Terminal Sterilization Influences the Efficacy of an Extracellular Matrix-Blood Composite for Treating Post-Traumatic Osteoarthritis in the Rat Model

Abstract

The objective was to determine if an intraarticular injection of an extracellular matrix (ECM) powder and blood composite (ECM-B) after anterior cruciate ligament (ACL) injury would have a mitigating effect on post-traumatic osteoarthritis, and if that effect would be different with terminal sterilization of the ECM powder prior to use. Eighty Lewis rats underwent ACL transection and were divided into four groups: 1) intraarticular injection with PBS (PBS; n = 20), 2) intraarticular injection of ECM-B using aseptically processed ECM (ASEPTIC; n = 20), 3) intraarticular injection of the ECM-B using ECM powder sterilized with 15 kGy electron beam irradiation (EBEAM; n=20), and 4) intraarticular injection of the ECM-B using ECM powder sterilized with ethylene oxide (EO; n=20). Twenty additional animals received capsulotomy only (SHAM). Animals were followed for 6 weeks and evaluations of gait, radiographs and joint cartilage histology were performed. At 6 weeks, when compared to the SHAM group, the group treated with PBS had significantly worse gait and histologic changes, while the ASEPTIC group was not different from SHAM for either of these outcomes. When compared to the SHAM group, the EO group had similar gait outcomes, but greater histologic damage, and the EBEAM group had significantly worse gait and histological outcomes. The ECM-B composite produced using aseptically processed ECM powder mitigated the gait and histologic changes associated with osteoarthritis after ACL transection in the rat; however, care must be taken when selecting a terminal sterilization method as this may affect the effectiveness of treatment.

INTRODUCTION

Post traumatic osteoarthritis (PTOA) is a common sequela of an anterior cruciate ligament (ACL) tear with a reported incidence of up to 50% between 10 and 20 years following reconstruction surgery.^1,2 Longitudinal multicenter prospective cohort studies have identified ACL injury as a significant risk factor for the development of PTOA.^1,3 Chronic knee pain attributed to articular degeneration in patients suffering from symptomatic PTOA hinders activity and overall quality of life, especially those in younger demographics, eliciting a need for research into new therapeutic methods of treatment.⁴

The etiology for PTOA is thought to include mechanical factors, including the impact forces induced at time of injury,⁵ altered joint kinematics and altered loading patterns.^6,7 Acute mechanobiologic responses to joint trauma are believed to initiate a range of chronic pathogenic processes from increased catabolic activity in articular cartilage to stress induced apoptosis of chondrocytes and altered gene expression profiles.^8,9 Biologic factors have also been proposed, including elevated levels of inflammatory cytokines and proteases,^10,11 and reduced lubrication post injury.¹² Considering that many irreversible changes occur within the first weeks after injury,^13,14 treatments to prevent cartilage damage and joint arthrosis, which are applicable at the time of injury, are of clinical interest.

We previously demonstrated that a non-terminally sterilized injectable extracellular matrix (ECM) powder and blood composite (ECM-B) provides chondroprotection to the rat knee following ACL transection.¹⁵ As our goal is to translate the ECM-B to clinical use, a terminal sterilization procedure is required to ensure patient safety.¹⁶ However, it is well known that terminal sterilization processes involving radiation can breakdown and/or cross-link collagen, which in turn could reduce its effectiveness.¹⁷

The study objective was to determine if sterilization of the ECM powder using two different terminal sterilization processes [electron beam irradiation (EBEAM) and ethylene oxide gas treatment (EO)] has a significant effect on the ECM-B effectiveness for minimizing PTOA in the rat ACL transection model. We hypothesized that ACL transection in the rat knee followed by phosphate buffered saline (PBS) injection would result in significantly greater PTOA than capsulotomy alone, as measured by gait, radiographic and histologic changes. We further hypothesized that these changes would be significantly less when the ACL transection procedure was immediately followed by an injection of the ECM-B. Our final hypothesis was that the effects of the ECM-B injection would not be significantly different when the ECM powder was terminally sterilized with EBEAM irradiation or EO treatment.

