Intra-articular implantation of collagen scaffold carriers is safe in both native and arthrofibrotic rabbit knee joints

Article focus

• A total of 96 rabbits were randomly allocated to four experimental groups: arthrotomy alone; arthrotomy and collagen scaffold placement; contracture surgery; and contracture surgery and collagen scaffold placement.

• Animals were killed at 72 hours and two, eight and 24 weeks.

• Joint contracture, chondral damage and synovitis were assessed.

Key messages

• Intra-articular implantation of a collagen sponge does not induce synovitis, cartilage damage or fibrosis with contracture in the rabbit joint.

• All collagen sponges were resorbed in this model by eight weeks.

• Collagen sponges may be considered a safe carrier for intra-articular administration of pharmacological agents.

Strength and limitations

• Strengths: large sample size, four study groups, multiple time points, validated scales for grading.

• Limitations: assessment of chondral damage was difficult due to artefacts at the time of sectioning.

Introduction

Arthrofibrosis, or fibrosis of the tissues around synovial joints, remains a difficult clinical problem in orthopaedics, despite numerous treatment modalities.^1,2,3 In order to study the pathophysiology behind arthrofibrosis, animal models have been developed using the New Zealand White rabbit knee.^4,5 These models involve an intracapsular knee injury followed by Kirschner-wire (K-wire) immobilisation for eight weeks, and result in substantial contracture that resembles the human biomechanical, cellular, and molecular response to injury, with increased myofibroblasts and transforming growth factor beta (TGF-β) activity.^6-12

Previous studies have demonstrated significant upregulation of myofibroblasts early in the process of joint contracture (within two weeks of injury),¹² as well as notable changes in messenger ribonucleic acid (mRNA) expression within the first 72 hours of injury. Furthermore, microarray analysis of mRNA has demonstrated that the most notable changes in mRNA expression in capsular tissues occurred within the first 24 to 72 hours after injury.^13,14 Other studies have suggested the possible influence of mast cells on the development of arthrofibrosis in the four weeks after injury.¹¹ The early onset of changes, both on the cellular and molecular level, seems to indicate the need for early intervention to prevent or abort the development of arthrofibrosis. Using these animal models, attempts to find an effective anti-fibrosis medication are underway. Ketotifen, a mast cell stabiliser, has already shown some promise in reducing contracture severity in a rabbit model.¹⁵ Decorin, while demonstrating change in the local mRNA expression, did not affect the overall degree of contracture.¹³

If administration of a pharmacological agent is proven to be effective in the prevention or treatment of joint contracture, the route of administration becomes critical. Systemic administration may lead to unpredictable pharmacological levels at the joint, and could be associated with side effects. Local intra-articular administration is very appealing, but would require the use of a carrier for sustained administration and release. However, intra-articular implantation of a carrier could potentially have adverse effects, such as cartilage damage, an inflammatory synovial response, or fibrosis.

Collagen, a naturally occurring polymer, presents an attractive option for intra-articular drug delivery. Currently, collagen scaffolds are clinically employed for (or under investigation for use in) ophthalmologic applications,¹⁶ local antibiotic delivery,^17-24 local wound care,^25-27 and healing of bone defects or generation of bone fusion.^28-32 Despite the ongoing research into the clinical applications of collagen scaffold implantation, the safety of intra-articular implantation of a collagen scaffold is largely unknown.

We hypothesised that a collagen scaffold could be placed in an intra-articular location in the rabbit knee, without causing alteration of joint range of movement (ROM), significant cartilage damage, or synovial inflammatory reaction, as assessed by mechanical and histological analysis. Secondly, we hypothesised that intra-articular placement of a collagen scaffold in a rabbit model of arthrofibrosis would not alter the expected joint contracture, nor cause damage to the articular cartilage, nor create a synovial inflammatory reaction, as assessed by mechanical and histological analysis. Thirdly, we hypothesised that collagen scaffold absorption would occur within eight weeks.

