Animal Model Standardization for Studying Avascular Necrosis of the Femoral Head in Legg-Calvé-Perthes Disease

Keywords

femur head necrosis - ischemia - Legg-Calvé-Perthes disease - models, animal

Introduction

The Legg-Calvé-Perthes disease (LCPD)[1] [2] affects children, causing sequelae in the hip joint. There is no treatment to discontinue progressive deformity of the femoral head (FH). The scarcity of human material for the study of LCPD makes it necessary to use experimental animal models.[3] [4] [5] [6] [7] [8] [9] [10]

Among emerging countries, only Argentina has a study published in this area.[10]

Our objective is to standardize an experimental model of femoral head ischemic necrosis (FHIN) for the study of feasible LCPD in Brazil. Also, we proposed to introduce gait evaluation tests for functional analysis.[11] [12] [13] [14] [15] [16] [17] [18]

Material and Methods

This work was approved under the Commission of Ethics in the Use of Animals of our institution.

Sample

The sample was chosen according to the literature,[11] [12] [13] [14] [15] [16] [17] [18] [19] [20] under the guidance of the Ethics Committee. Piglets were divided into two groups: group A, with 8 animals, of which 2 animals were euthanized in the 2^nd, 4^th, 6^th, and 8^th week after surgical induction of necrosis, respectively; group B had 2 animals submitted to the surgical procedure without cercling the right femoral neck (RFN), to prove it as a FHIN (sham) inducing factor. The piglets of this group were euthanized in the 6th week because the FH deformity is more evident starting from this period of ischemia.[11]

Eleven piglets were operated, commercial hybrids (crossing of Large White and Landrace breeds), males, weighting from 4 to 6 kg, age of 3 to 4 weeks. In group A, a piglet was removed from the study due to postoperative death from sepsis and replaced. The substitute was euthanized in the 4^th week after surgery.

Surgical Technique

The anesthetic model used intravenous acepromazine. Then, Ketamine hydrochloride along with diazepam were applied also intravenously. Lidocaine 2% was applied via the lumbosacral epidural.

The same surgeon performed all the procedures in the operating room. The femur operated was the right one using the left one as control. The piglet was positioned in left lateral decubitus. The posterior approach was used, using the greater trochanter as a parameter ([Fig. 1A]). Dissection was performed by planes with incision of the gluteus maximus muscle and removal of the gluteus medius muscle for capsule exposure; then, capsulotomy and longitudinal traction for hip dislocation and ligamentum teres section ([Fig. 1B]) to avoid irrigation through the artery of the ligamentum teres. The procedure previously described was performed in groups A and B. Only, in group A double cerclage was performed on RFN with Prolene 2 Ethicon wire (Ethicon Inc., Raritan, NJ, USA) using a “wire pass instrument” to induce FHIN ([Figs. 2A] and [2B]). The mononylon 2.0 Ethicon yarn was closed in both groups.

Fig. 1 (A) Description of posterior approach parameters. Posterior approach (black arrow). Apex of the greater trochanter (white arrow). (B) Posterior approach: deep plans. Clearance of the gluteus medius muscle with a Farabeuf (larger white arrow). Identification of the femoral head (smaller white arrow). Capsulotomy (larger black arrow). Identification of the ligamentum teres (smaller black arrow).

Fig. 2 (A) Passage of two Prolene 2 Ethicon wires (white arrow) around the femoral neck with a “wire pass instrument” (black arrow). (B) Double cerclage around the femoral neck with Prolene 2 Ethicon wires.

Tramadol and the anti-inflammatory flunixim meglumine were applied intramuscularly for analgesia.

Benzaine penicillin was used, also intramuscularly.

Gait Assessment

The piglets were observed walking, the moment before anesthesia, on a flat surface. Gait alterations were compared in groups A and B (sham), in the various study times to verify the correlation of FHIN images with the presence of claudication. The gait classification for pigs proposed by Etterlin et al.[21] was used, ranging from 0 to 3, with grade 0: normal; grade 1: irregular gait with stride shortening and uneven load in one or more limbs; grade 2: moderate claudication with evident deviation of the load of one or more limbs and clear difficulty of ambulation; and grade 3: severe claudication without support in the affected limb or inability to move.