METHODS

Study Design

The study was designed following the ARRIVE guidelines. After receiving IACUC approval, one hundred female Lewis Rats (Charles River Laboratories, Wilmington MA), ages 12±0.4 weeks, were randomized into five groups (20 animals per group, Figure 1): 1) capsulotomy but no ACL injury/treatment (SHAM), 2) ACL transection followed by an intraarticular injection with PBS (PBS group), 3) ACL transection followed by intraarticular injection with ECM powder manufactured aseptically without terminal sterilization and mixed with autologous blood (ASEPTIC group), 4) ACL transection followed by an intraarticular injection with ECM powder terminally sterilized with 15kGy EBEAM mixed with autologous blood (EBEAM group), and 5) ACL transection followed by an intra-articular injection of ECM powder sterilized with EO mixed with autologous blood (EO group). The sample size of 20 rats/group was determined a priori to have 90% power for detecting an 11% difference in the cartilage degeneration sum score and an 8-point change in differential hindlimb loading at 6 weeks.¹⁵ Gait analyses were performed pre-operatively, 2 and 6 weeks after surgery. At 6 weeks, the animals were euthanized (via CO₂ asphyxiation and exsanguination) and both hindlimbs were harvested. With the knee capsule intact, plane radiography was used to evaluate bony changes post-mortem. The specimens were then prepared for histology to evaluate the presence and extent of articular cartilage damage.

Figure 1.

Schematic diagram of the experimental design. ACLT=ACL Transection; Sx=Surgery; ECM-B=extracellular matrix blood composite; EO=ethylene oxide; PBS=phosphate buffered saline.

ECM Powder Production

Briefly, the ECM powder was aseptically manufactured from reconstituted decellularized bovine connective tissue (Maverick BioSciences, New Zealand) by decellularizing, solubilizing and lyophilizing the tissue. The bovine tissues were shipped to the lab on dry ice to keep the temperature below −20°C for the duration of the transfer. At the lab the tissues were decellularized using Triton X-102 (Sigma-Aldrich, St Louis, MO),¹⁸ then solubilized in an acidic pepsin solution, lyophilized and rehydrated with a specified amount of water to create a solution with a total protein content >5mg/ml.¹⁵ The concentrated slurry was brought to an alkaline pH using sodium hydroxide (Sigma-Aldrich) to inactivate pepsin, neutralized in a HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer (Mediatech Inc, Herndon, VA), and brought to iso-osmolarity with calcium chloride (Sigma-Aldrich) before a final lyophilization step. The resulting dry ECM material was milled into a powder and loaded into 1 mL Luer lock syringes (20 mg per syringe). Each syringe was then placed into double peel-pack package and stored at −20°C protected from light until use.

Sterilization

Two thirds of the loaded syringes were sent out to undergo the following terminal sterilization regimens: 1) low temperature EO (EO group), and 2) 15 kGy EBEAM (EBEAM group). The remaining third was not terminally sterilized and served as a control (ASEPTIC group). Low temperature EO sterilization was performed at a commercial facility (Nelson Laboratories, Salt Lake City, UT) at 30°C in 30% relative humidity and a gas concentration of 650 mg/L. A gas dwell time of 360 minutes and subsequent steam dwell time of 60 minutes were used. EBEAM sterilization at 15 kGy was conducted at a commercial facility (Sterigenics, San Diego, CA). Sterility testing was then performed on the EBEAM and EO sterilized powder at a contract research organization (Avista Pharma, Agawam, MA) to confirm that each treatment resulted in sterile scaffolds. Bioburden of the ASEPTIC product was also tested at a contract laboratory (Nelson Laboratories, Salt Lake City, UT) and no bioburden was detected

Animal Surgeries

All animal care procedures were performed at Rhode Island Hospital and Institutional Animal Care and Use Committee approvals from Rhode Island Hospital and the Department of Defense were obtained prior to initiating the study. Anesthesia was induced by chamber exposure to inhalation of isoflurane (3–5%) and then maintained using a nose cone (1–5%). Eye drops were applied pre-operatively to prevent drying during surgery, and cefazolin (20 mg/kg) was administered via intramuscular injection as a prophylactic antibiotic. Pre-operatively, animals received one subcutaneous injection of sustained-release buprenorphine [1.2 mg/kg bodyweight] to provide 72 hours of analgesia. Post-operatively, the animals were permitted to bear weight as tolerated. Two animals were housed per cage with free access to food and water.

The ACL transection and intraarticular injection procedures were performed as previously reported.¹⁵ In brief, an anteromedial approach was used to expose the ACL, which was transected sharply under direct vision and confirmed by a positive anterior drawer test. The incision was closed in two layers using 6–0 absorbable suture (Vicryl; Ethicon Inc., Bridgewater, NJ) and secured with a skin adhesive (Vetbond; 3M, St. Paul, MN). For each animal in the ECM-B injection groups, 20 mg of ECM powder was initially rehydrated with 400 μL water and mixed between two syringes to create a suspension. To make the ECM-B, two hundred μL of the ECM suspension was then mixed with 200 μL of autologous blood drawn without anticoagulant from the saphenous vein of the contralateral limb by mixing between the two syringes. The time between the blood draw to ECM-B injection was approximately 90 seconds. After closure of the rat knee joint capsule but prior to closing the skin, 100 μL of the ECM-B was injected through a 25-gauge needle into the rat knee prior to clot formation. This amount produced a small palpable extension of the capsule without leakage from the closed incision.

