.
Analysis of the density of bone trabeculae in the spongy substance
To evaluate the density of bone trabeculae in the spongy substance, the radiographic images captured at different times were processed in an image editing software (Adobe Photoshop CS6, version 6.0, Adobe Systems Inc., San Jose, CA, USA) to delimit the area of interest in standardized size, according to Mahl et al. (2009).
After the delineation of the cuts representing the spongy substance in the region of the osteotomy site after the surgical procedure, the analyzed images of the groups were processed in Image-Pro Plus 4.1 image analysis software (Media Cybernetics Inc. Silver Springs, MD) according to Blatt et al. (2004).
Statistical analysis
A descriptive analysis was performed to obtain the mean and standard deviation, followed by the Kolmogorov-Smirnov test. The radiographs were divided into groups in the postoperative evaluation periods so that the images were classified as proposed by Hoffmann et al. (2006) in different scores and different periods from the application of the Chi-square test. The density of the bone trabeculae arranged in the spongy substance was evaluated in each of the periods for the two groups by applying the Mann-Whitney test. The density was also compared between the different periods from the application of Tukey’s multiple comparison tests between the two groups. Statistical significance was set at p ≤ 0.05 for all tests, and the Graph Pad Prism 6.0 program was used.