Mesenchymal stem cells in osteotomy repair after tibial tuberosity advancement in dogs with cranial cruciate ligament injury

The main function of the cranial cruciate ligament is the stabilization of the knee, where it prevents cranial displacement of the tibia in relation to the femur, in addition to aiding the rotational movements of the tibia (Calvo et al. 2014; Fossum 2015; Venzin et al. 2004). Cranial cruciate ligament rupture (CCLR) is the orthopedic knee condition most common in dogs (Calvo et al. 2014; Marino and Loughin 2010). The causes that lead to CCLR are still not completely defined (Vedrine et al. 2013), but a number of presumed factors lead to a common pathway in the origin of these lesions (Cook 2010). Although the lesions are primarily traumatic, other degenerative factors have been noted in the pathogenesis of this condition, such as age, sex, and obesity (Venzin et al. 2004), as well as racial predisposition, inflammatory and conformation alterations, and immune-mediated mechanisms (Griffon 2010).

The extracapsular techniques usually applied for the treatment of this condition are the fabelo-tibial suture, osteotomy of the tibial plateau, and advancement of the tibial tuberosity (TTA). In addition, there are intracapsular techniques that most commonly use the autogenous fascia lata as material. Synthetic materials have not been widely used due to the possibility of distension, rupture, or infection (Fossum 2015).

The TTA demonstrates excellent results due to lack of alteration of the original geometry of the knee joint and its promoting of the distribution of pressure in the cartilage (Hoffmann et al. 2006). In addition, this technique is a simple surgical procedure with less potential to generate adverse effects due to technical problems (Boudrieau 2009). The use of the TTA technique in combination with the application of stem cells provides a new option for the treatment of CCLR.

Bone formation and repair are performed by osteoprogenitor cells derived from mesenchymal stem cells (MSC) that act in the lesion area by signaling cytokines that stimulate the replication and differentiation in active osteoblasts. The osteoblasts deposit an extracellular matrix mineralized with hydroxyapatite (Kraus and KIRKER-HEAD 2006). In addition, MSC also maintain viability and potential multi-lineage after cryopreservation, so MSC can be effectively isolated, expanded, preserved and implanted (Kraus and KIRKER-HEAD 2006).

Kang et al. (2012) found that MSCs from adipose tissue have a higher proliferation potential than the other types of MSCs and that their main advantages over other types of MSCs include easy availability in substantial quantities and that they can be collected without risk to the donor (Schäffler and Büchler 2007).

The aim of this study was to analyze the feasibility of using MSCs implanted at the osteotomy site obtained during the TTA technique in dogs with a CCLR diagnosis and to verify the clinical recovery of the dogs after the use of the TTA technique and implantation of MSCs at the osteotomy site. Our hypothesis is that the use of MSC will accelerate the cicatricial process after TTA in dogs.

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.

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:

• zero - indicates that there was no bone healing;

• I - represents early bone production without bridging the tibial tuberosity and the axis of the tibia;

• II - significant bone bridge in one site;

• III - indicates bone bridge in two sites;

• 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).

In order to ensure that the isolated cells are mesenchymal stem cells, we used the CD44, CD29 and CD90 markers, which were positive, indicating that the sample consisted of mesenchymal cells (Fig. 3).

Through flow cytometry results, we observed that 97.9% of the cells were OCT3/4 positive and 99.9% of the cells were SOX2 positive, thereby ensuring that the cells maintained the undifferentiated state of the stem cells. In addition, the OCT3/4 and SOX2 transcription factor are involved in the maintenance of the proliferative state of the stem cells, it being desirable that such cells present these factors (Fig. 4).

The radiographs of the groups were divided over the postoperative evaluation periods (Fig. 5) so that the images were classified according to the table below, and the scores were evaluated before the different periods according to the application of the Chi-square test (Table 1).