Gait Analysis

Gait was evaluated using a pressure mat (VH3 Very High-Resolution Walkway System; Tekscan Inc, Boston, MA) with an active sensing area of 33.5 × 11.2 cm.¹⁹ Animals were trained to walk on the pressure mat in one direction using a dark box as a safe haven at the end of the walkway to encourage them to move across the instrumented platform. A custom plexiglass tunnel placed over the platform was used to aid the animal to walk in a straight line. Based on the pressures observed for our cohort, a step calibration was performed over the sensing tile of the platform using a 200 g custom three-legged phantom as recommended by the manufacturer. Data were collected at a frame rate of 104 Hz using the first hoof contact as a trigger until the animal stepped off the mat. Commercial software (Walkway 7.0; Tekscan, Inc) was used for data acquisition and analysis, and 5 trials were obtained for each animal at each of the 3 timepoints (pre-operatively, 2 and 6 weeks post-operatively). The 2- and 6-week timepoints were selected to coincide with the end of the acute inflammatory response and the end of the study in which structural damage of the cartilage was present. For analysis, hoof strikes were automatically identified, and partial strikes were discarded. The primary outcome measure for the gait assessment was the difference in peak vertical load bearing relative to baseline (pre-operative) between the experimental and control limbs within each experimental group. Additional secondary measures included stance time, stride time, stride length, and stride velocity for each hind limb.²⁰ The average from the three best trials, those in which the rat walked across the platform without stopping and with at least 2 complete gait cycles over the sensing portion of the walkway, was reported for each outcome measure.

Radiographs

After the hindlimbs were harvested, lateral and posterior-anterior radiographs were obtained using a high-resolution (14 bit) small animal radiography system (MX-20; Faxitron,Wheeling, Illinois). The images were obtained at 28 kV, with adjustments in kilovolts, as needed, to obtain adequate penetration and image quality. Osteoarthritic changes including osteophytes in the lateral intercondylar notch and medial tibial spine were assessed by two independent observers (MMM and PK), who were blinded to the animal number, surgical limb, and treatment group. A modification of the Kellgren-Lawrence grading system²¹ was adapted for use in the rat.²² Consistent with the original grading system, a score of 0 was assigned for knees with no osteophyte formation, a score of 1 if possible osteophytes were observed, and a score of 2 if there were definite osteophytes present. Joint space narrowing was not assessed as the limbs were imaged non-weightbearing.

Histology

After imaging was completed, all knees were decalcified, and embedded as previously described.¹⁵ Coronal sections (6 μm thick) through the center of the knee, which were then stained with Hemotoxylin and Eosin or Safranin-O, were analyzed. Scoring of the medial femoral condyle, medial tibial plateau, lateral femoral condyle and lateral tibial plateau were conducted using the modified Mankin cartilage structure and cellularity scores (Supplemental Table 1).²³ Analysis of the synovium was performed according to the OARSI synovial membrane inflammation score for rats.²⁴ Synovial scoring was done on synovium adjacent to the medial meniscus at the most affected area, with a score of 0 indicating no change in the synovium and a score of 4 indicating a significant increase in synovial lining, cell proliferation and infiltration of inflammatory cells. In addition to the Mankin sub-scores of the cartilage, analysis of additional microscopic features of each cartilage zone of the four articular surfaces were also performed as in Table 1. All histological scoring was performed by three experienced graders (BCF, MMM and PK) and determined by consensus. All readers were blinded to the animal number, surgical treatment and hindlimb.

Table 1.

Additional feature scoring used to assess the cartilage structure of the medial tibial plateau (MTP), lateral tibial plateau (LTP), medial femoral condyle (MFC) and lateral femoral condyle (LFC) in each knee.