Imaging Exams

Immediately after euthanasia, the femurs were dissected and stored in a common domestic refrigerator at an average temperature of -20°C.

In both groups, digital radiography (DR) was performed at anteroposterior incidence and computed tomography (CT) was performed in frontal, axial and three dimensional (3D) sections in all entire femoral heads. The left FH was used as control. The RD Toshiba 12M 500MAS radiography device (Minato, Tokyo, Japan) and the GE Hispeed Dual model CT scanner (General Electric Company, Boston, MA, USA) were used.

Description of Femoral Head

The normal FH of the immature piglet is divided into ([Fig. 3]):

Fig. 3 Description of the femoral head of the piglet. Front cut of the femoral head at midpoint. Secondary center of ossification (SCO) surrounded by epiphyseal cartilage (EC). Growth plate (GP) located between EC and SCO. Metaphyseal physis (MP) located at the base of the SCO. Methaphysis (M) of the proximal femur, located below MP.

• Secondary center of ossification (SCO) with a semiespheric format;

• Epiphyseal cartilage (EC) bypassing the SCO;

• Growth plate (GP) between the EC and the SCO, responsible for circumferential growth;

• Metaphyseal physis (MP) covering the entire proximal metaphysis, being responsible for the longitudinal growth of the proximal portion of the femur.

Description of the Measures Used in the FH

After imaging, the femurs were cut in their proximal 1/3, in the central region of the frontal plane using a nitrogen surgical saw. Immediately, the height and width of the epiphysis were measured in the sectioned part in centimeters. The height (H) was measured from the apex of the articular surface to the midpoint of the metaphysary physis. The width (W) of the medial maximum point to the lateral maximum was measured in the metaphysary head-physis transition. The epiphysary coefficients (EC) were calculated using the ratio between A and D; they indicate the prognosis of the LCPD. The decrease in EC values indicates a worsening in prognosis.[22] [23] [24] ([Fig. 4]).

Fig. 4 Description of the height and width of the epiphysis and epiphyseal coefficient (EC). The height of the epiphysis is represented by the closed arrow (H). The width of the epiphysis is represented by the dotted arrow (W). The EC is calculated by the ratio between H and W.

Light Microscopy

The slides were colored in hematoxylin eosin (HE) to evaluate the alterations in GP. Light microscopy (LM) was analyzed with magnifications of 40, 100, 140, 240, and 340 times.

Immunohistochemical Analysis

In all slides, the primary antibodies Transforming Growth Factor Beta 1 (TGF-β1, 1:300, Santa Cruz Biotechnology, Dallas, TX, USA) were applied.

Statistical analysis

The descriptions included for categorical variables were: frequency calculation and respective percentage; and for the scaling variables: calculation of mean and respective standard deviation, maximum, minimum, and percentiles (25%, median – 50%, and 75%). The Wilcoxon Signed Rank Test was applied to verify possible differences between both sides, in each studied group, for the variables of interest (W, Wond, EC) with significance level p = 0.050.

The results of immunoexpression of TGF-β1 were compared two by two, i.e., the right side against the left side, at certain study times. The student's t-test, with significance level p = 0.050, was used.

Results

There was one death from sepsis on the second day in group A, being replaced, totaling 11 animals in the experiment.

In the gait evaluation, all piglets in group A presented moderate claudication with clear difficulty of ambulation (grade 2 of Etterlin et al.[21]). In group B, piglets presented normal gait. In group A, in DR and CT scans ([Figs. 5] and [6] and [Table 1]), and in the macroscopic evaluation ([Fig. 7] and [Table 2]), all the right FHs presented characteristic alterations of FHIN. In group B, no changes were observed ([Fig. 8]).

Table 1

Group A	Digital radiography	Coronal and axial CT	3D CT
2 weeks	Discreet FH flattening	Discreet FH flattening Presence of sclerotic areas, suggestive of FHIN - Enlargement of the RFN	Discreet FH flattening
4 weeks	Loss of the right FH semiespheric shape and presence of depression areas of the articular surface	Loss of the right FH semiespheric shape and presence of depression areas of the articular surface	Loss of the right FH semiespheric shape and presence of depression areas of the articular surface
6 weeks	Total collapse and fragmentation of the right FH with loss of the semiespheric shape and great enlargement of the femoral neck	Total collapse and fragmentation of the right FH with loss of the semiespheric shape and great enlargement of the femoral neck	Total collapse and fragmentation of the right FH with loss of the semiespheric format and great enlargement of the femoral neck
8 weeks	Loss of the semiespheric shape and right FH flattening. Suggestive sclerotic areas of FHIN	Loss of the semiespheric shape and right FH flattening. Suggestive sclerotic areas of FHIN	Loss of the semiespheric shape and right FH flattening.
Group B	Digital radiography	Coronal and axial CT	3D CT
6 weeks	No changes	No changes	No changes