Materials and Methods

After obtaining approval from our departmental Review Board and Institutional Animal Care and Use Committee, 102 skeletally mature female New Zealand White rabbits (using the same gender rabbits increased likelihood of homogeneity), were randomly and equally divided into four study groups: sham arthrotomy (SA); collagen scaffold arthrotomy (CSA); sham Mayo contracture model (MCM); and collagen scaffold Mayo contracture model (CMCM), with six animals assigned as non-operative histological controls (Fig. 1). National Institutes of Health guidelines for the care and use of laboratory animals were observed at all times. Rabbits in the arthrotomy groups underwent an arthrotomy, as described below. Rabbits assigned to the arthrofibrosis model underwent the same initial procedure to develop a contracture as described in previous studies and briefly outlined below.^5,12-14,33 Collagen scaffolds used in this study were commercially available collagen sponges, derived from purified bovine tendon, currently used in dental applications. These scaffolds are pre-sterilised, eliminating the need for sterilisation. The sterile scaffolds were fashioned into 1 cm × 1 cm implants prior to implantation.

Fig. 1

Organisational chart demonstrating study design.

Results

Animals that underwent joint arthrotomy and placement of a collagen scaffold (CSA) demonstrated equivalent ROM when compared with animals that underwent arthrotomy alone (SA), with equivalence limits set at 15° (Fig. 2). No contracture was noted in either group of animals: in fact, the mean ROM for the operative knee was found to have increased by 13.9° and 10.9° (SA and CSA groups, respectively) compared with the non-operative extremity. The difference in the mean contractures between the two arthrotomy groups (SA and CSA) was -3.1°, with a 95% CI of -6.9° to 0.8°, well within the equivalence limits of -7.5° to 7.5°.

Fig. 2

Final joint contracture for all time points at death (mean ± 95% confidence intervals). The mean contractures of the arthrotomy and collagen + arthrotomy groups were statistically equivalent, with an equivalence limit of 15°.

Animals that underwent Mayo contracture model surgery and placement of a collagen scaffold (CMCM) did not have an equivalent joint contracture formation compared with MCM animals, with equivalence limits set at 15°. When comparing the two groups at all time points, CMCM animals demonstrated a mean joint contracture of 41.8° (95% CI 22.8° to 60.7°), compared with 53.9° (95% CI 31.8° to 76°) in MCM animals. The mean contracture difference was calculated to be 12.2°, with the 95% CI of -11.5° to 35.8° falling outside the equivalent limits of -7.5° to 7.5°.

Absorption of the collagen scaffold occurred within eight weeks in all animals (Table I). In CSA animals, the collagen scaffold was found at the time of death in all animals (six of six) killed 72 hours following the index procedure, in five of six animals killed two weeks following index procedure, and in no animals killed eight or 24 weeks following the index procedure. In CMCM animals, the collagen scaffold was found at the time of death in five of six animals at 72 hours following the index procedure, in two of six (33%) animals killed two weeks after the index procedure, and in no animals killed eight or 24 weeks after the index procedure. The difference between the 72-hour (five of six) and two-week (two of six) time points was not found to be significant (p = 0.24). When comparing the scaffold absorption rate in CSA animals and CMCM animals, the scaffold was found at two weeks in five of six CSA animals compared with only two of six CMCM animals. This difference was also not found to be significant (p = 0.24).

The vast majority of animals suffered no chondral damage greater than that due to sectioning artefact (Figs 3 and 4, Table II). Only three animals (two of which had collagen scaffolds implanted) suffered chondral changes in the operative knee greater than those due to artefact in all three samples (medial and lateral femoral condyles and medial tibial plateau). Collagen scaffold implantation resulted in an 8% increase (95% CI -12% to 28%) in the risk of any cartilage damage greater than artefact in animals that underwent arthrotomy, and a 4% decrease (95% CI -28% to 20%) in animals that underwent Mayo contracture model surgery (Fig. 5). Additionally, ten of 96 (10%) of the non-operative knees demonstrated chondral changes greater than those due to artefact. The overall risk of chondral damage in the operative limbs of CSA and SA animals was not significantly different (p = 1.0) to the risk of chondral damage in the non-operative limbs. Conversely, the risk of chondral damage in the operative limbs of MCM and CMCM animals was significantly greater (p = 0.02) than the risk of chondral damage in non-operative limbs.

Fig.