Parameter	Grade	Description
Superficial zone	0	Lamina splendens intact and cellular, color of cancellous bone on H and E
	1	Lamina splendens intact and cellular, color of calcified cartilage on H and E
	2	Acellular lamina splendens
	3	Disrupted lamina splendens
Transitional zone	0	Cells present in groups of 1–4 cells
	1	Cloning in clusters of 6+ cells with small, dense nuclei
	2	Loss of cartilage ECM in the transitional zone
Radial Zone	0	Present in MTP with columns of 2–8 cells
	1	Enlarged in MTP or present in LTP
	2	Loss of cartilage ECM in the radial zone
Tidemark	0	Present and continuous
	1	Blurred or missing for more than half of the width of the condyle or plateau
	2	Loss of extracellular matrix to the tidemark
Calcified Cartilage	0	Columns of 2 or fewer cells
	1	Columns of greater than 2 cells, thickening of this zone
	2	Loss of extracellular matrix extends to this zone

Statistical Methods

Generalized estimating equations (GEEs) were used (1) to model the gait parameters as a function of animal within follow-up timepoint and experimental group and (2) to model the Kellgren-Lawrence grading scores, the Mankin structure scores, the Mankin cellularity scores, the synovitis scores, the superficial and transitional zone scores, the radial, tidemark, and calcified cartilage zone scores as a function of knee within animal and experimental group. Classical sandwich estimation was used to adjust for any possible model misspecification. Pairwise comparisons between groups were conducted via orthogonal contrasts. The Holm-test was used to adjust for multiple comparisons to maintain a two-tailed familywise alpha at 0.05. A p-value of 0.05 was used to determine statistical significance.

RESULTS

Gait Outcomes

There were no significant effects due to surgical group on any of the gait parameters at baseline (pre-operatively) (Supplemental Data Table 2).

At two weeks, the change in maximum force (%BW) relative to baseline for the surgical limb was significantly different from that in the contralateral limb (P<.001) for all treatment groups except for the SHAM group (Figure 2A). On average the animals in the ASEPTIC, EBEAM, EO and PBS groups off loaded the ACL transected limb and increased the load on the contralateral control knee relative to baseline (Table 2). There were no significant effects of surgical group on stance time, stride time, stride length, or stride velocity at the 2-week time point (Supplemental Data Table 2).

Figure 2.

Mean difference in maximum force relative to baseline (%BW) at (A) 2 weeks and at (B) 6 weeks between the surgical and contralateral control knees. A negative value indicates more offloading of the surgical limb. Error bars represent the 95% confidence intervals. BW=body weight; PBS=phosphate buffered saline; EO=ethylene oxide; EBEAM=electron beam; ASEP=ASEPTIC.

Table 2.

Mean (95% confidence intervals) values of the maximum force expressed as %BW for the surgical and control limbs at baseline, 2 and 6 weeks for each group. Tx=treatment; PBS=phosphate buffered saline; EO=ethylene oxide.

Tx Group	Surgical Limb (Pre-op)	Control Limb (Pre-op)	Surgical Limb (2 week)	Control Limb (2 week)	Surgical Limb (6 week)	Control Limb (6 week)
SHAM	51.2 (49.0, 53.4)	52.6 (50.7, 54.4)	51.1 (48.4, 53.8)	53.0 (50.6, 55.4)	52.5 (49.8, 55.2)	54.3 (52.7, 55.9)
ASEPTIC	50.6 (49.1, 52.1)	51.8 (49.5, 54.1)	43.6 (40.5, 46.7)	57.8 (54.3, 61.3)	51.8 (49.4, 54.2)	54.1 (51.8, 56.4)
EBEAM	50.3 (48.0, 52.6)	50.3 (48.2, 52.4)	41.6 (39.9, 43.3)	56.9 (54.1, 59.7)	52.2 (49.9, 54.5)	54.4 (51.9, 56.9)
EO	51.6 (48.7, 54.5)	51.2 (49.4, 53.0)	41.6 (38.8, 44.4)	55.2 (52.3, 58.1)	51.8 (49.7, 54.0)	56.3 (53.5, 59.1)
PBS	50.3 (47.6, 53.0)	52.2 (50.5, 53.9)	38.8 (36.4, 41.2)	58.9 (55.9, 61.9)	48.0 (44.7, 51.3)	54.4 (52.0, 56.8)

At 6 weeks, the change in maximum force (%BW) relative to baseline for the surgical limb was significantly different from that in the contralateral limb for the EO and PBS treated groups but not the EBEAM, ASEPTIC and SHAM groups (Figure 2B). On average the animals in the EO and PBS groups continued to off load the ACL transected limb relative to baseline (Table 2). There were no significant effects of surgical group on stance time, stride time, stride length, or stride velocity at the 6-week time point (Supplemental Data Table 2).

