Intra-articular collagenase injection increases range of motion in a rat knee flexion contracture model

Figures & data

Table 1 Mean extension angles between tibia and femur for both legs of CCH-injected and buffer-injected rats, with muscle on and muscle off and at four torques

Muscle	Torque (N⋅cm)	CCH (n=9)				Buffer (n=11)
		Immobilized		Contralateral		Immobilized		Contralateral
		Mean (°)	SEM	Mean (°)	SEM	Mean (°)	SEM	Mean (°)	SEM
On	2.5	93.3	6.7	134.7	4.5	89.5	2.8	125.1	2.7
	7.5	98.4	7.2	143.8	4.3	95.7	3.2	134.5	2.6
	12.5	116.2	7.7	156.3	3.0	105.6	3.7	145.4	2.4
	23.4	133.4	6.4	169.4	2.6	119.1	4.1	157.0	2.3
Off	2.5	128.5	6.4	156.0	2.1	111.8	8.8	147.2	2.3
	7.5	134.8	6.8	162.0	2.1	117.7	2.6	153.2	2.2
	12.5	142.6	7.6	173.6	2.0	126.8	2.7	161.8	2.5
	23.4	175.5	9.3	189.6	5.4	140.5	4.2	173.7	3.6

Notes: After 4 weeks of immobilization, experimental knees were intra-articularly injected with CCH (50 µL, 0.6 µg/µL) or buffer (50 µL). After 2 weeks of spontaneous remobilization, ROM was measured with an automatic arthrometer at torques 2.5, 7.5, 12.5, and 23.4 N⋅cm, with muscle on and muscle off. Mean extension angles are presented with standard error. (Statistical analysis is provided in Table 2.)

Abbreviations: CCH, collagenase Clostridium histolyticum; ROM, range of motion; SEM, standard error of mean.

Table 2 Statistical linear mixed effect modeling to determine the effect of CCH injection compared to buffer injection on the ROM of a rat knee with contracture

Linear model: ROM ~ injection + torque + muscle + leg + (error: rat.ID)
Estimated increase (°)	Standard error	p-value	Estimated increase (°)	Standard error	p-value
Buffer to CCH			Torque 2.5 N cm to torque 23.4 N⋅cm
8.0	3.8	0.046	33.8	1.7	1.0×10^−58
Muscle on to muscle off			Immobilized to contralateral
23.8	1.2	3.9×10^−55	37.7	1.2	4.3×10^−95

Notes: CCH injection with 2 weeks of spontaneous remobilization increases ROM by 8.0° (±3.8°) with a p-value of 0.046. CCH, n=9; buffer, n=11.

Abbreviations: CCH, collagenase Clostridium histolyticum; ROM, range of motion.

Figure 1 Pictures of the automatic arthrometer developed by The Bone and Joint Laboratory and used to measure the knee ROM in extension.

Notes: Images show a rat leg without skin and with muscle mounted on the arthrometer with measures of the ROM in extension at (A) torque 1 (131°), (B) torque 2 (149°), and (C) torque 3 (161°).
Abbreviation:DDDT_A_12181558_F0001

Figure 2 Mean extension angles of CCH-injected and buffer-injected experimental rat knees.

Notes: After 4 weeks of immobilization, experimental knees were intra-articularly injected and given 2 weeks for spontaneous remobilization. Mean angles were calculated after measurement of ROM at four torques (2.5, 7.5, 12.5, and 23.4 N⋅cm) with muscle on and then after sectioning the muscle (muscle off). Mean ROM values are given within each bar along with the mean gain in ROM with CCH treatment (number above buffer bar). The difference between the groups was most noticeable with muscle off and at the highest torque, where ROM in the treated legs was higher than the control legs. Means are presented with standard error. Collagenase, n=9; buffer, n=11. (Statistical analysis is presented in Table 2.)
Abbreviations: CCH, collagenase Clostridium histolyticum; ROM, range of motion.

Figure 3 Box plots showing the degree of variation for ROM for experimental and contralateral knees of buffer-injected and CCH-injected rats.

Notes: The black lines represent median values for each group: experimental (Exp) or contralateral (ConLat) with muscle on (on) or sectioned (off). Each box represents the interquartile range, whiskers indicate the range of highest and lowest extension angles that are nonoutliers, and outliers (outside 1.5 times the interquartile range) are plotted as individual points. CCH, n=9; buffer, n=11.
Abbreviations: CCH, collagenase Clostridium histolyticum; ROM, range of motion.

Figure 4 Immunostaining of collagen type I and collagen type III in the posterior capsule from contralateral knees and experimental knees intra-articularly injected with CCH or buffer.

Notes: (A) The extracellular matrix staining was quantified using ImageJ. Intensity of staining as a percentage is presented in the bar graphs (with total black considered as 100%). Experimental capsules (both CCH and buffer) showed significantly more staining than contralateral for collagen type I. Quantification of collagen type III staining for the same capsules showed a significant increase in staining in the CCH-treated capsule compared with buffer-treated and contralateral capsules (n=44 fields from buffer knees, n=36 fields from CCH knees, and n=64 fields from contralateral knees). (B) Representative micrographs of sections from the posterior knee capsule from joint injected with either buffer or collagenase (CCH) and stained with collagen type I or type III are shown.
Abbreviation: ns, not significant.

Figure 5 Hematoxylin and eosin staining of contralateral knees and experimental knees intra-articularly injected with CCH or buffer.

Notes: Original magnification of 13.2× and 33×. The articular cartilage along the femoral condyles and tibial plateaus of the contralateral joints is smooth and undamaged. The cartilage of joints with contractures is uneven; however, CCH-injected knee joints show no further damage compared to the buffer-injected joints.
Abbreviation: CCH, collagenase Clostridium histolyticum.