Cartilage sample of normal appearance from an animal that underwent arthrotomy alone, 1.25× magnification shown above (a), 4× magnification below (b).

Fig.

Cartilage sample from the operative knee of an animal undergoing Mayo contracture model surgery. These illustrations demonstrate changes due to artefact.

Fig.

Cartilage samples (1.25× above, 4× below) from an animal undergoing Mayo contracture model surgery and collagen scaffold placement, demonstrating changes greater than those due to artefact.

When the synovial samples were graded, no sample received a score higher than three of nine (indicative of mild synovitis). No significant difference was noted between the mean synovitis scores for the animals that underwent arthrotomy and those that underwent arthrotomy + collagen scaffold implantation (0.5 versus 0.5, p = 1.0) (Fig. 6, Table III). Similarly, no significant difference was noted between the mean synovitis scores for animals undergoing Mayo contracture model surgery and those undergoing Mayo contracture model surgery + collagen scaffold implantation (0.63 versus 0.75, p = 0.69). The mean synovitis score for the operative knee of animals that underwent Mayo contracture model surgery + collagen scaffold implantation was 0.75. The mean synovitis scores for animals that underwent arthrotomy + collagen scaffold placement (0.5), and those that underwent Mayo contracture model surgery + collagen scaffold placement (0.75), are indicative of “no synovitis” (values 0 to 1, not significant).

Fig. 6

Mean synovitis score by time and group (range 0 to 9). No significant difference was noted between any groups.

Interestingly, some animals undergoing arthrotomy ± placement of a collagen scaffold demonstrated signs at time of death of chronic medial patellar instability, with formation of a “pseudotrochlea” (Table IV). Such changes were not observed in any of the animals undergoing Mayo contracture model surgery. In CSA animals, these changes were noted in three of six animals at eight weeks and three of six animals at 24 weeks (six of 24 animals). In SA animals, such changes were found in three of six animals killed two weeks following the index procedure, and one of six animals at eight weeks after the index procedure (four of 24 animals). Overall, ten (20.1%) of the 48 animals that underwent arthrotomy ± scaffold implantation displayed signs of chronic medial patellar instability and pseudotrochlea formation. The rate and grade of synovitis for animals in which a pseudotrochlea was found (ten of 48 animals) were compared against those of animals in which no pseudotrochlea was found (38 of 48), and no significant differences were found by chi-squared analysis (p = 0.46).

Discussion

In this study, we intended to investigate the safety of an intra-articular collagen scaffold drug delivery device on native rabbit knee joints, and on the knee joints of rabbit knees in a contracture model by assessing: stiffness secondary to joint fibrosis by comparing joint contracture development in study animals (CSA, CMCM) and sham animals (SA, MCM); the absorption kinetics of the intra-articular collagen scaffold by ex vivo observation at 72 hours and two, eight, and 24 weeks; and the histological effects of intra-articular collagen scaffold placement by comparing cartilage and synovial tissue samples of study animals and control animals.

Biomechanical testing of movement revealed statistical equivalence between CSA and SA animals, but did not demonstrate equivalence between CMCM and MCM animals. These data indicate that scaffold placement into a native knee by lateral arthrotomy poses equivalent risk of joint contracture as surgery alone, suggesting that implantation of a collagen scaffold in a non-contracted joint does not generate arthrofibrosis. Interestingly, when implanted in joints developing contracture, collagen scaffold implantation seemed to be protective of joint contracture. From a scientific perspective, this protective effect could make it difficult to determine whether using a collagen sponge for administration of a pharmacological agent would confound the assessment of reduction in contracture (due to the collagen sponge versus the pharmacological agent itself). On the other hand, from a practical perspective it is very attractive to consider the potential additive effects of collagen and antifibrotic drugs.

Absorption of the scaffold consistently occurred prior to the eight-week time point. Interestingly, our data may suggest a trend towards slower absorption in SA animals compared with CMCM. This is not unexpected given the increased surgical trauma suffered by animals in the Mayo contracture model, which likely leads to a greater inflammatory response, and consequently faster breakdown of the scaffold in the injury model animals. The suggestion of increased synovitis in all animals at the two-week mark would support this theory. In both groups of animals, however, the scaffold demonstrated slower breakdown than expected based on product literature (Integra Miltex Collagen Products, York, Philadelphia). This may be due to the relatively decreased vascularity of the joint space compared with other tissues in which these scaffolds are typically placed.