Radiography - Kellgren-Lawrence Grading

There were no definite osteophytes noted in any of the knees. ACL transection followed by EBEAM injection resulted in a higher mean Kellgren-Lawrence score than the SHAM group (Figure 3, P=.01). The ASEPTIC group was not significantly different from the PBS group (P=0.99) and/or the SHAM group (P=0.35).

Figure 3.

Mean Kellgren-Lawrence scores for the knees in each treatment group. Error bars represent the 95% confidence intervals. Only the EBEAM group had significantly higher scores than the SHAM group (*P=.01). All groups had mild changes with mean scores between 0 (normal) and 1 (possible osteophytes). PBS=phosphate buffered saline; EO=ethylene oxide; EBEAM=electron beam.

Histology - Mankin Structure Score

ACL transection followed by PBS injection led to significantly greater structural cartilage damage scores in the medial femoral condyle on the surgical knee than in the SHAM group (Table 3, Figure 4; P=.001). Likewise, ACL transection followed by EBEAM or EO injections also led to significantly greater structural cartilage damage scores in the medial femoral condyle (P<.01), while there were no significant differences between the ASEPTIC and the SHAM groups (P=.55). No significant differences in the structural cartilage damage scores were found between groups on the medial tibial plateau nor in the lateral compartment (Table 3).

Table 3.

Mean Mankin Structural Score (95% confidence intervals) as a function of treatment group and location within the knee. PBS=phosphate buffered saline; EO=ethylene oxide, MFC=medial femoral condyle; LFC=lateral femoral condyle; MTP=medial tibial plateau; LTP=lateral tibial plateau.

Mankin Structure	SHAM	PBS	ASEPTIC	EBEAM	EO
Surgical Side
MFC	0.3 (0.0, 0.5)	2.4 (1.0, 3.7)#	0.7 (0.03, 1.4)	2.1 (0.8, 3.3)#	2.1 (0.8, 3.4)#
MTP	0.7 (0.0, 1.5)	1.8 (0.9, 2.6)	0.9 (0.4, 1.4)	2.3 (1.1, 3.4)	1.6 (0.7, 2.4)
LFC	0.6 (0.3, 0.8)	1.0 (0.4, 1.6)	0.9 (0.2, 1.5)	0.8 (0.5, 1.1)	0.6 (0.2, 1.0)
LTP	1.0 (0.2, 1.7)	1.4 (0.8, 2.0)	1.0 (0.6, 1.4)	1.2 (0.6, 1.7)	0.6 (0.2, 0.9)
Severe change (any surface)	2/19	4/19	1/19	4/20	3/20
Contralateral Side
MFC	0.3 (0.01, 0.5)	0.8 (0.0, 1.6)	1.3 (0.2, 2.3)	1.2 (0.1, 2.2)	1.5 (0.2, 2.7)
MTP	0.3 (0.1, 0.5)	0.6 (0.1, 1.0)	1.8 (0.9, 2.7)#	1.2 (0.4, 2.0)	1.4 (0.6, 2.2)
LFC	0.6 (0.2, 0.9)	0.4 (0.0, 0.9)	0.6 (0.2, 1.0)	0.6 (0.2, 0.9)	0.6 (0.1, 1.0)
LTP	0.4 (0.1, 0.7)	0.7 (0.3, 1.0)	1.0 (0.4, 1.5)	1.1 (0.5, 1.6)	0.4 (0.1, 0.7)
Severe change (any surface)	0/19	1/19	3/19	2/19	4/19

^#significantly different from SHAM (P<.05, Holm test)

Figure 4.

Representative images showing the cartilage damage (black arrows) in the medial compartment of the surgical limbs for each treatment group. PBS=phosphate buffered saline; EO=ethylene oxide; EBEAM=electron beam.

Severe arthritic changes (defined as a Mankin Structural score of 7 or 8) were noted in 11% of the knees in the SHAM group, 21% of the knees treated with PBS, and 5% of the knees in the ASEPTIC group (Table 3). Knees treated with ECM-B where the ECM powder had been sterilized by either EBEAM or EO had severe arthritic changes in 20% and 15% of knees, respectively. The severe changes were more commonly noted in the medial femoral condyle (11 knees), followed by the medial tibial plateau (6 knees), lateral femoral condyle (1 knee) and lateral tibial plateau (1 knee).

On the contralateral knee, the ASEPTIC group had a higher mean structural score than the PBS and SHAM groups (Table 3). However, the mean values for all groups were less than 2. When a damage score greater than 7 was seen in the contralateral knee, it was always in the medial compartment (medial tibial plateau or medial femoral condyle) and more commonly in the medial femoral condyle (8 knees) than medial tibial plateau (3 knees).