SCORE	Day 15		Day 30		Day 60		Day 90		Day 120		p
	GC	GT	GC	GT	GC	GT	GC	GT	GC	GT
ZERO	50	75	20	50	10	–	–	–	–	–	< 0,0001
I	30	12,5	10	12,5	10	12,5	–	–	10	12,5	0,07
II	–	12,5	40	25	50	25	30	–	20	–	< 0,0001
III	20	–	20	12,5	–	50	30	62,5	30	50	< 0,0001
IV	–	–	10	–	30	12,5	40	37,5	40	37,5	0,009

The Chi-square test with a significance level of p ≤ 0.05 was performed between the same periods for the different groups

Relatively and partially, and based only on the assessment of claudication, it was observed that the GT animals had a similar response to GC animals at the end of the study. Thus, it was possible to extrapolate that the application of mesenchymal stem cells was not as beneficial as expected with regard to ambulation and reduction of immediate postoperative discomfort for the treated group. This study did not agree with Hoffmann et al. (2006); Lafaver et al. (2007), in which the evaluated animals had an effective response even in the short term.

In both objective and subjective evaluations, it was observed that the GC obtained results were equal to the GT in relation to the ossification process at the end of the experiment. However, the GC may have obtained a delay in the period of differentiation, which can be justified due to the necessity of reorganization due to the inflammatory process after the postoperative introduction of the stem cells eight days later, unlike the study by Zamprogno (2007), which found efficacy in therapy with stem cells introduced after 15 days of the first intervention in fractures and promoted bone union by the end of the study.

As a result, the application of cells should perhaps have occurred earlier, since the process of cell differentiation should have started from the moment of their application. Contrary to this assessment, the cells applied were probably diverted, in part, due to the reorganization of the inflammatory process present after their introduction into the medium. Taking into account that the first inflammatory stage occurs from the first day of injury to the third day (Cho et al. 2002) and that during this period, the cleaning of damaged areas and migration of pluripotent cells are performed, immunosuppressive functions and reduction of the immune cell response are activated (El-Jawhari et al. 2016), thus justifying the observed response.

In the second phase, called the repair or chondrogenic phase and characterized by the peak of collagen expression from the seventh to the fourteenth day (Cho et al. 2002), differentiation of pluripotent cells into osteoblasts or chondroblasts was observed. This phenomenon was induced by macrophages, which also regulated the differentiation of pluripotent cells and thus the action of other immune cells, including T and B lymphocytes located near the osteoblasts, thus aiding in the differentiation of cartilage and bone (El-Jawhari et al. 2016).

Bone fracture promotes disruption of blood supply and subsequent platelet aggregation with the release of proinflammatory cytokines. These cytokines stimulate the return of lymphocytes and monocytes/macrophages to the site of the fracture, initiating the process of bone healing (El-Jawhari et al. 2016).

The third and final phase, called osteogenic or remodeling, begins in the first 24 h after the injury, and the maximum differentiation occurs between days 14 and 21 after the fracture (Cho et al. 2002). Thus, in the two GT cases where there was a fracture at 15 days, this may have influenced the observed healing delay in relation to GC animals that presented the fracture late, suggesting that the introduced stem cells may not have differentiated into osteoblasts, as expected, but again diverted to the new inflammatory process installed after the fracture of the tibial tuberosity. In addition, there is the fact that the timing of the therapeutic intervention in relation to the inflammatory state is an essential challenge to be addressed according to El-Jawhari et al. (2016). In any case, both groups achieved complete ossification within the postoperative period of 8 to 10 weeks, as proposed by Hoffmann et al. (2006).

Although there was complete ossification within the estimated period, the therapeutic use of MSC for bone loss recovery did not achieve statistically significant results as expected in relation to minimizing the estimated gap recovery time. The microenvironment of the lesion, and particularly the local inflammation, interfered with the action of the stem cells applied in the region of the osteotomy.

The results obtained in this study were not those expected by the hypothesis we outlined at the beginning, but we must be careful not to jump to conclusions. This study used a great variability of ages and dog breeds. Perhaps we would have obtained a different result if the sample was more homogeneous, but the sample used in this study represents the variability that we verified in the clinical routine. In the future, we intend to carry out new research in order to overcome these limitations. However, we believe that we use a very important technique in the routine, so these results, even if partially inconclusive, should be shown to delineate research and future uses in the clinic.