Table 2

Group A	Macroscopic analysis
2 weeks	RFN enlargement
4 weeks	FH flattening and RFN enlargement
6 weeks	Total collapse of FH and RFN enlargement Increased EC thickness
8 weeks	FH flattening Increased EC thickness
Group B	Macroscopic analysis
6 weeks	No changes

Fig. 5 Digital radiography (DR) and computed tomography (CT) of the proximal 1/3 of the right and left femurs of piglets euthanized after 6 weeks of ischemic induction. Total collapse and fragmentation were observed on right femora head, in DR and CT, with loss of the semiespheric format. A large enlargement of the femoral neck (arrows) was observed. Collapse and fragmentation were detailed in 3D CT.

Fig. 6 Digital radiography (DR) and computed tomography (CT) of the proximal 1/3 of the right and left femurs of piglets euthanized after 8 weeks of ischemic induction. In RD and CT, a loss of the semiespheric shape and flattening of the right femoral head (FH) and sclerotic areas suggestive of necrosis of the femoral head (arrows) were observed.

Fig. 7 (A) Photograph of the femoral heads (FHs) right and left of the piglet euthanized after 6 weeks of ischemia. Flattening of the right femoral head and tight cerclage to the femoral neck (black arrow) are observed. (B) Frontal cut photograph of femoral heads (FHs) of the piglet euthanized after 6 weeks of ischemia. The right femoral head (FH) has a smaller height (H) and a width (W) greater than the control left side. The secondary center of ossification (SCO) of the right FH is deformed and decreased. It is observed the loss of the semiespheric format of the SCO. (C) Front cut photograph of the FHs from a piglet euthanized after 8 weeks of ischemia. The right FH has H smaller and W greater than the left control side. (D) Front cut photograph of the FHs from a piglet euthanized after 8 weeks of ischemia. The epiphyseal cartilage (EC) on the right side is thicker than the EC on the left side.

Fig. 8 Digital radiography (DR) and computed tomography (CT) of the proximal 1/3 of the right and left femurs of piglets of group B. The right femoral heads (FHs) did not present differences compared to the control left FHs, without radiographic signs of ischemic necrosis of the femoral head (FHIN), remaining semiespheric without areas of depression and sclerosis.

In group A, all piglets presented values for the right FH epiphysis height and the EC lower than those for the left FH. In this group, the width of the right femoral epiphysis was greater than that of the left in 7 piglets (87.5%). The measurements of the height and width of the epiphysis and EC of the right and left FHs showed a statistically significant difference (p < 0.050) ([Table 3]).

Table 3

Group	Timepoints	Measures (cm)	Piglet 1		Piglet 2
A	2 weeks		Side		Side
			R	L	R	L
		H	0.8	1.0	1.0	1.1
		W	1.7	1.6	1.6	1.6
		EC	0.5	0.6	0.6	0.7
	4 weeks	H	1.0	1.2	1.0	1.1
		W	1.9	1.8	2.0	1.9
		EC	0.5	0.7	0.5	0.6
	6 weeks	H	0.9	1.0	0.4	1.2
		W	2.0	1.9	1.9	1.8
		EC	0.4	0.5	0.2	0.7
		H	0.9	1.1	1.0	1.2
	8 weeks	W	2.2	1.9	2.3	2.1
		EC	0.4	0.6	0.4	0.6
B	6 weeks	H	1.0	1.0	1.0	1.0
		W	1.9	1.9	1.9	1.9
		EC	0.5	0.5	0.5	0.5

In group B, no differences in measurements were observed between the sides of the right and left FHs (p³  = 0.050) ([Table 2]).

There was no development of right FHIN in group B; consequently, the EC values remained equal on both sides. This corroborates that the factor inducing necrosis is RFN cerclage ([Table 2]).