Histology - Mankin Cellularity Score

The cellularity score in the medial femoral condyle of the surgical knee was greater (worse) in the PBS, EO, and EBEAM groups compared to the SHAM group (Table 4, P<.04). There were no significant differences in the cellularity scores in the medial femoral condyle of the ASEPTIC group when compared to the SHAM group (P=.23). There were also no significant effects of treatment on the cellularity scores of the medial tibial plateau, lateral femoral condyle or lateral tibial plateau on the surgical side with respect to the SHAM or PBS groups. On the contralateral side, only the ASEPTIC group had a higher mean cellularity score than the SHAM group on the lateral tibial plateau (P=.04). There were no differences between groups for the medial tibial plateau or lateral femoral condyle of the contralateral knee.

Table 4.

Cellularity Scores for the five groups [mean (95% confidence intervals)] on each of the four cartilage surfaces of both the surgical and contralateral knees. PBS=phosphate buffered saline; EO=ethylene oxide, MFC=medial femoral condyle; LFC=lateral femoral condyle; MTP=medial tibial plateau; LTP=lateral tibial plateau.

Parameter	SHAM	PBS	ASEPTIC	EBEAM	EO
Surgical Side
MFC	0.2 (0.0, 0.4)	1.2 (0.7, 1.7)#	0.7 (0.2, 1.1)	1.2 (0.7, 1.6)#	0.9 (0.4, 1.4)
MTP	0.4 (0.0, 0.8)	0.6 (0.3, 0.9)	0.4 (0.1, 0.6)	0.9 (0.4, 1.3)	0.5 (0.1, 0.8)
LFC	0.3 (0.0, 0.5)	0.7 (0.4, 0.9)	0.6 (0.2, 0.9)	0.6 (0.2, 0.9)	0.3 (0.0, 0.6)
LTP	0.6 (0.2, 0.9)	0.7 (0.3, 1.0)	0.6 (0.2, 0.9)	0.7 (0.3, 1.1)	0.4 (0.1, 0.6)
Contralateral Side
MFC	0.1 (0.0, 0.2)	0.5 (0.0, 0.9)	0.8 (0.3, 1.2)	0.7 (0.2, 1.2)	0.5 (0.1, 0.9)
MTP	0.2 (0.0, 0.5)	0.2 (0.0, 0.4)	0.7 (0.3, 1.0)	0.3 (0.0, 0.5)	0.3 (0.1, 0.5)
LFC	0.1 (0.0, 0.1)	0.2 (0.0, 0.3)	0.3 (0.1, 0.5)	0.2 (0.0, 0.4)	0.3 (0.1, 0.5)
LTP	0.1 (0.0, 0.1)	0.2 (0.0, 0.5)	0.4 (0.2, 0.6)#	0.2 (0.0, 0.3)	0.2 (0.0, 0.3)

^#significantly different from SHAM (Holm test, P<.05).

Histology - Synovitis Scores

Many of the knees that underwent ACL transection had synovitis scores that consisted of mild synovial thickening but no inflammatory cells, regardless of treatment group. ACL transection followed by PBS injection had a higher degree of synovitis than the SHAM group (Figure 5, P<.001). Injection of ASEPTIC or terminally sterilized ECM-B did not affect the degree of synovitis (Figure 5). All treatment groups, other than the SHAM group, had significantly more synovitis when compared to their contralateral controls (Data not shown, P<.001 for all comparisons).

Figure 5.

Histological analysis of synovitis in the medial compartment of the treated knees. Synovitis was scored adjacent to the medial meniscus according to a synovial membrane inflammation score. The mean values and the 95% confidence intervals are provided. Knees that underwent a capsulotomy with no ACL transection (SHAM) had significantly less synovitis when compared to all other groups (*** P<.001). PBS=phosphate buffered saline; EO=ethylene oxide; EBEAM=electron beam.

Histology - Superficial and Transitional Zones

ACL transection followed by PBS injection led to increased damage in the superficial zone of the medial femoral condyle and medial tibial plateau when compared to the SHAM group (Table 5; P<.05). However, no significant differences in superficial zone damage were found between the ASEPTIC and SHAM groups (P>.75). There were no significant effects of treatment group on superficial zone cartilage damage in either of the lateral compartment surfaces. The percentage of knees with a disrupted lamina splendens was approximately 30% for the PBS and EBEAM groups, while it was 0% for the SHAM group and 5% for the ASEPTIC group (Table 5).

Table 5.

Mean (95% confidence intervals) histologic scores of the superficial and transitional zones. Also, the percentages of knees in each group for the lamina splendens disruption are presented. PBS = phosphate buffered saline; EO=ethylene oxide, MFC=medial femoral condyle; LFC=lateral femoral condyle; MTP=medial tibial plateau; LTP=lateral tibial plateau.

Parameter	SHAM	PBS	ASEPTIC	EBEAM	EO
Superficial Zone
MFC	0.0 (0.0, 0.0)	1.1 (0.5, 1.6)#	0.4 (0.0, 0.7)	1.3 (0.7, 1.8) #	0.9 (0.3, 1.5)#
MTP	0.2 (0.0, 0.5)	1.4 (0.9, 1.9)#	0.7 (0.2, 1.1)	1.6 (1.0, 2.1) #	1.1 (0.5, 1.7)#
LFC	0.3 (0.0, 0.6)	0.7 (0.2, 1.2)	0.6 (0.1, 1.0)	0.4 (0.0, 0.8)	0.5 (0.1, 0.8)
LTP	0.7 (0.2, 1.1)	0.8 (0.2, 1.3)	0.8 (0.3, 1.3)	0.5 (0.1, 0.8)	0.6 (0.1, 1.0)
Transitional Zone
MFC	0.0 (0.0, 0.0)	0.7 (0.3, 1.1)#	0.3 (0.0, 0.5)	0.8 (0.4, 1.1)#	0.6 (0.2, 0.9)#
MTP	0.2 (0.0, 0.4)	0.3 (0.0, 0.5)	0.1 (0.0, 0.3)	0.4 (0.0, 0.7)	0.3 (0.0, 0.5)
LFC	0.1 (0.0, 0.1)	0.4 (0.1, 0.7)	0.2 (0.0, 0.4)	0.1 (0.0, 0.2)	0.1 (0.0, 0.2)
LTP	0.2 (0.0, 0.4)	0.3 (0.0, 0.6)	0.1 (0.0, 0.3)	0.1 (0.0, 0.1)	0.2 (0.0, 0.4)
Percentage of knees with lamina splendens disruption
MFC	0	27.8	5.3	30.0	21.1
MTP	5.3	22.2	10.5	25.0	21.1
LFC	0	15.8	5.3	0	0
LTP	5.3	15.8	5.3	5.0	5.3

^#significantly different from SHAM (P<.05, Holm test)

The PBS group had higher (worse) scores in the transitional zone of the medial femoral condyle when compared to the SHAM group (Table 5; P<.001). Likewise, the EO and EBEAM groups had significantly greater transitional zone damage scores relative to the SHAM group (P<.001). There were no effects of treatment group on the transitional zone scores in the medial tibial plateau, lateral femoral condyle or lateral tibial plateau (Table 5).

Histology - Radial, Tidemark and Calcified Cartilage Zones

The scoring of the medial tibial plateau radial zone histology did not reveal any statistically significant differences between the groups (Supplemental Table 3). In the lateral tibial plateau, ACL transection with PBS injection led to approximately 50% of the knees having a radial zone (which was not seen in the SHAM group, P<.001). The injection of ASEPTIC, EO or EBEAM ECM-B instead of PBS did not lead to a significant improvement of the radial zone scores (P>.99 for all three comparisons). There were no significant differences between groups on the tidemark histology in the medial compartment or the lateral femoral condyle (P>.11 for all comparisons). However, the EBEAM group did exhibit a higher tidemark score than the ASEPTIC group in the lateral tibial plateau (P=.01).

DISCUSSION

ACL transection followed by an injection of PBS led to significant changes in parameters consistent with posttraumatic osteoarthritis: greater limb offloading in the surgical limb relative to baseline, increased Kellgren-Lawrence radiographic score, and increased histological changes, including greater Mankin structural and cellular scores in the medial compartment of the knee, as well as increased synovitis. The injection of the ASEPTIC processed ECM-B composite significantly minimized the gait and histologic changes, and ECM-B sterilized using EO performed similarly to the ASEPTIC processed material with regards to Kellgren-Lawrence grading but not in histological grading. Terminal sterilization of the ECM powder with EBEAM irradiation resulted in greater Kellgren-Lawrence grading and histologic damage than the ASEPTIC processed group for the Mankin Structural score and damage scores in the superficial and transitional zones. It should also be noted that none of the knees treated with any form of the ECM-B composite (ASEPTIC, EO or EBEAM) had worse outcomes in any aspect when compared with the PBS group.