In the histological evaluation by LM, in the right FHs of group A piglets, chondrocytes were arranged in a disorganized way and separated by gaps. There was no clear separation between the physeal layers. In the left FHs, a normal aspect was observed, that is, organization of chondrocytes in columns and visible division between the layers of the physis: proliferative, hypertrophic, and calcification zones. In group B, the same normal aspect was observed, described above, in the right and left FHs ([Fig. 9] and [Table 3]).

Fig. 9 Photograph of growth plates (GP) of the femurs from piglets euthanized after 6 weeks of surgery. Photomicrography in 100-fold increase from growth plates of the femurs from piglets euthanized after 6 weeks of surgery. (A) In group A, in GP of the right femoral head (FH), the presence of chondrocytes grouped in a disorganized way (white arrow) and separated by large empty gaps was identified. (B) In the GPs of the left FHs (control), chondrocytes were well organized in columns arranged in parallel (red arrows). The proliferative, hypertrophic, and calcification zones are clearly divided and visible. Vascular proliferation was observed in the calcification zone (black arrows). (C. and D) In Group B, the growth plates of the right and left FHs showed no differences. Normal aspect, with the proliferation, hypertrophic, and calcification zones clearly identified, and chondrocytes were organized in columns (red arrows).

In the evaluation by immunohistochemical reaction in group A, a decrease in TGF-β1 expression was observed in the slides of the right FHs with 2 and 6 weeks of ischemia, and in the left FHs with eight weeks of ischemia.

In group B, there was no difference in the expression of TGF-β1 between the right and left FHs (p³  = 0.050) ([Fig. 10]).

Fig. 10 Description of TGF-β1 expression in femoral heads (FHs) according to ischemia time. The columns indicate TGF-β1 expression. CE: Left FHs of Right FHs of Group B. CD: Group B. 2SE: Left FHs from piglets euthanized with 2 weeks. 2SD: Right FHs from piglets euthanized with 2 weeks. 4SE: Left FHs from piglets euthanized at 4 weeks. 4SD: Right FHs from piglets euthanized with 4 weeks. 6SE: Left FHs from piglets euthanized with 6 weeks. 6SD: Right FHs from piglets euthanized with 6 weeks. 8SE: Left FHs from piglets euthanized with 8 weeks. 8SD: Right FHs from piglets euthanized with 8 weeks. The test used was the Student t-test, significance level p = 0.050.

Discussion

The model proposed using frozen bone parts provided alterations of the FHIN in DR, CT, macroscopy, histology, and gait.

In all surgeries, during dissection, the presence of cerclage was still fair in the RFN, confirming it as the factor inducing necrosis.

Although nuclear magnetic resonance imaging (NMR)[9] [10] [13] is the most sensitive test for the diagnosis of FHIN,[12] CT[8] [9] [22] [25] and DR[10] [11] [12] [15] [18] [26] [27] [28] demonstrate the same alterations. Similarly, in this study with frozen parts we observed: decrease and increase, respectively, of the height and width of the femoral epiphysis, as well as flattening, collapse, and fragmentation of FH. All at a lower cost.

Taking into consideration that CT and NMR exams are limited to very few veterinary hospitals and the use of human laboratories by animals is prohibited by health surveillance standards, dissection and freezing of the femurs allowed for the examinations of these anatomical parts in these laboratories.

The use of NMR in dissected femurs is more difficult because it requires the presence of soft tissues, i.e., the whole animal should be examined and anesthetized. Alternatively, DR and CT could be performed on dissected bones.

Thus, the femurs were dissectised and frozen, and imaging tests were performed by a collaborating laboratory on the availability of vacancies.

It was observed that this storage method can reduce costs, does not affect the quality of the exams, and makes conducting it possible on a scheduled date, according to the possibility of care. This feature allows the use of DR and CT even without having them in your service.

Kim et al.,[11] in 2001, made radiographs of frontal cuts with a thickness of 3 mm with diamond saw for better image definition. We do not have the necessary material to perform millimetric cuts, so the whole FH was radiographed. Nevertheless, signs of FH necrosis were observed with only two weeks of RFN cerclage, and, after six weeks, fragmentation and collapse of FH, in the same way as the authors above. Additionally, 3DCT also allowed the visualization of the lesions in greater detail, also verifying that freezing did not hinder the method, nor the LM results. There were gaps splitting the disorganized chondrocytes and losing their arrangement in columns, without the clear identification of proliferative, hypertrophic, and calcification zones, thus characterizing the areas of necrosis.