ACL transection followed by an injection of PBS led to significant gait changes consistent with PTOA, including a lower maximum force relative to baseline in the surgical limb. These findings are consistent with our prior study of ACL transection in the rat model, where ACL transection followed by PBS injection provided a similar response.¹⁵ Furthermore, intra-articular injections of hydrogels by us and others have been shown to normalize limb asymmetries.^15,25

ACL transection followed by injection with PBS led to a mild increase in synovitis at 6 weeks after surgery when compared to the SHAM group at a level. This degree of synovitis in the current study is in contrast to the more severe reaction seen with historical materials previously used in the joint, including specific suture and synthetic devices which have resulted in the presence of multinucleated giant cells,²⁶ dense invasions of mononuclear round cells,²⁷ a more severe synovitic reaction,²⁸ or a granulomatous synovitis. The current findings are similar to those previously reported with non-surgical rupture of the ACL in the rat model without any biomaterial implantation,²⁹ and with surgical ACL transection in the porcine model,³⁰ with an increase in synovial cellularity but no significant inflammatory cell infiltrate.

The majority of the changes in the cartilage after ACL transection appeared to be in the superficial and transitional zones of the medial compartment, with minimal changes to the deeper zones, consistent with prior reports in the ACL transection model.^31–33 When looking at the distribution of the cartilage damage severity in the current study, the medial compartments were more severely damaged than the lateral compartments, which is also consistent with prior reports.³⁴ In prior studies looking at treatments or prevention of osteoarthritis, various injections^19,35 and oral therapeutics³⁶ have been reported to result in significant decreases in cartilage damage after ACL transection. However, as in this study, none of the cited therapeutic studies showed a complete suppression of cartilage damage after ACL transection as the instability induced by the ACL transection was not reduced during treatment. We also noted that the contralateral knee showed some histological evidence of relatively mild cartilage damage, with no difference seen among all the ACL transected groups. This could be due to the changes in limb loading the occurred following the ACL transection procedure.

EO and EBEAM irradiation represent two of the established Class A techniques in use for the terminal sterilization of medical devices to be introduced into the body, even though they are known to compromise the material properties of vulnerable protein-based biologics.¹⁶ These methods work in part by inducing cleavage and other chemical modifications to proteins in bacteria and viruses.^37,38 However, these same alterations can disrupt the polymeric extracellular matrix protein networks in ECM-based biomaterals,³⁹ leading to changes in mechanical and biophysical properties,^37,40 and thus should be selected with care. The aseptically processed material investigated in this study has previously been shown to slow the progression of histologic PTOA following unilateral ACL transections,¹⁵ and treatment with EO or EBEAM radiation can result in effective sterilization with only slightly altered in vitro matrix properties in similar extracellular matrix materials.^41,42 Despite these findings, the terminal sterilization of the ECM-B material with EBEAM resulted in decreased histologic impact in vivo in relation to the PBS group. However, the use of EO as a sterilization method resulted in a product that maintained its in vivo effectiveness in this model relative to the SHAM group.

There are several limitations that should be considered when interpreting the results. While the rat model is frequently used to study the pathogenesis of osteoarthritis and its treatment, it is not fully representative of the human condition as it is a small quadruped, exhibits structural cartilage differences, and develops osteoarthritis at a much faster rate.⁴³ Also, the ACL transection model is frequently used to evaluate PTOA therapeutics. However, these therapeutics typically address the biological action of the injury without treating the mechanical deficiency induced by the ligament injury. Thus, the mechanical instability may eventually over-ride any biological benefit of the initial treatment and complete restoration of joint health would not be achieved. There are also limitations inherent to the assays including the kinetic gait assessment without directly measuring joint kinematics or the shear forces at the foot, the post-mortem acquisition of the radiographs in the non-weightbearing knee, and the semi-quantitative histological assessments of cartilage health. However, these methods are frequently used in small animal models. Despite these limitations, the data presented here suggest that the ECM-B composite shows promise and could be evaluated in larger animal models and eventual clinical trials if the large animal work replicate the findings in rats.

In conclusion, the aseptically processed ECM-B as well as ECM-B made with ECM powder terminally sterilized using ethylene oxide reduced some of the gait and histologic changes that are associated with PTOA after ACL transection in the rat. However, terminal sterilization of the ECM powder with EBEAM irradiation resulted in greater (i.e., worse) histologic damage than that seen in the aseptically processed group.