The EC of the right femoral heads of group A were smaller than the EC on the contralateral side, demonstrating FH flattening by necrosis, and the lower the EC, the greater the deformity and the worse the prognosis.[16] [22] [23] [24] However, the greatest deformity was observed at six weeks of ischemia and not at eight. A possible explanation would be that in LCPD, the longer the evolution time, the worse the FH deformity, with the collapse of FH varying directly with the amount of weight bearing on the joint.

Kim et al.[22] demonstrated, in a swine model, that the weight bearing on the hip worsens the prognosis of the disease. Our piglets were housed with free movement, so the amount of load on the ischemic limb was dependent on the degree of voluntary activity of each test subject. A higher degree of activity in the piglet with 6 weeks of ischemia could justify the greater deformity. Also, Etterlin et al.,[21] evaluating the gait of piglets with arthrosis, observed that more active piglets had a better gait pattern than inactive, even with severe changes in the joints, attributing this to the musculature better developed by exercise. Thus, a more active piglet wanders more, imposes more weight bearing on the joint, deforming the FH more markedly.

Another hypothesis would be that the femoral heads with eight weeks of ischemia had developed a neocirculation, but this is unlikely because in the FHIN repair process, accessories secondary centers of ossification nucleis emerged that promoted disordered and irregular growth that gave the aspect of fragmentation on radiographs.[11] Perhaps a longer study time, with controlled movement and a greater number of piglets could help clarify these findings.

Frequently, FHIN evolves into arthrosis, causing functional impairment of this joint[1] and, consequently, claudication. There are no experimental models using functional gait assessment in LCPD studies. In this study, it was possible to study gait and observe very evident alterations without the need for sophisticated instruments that could hinder the performance of functional evaluation. Santangelo et al.,[29] in 2014, used a breed of guinea pigs that spontaneously developed knee arthrosis to test the effects of flunixin meglumine. The authors used a computerized gait platform and observed an improvement in the gait pattern of the animals using the drug.

Thus, a swine experimental model with the functional evaluation of effective and low-cost gait can be an advent to test new drugs for FHIN, making it possible to associate the effects of treatment on morphological and biochemical tests with functional clinical changes in gait.

The surgical technique was described by Kim et al.,[11] in 2001, when these authors had four successes in 18 piglets, of which 1 piglet developed septic arthritis and 3 did not develop necrosis by failure of the surgical technique in cerclage. In our experiment with 11 piglets, there was only one death from sepsis, with success in the ischemia process in all piglets. There is no in-depth demonstration of surgical steps with sufficiently detailed images in the literature.

It is believed that our result can be justified by the surgeries being performed by the same experienced surgeon, who is a specialist in hip surgery. Although we had a simple surgical center, with minimal conditions, it was observed that these structural conditions were not limiting. And it is considered that the main factor for the success of the procedure is technical knowledge.

It is our opinion that the thorough knowledge of the regional anatomy and surgical technique are necessary, being fundamental to train with cadavers to acquire skill. Preoperative evaluation and confinement, as well as postoperative veterinary care, can also avoid losses.

The TGF-β1, therefore, can be used as an indirect measure of damage and immune alteration triggered by FHIN, because it is involved in bone regeneration.[30]

Tao et al.,[31] in 2017, observed a decrease in TGF-β1 expression in femoral heads with ischemic necrosis, taken from patients submitted to total hip arthroplasty for this reason.

In this study, there was no decrease in TGF-β1 expression in all right femoral heads of group A, although macroscopic, microscopic, radiographic, and clinical signs of FHIN were identified. This finding may be related to the small number of slides performed, limited by the need of the part for other tests. For future experiments, we suggest allocating half of the sectioned FH for immunohistochemistry examination and the other half for macroscopic and histological analysis. Thus, it would be possible to use larger fragments with a larger amount of bone tissue. A greater number of guinea pigs will allow greater conclusions.

Conclusions

The changes in FH in the LCPD were reproduced in macroscopic analysis, DR, CT, and LM.

Gait evaluation showed a good correlation with macroscopic changes and imaging.

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