A limitation of this study is the sensitivity of the histological analysis, particularly of the histological analysis of the bone and cartilage samples. The generation of sectioning artefact in many samples limited our ability to detect subtle histological changes in cartilage. This limited the depth of our histological analysis of cartilage samples. Additionally, changes thought to be greater than those explained by artefact were noted in 10% of the non-operative limbs, for which artefact or routine age-related wear and damage were the only plausible explanations. This suggests that some of the changes in the operative limbs thought to be pathologic may indeed be related to artefact or routine wear rather than to the device or to iatrogenic (surgical) trauma. An additional limitation of the histological analysis was the difficulty in capturing synovial tissue on slides for examination. In samples from nine of 90 operative knees, synovial cells could not be isolated. This does limit our analysis of the synovium somewhat, as the synovial architecture could not be evaluated in every animal. Despite this, all samples were reviewed for inflammatory changes, and were scored accordingly, to the best of the veterinary pathologist’s (RM) ability.

Despite the limitations in our histological analysis, both collagen (CSA, CMCM) and sham (SA, MCM) animals were found to have similar rates of chondral injury. These data suggest that the chondral damage noted was likely sequelae of injury or surgery, or iatrogenic at time of death, rather than secondary to mechanical wear or inflammatory changes due to collagen implantation. Additionally, the relative rarity of changes in all anatomic locations would suggest that collagen implantation did not cause wear throughout the joint, as would be expected in inflammatory arthritis. This is consistent with data from other experimental models, which do not suggest an adverse local reaction in collagen membranes or scaffold used for cartilage repair.^35-37 Additionally, collagen scaffolds have been noted to demonstrate faster release of pro-inflammatory cytokines than other scaffold materials such as polylactic acid, which may create a more chondrocyte-friendly environment.³⁸

Animals in all groups (SA, CSA, MCM, and CMCM) experienced some degree of peri-articular synovial inflammation, particularly at the 72-hour and two-week time points. By the 24-week time point, this response had largely abated. No significant difference was noted in the rate of synovial inflammation in the study animals (CSA, CMCM) when compared with the sham surgery animals (SA, MCM). This is encouraging given that some studies investigating the intra-articular injection of drug delivery scaffolds have noted synovial inflammation as a result³⁹ since some inflammatory infiltrate is expected for breakdown of the scaffold.¹⁶ These data suggest that implantation of the collagen scaffold itself does not lead to an increase in synovitis.

At the point of death of the animals undergoing arthrotomy ± scaffold placement, an interesting phenomenon was noted; several animals suffered chronic medial dislocation of the patella following the lateral arthrotomy. At the time of death, these animals were noted to have dislocated patellae, along with the formation of a fibrous pseudotrochlea medial to the actual trochlea. Interestingly, this did not occur in any of the Mayo contracture model animals, regardless of time of immobilisation, remobilisation status, or placement of a collagen scaffold. Given that this occurred in both sham arthrotomy animals as well as animals undergoing arthrotomy and collagen scaffold placement, it is likely to be related to the mechanical alignment of the rabbit knee and surgical disruption of the lateral structures which clearly play a role in stabilisation of the rabbit patella. Such changes have been noted in a rabbit model in the past, although by a different mechanism.⁴⁰ While interesting and a potential confounding factor, these findings do not limit the findings of this study, as they occurred with similar frequency in both collagen and sham arthrotomy animals, and did not demonstrate a significant effect on the rate or severity of synovitis.

In conclusion, intra-articular implantation of a collagen scaffold does not seem to lead to contracture, synovial inflammation, or chondral damage. Using equivalence analysis, collagen scaffolds implanted in joints undergoing contracture seem to be protective, which could be interpreted as a confounding factor if a collagen sponge is considered for testing of antifibrotic drugs in the future, but could also provide additive beneficial effects in the prevention and treatment of arthrofibrosis. Further studies are warranted to assess the potential of collagen scaffolds as an intra-articular carrier of antifibrotic pharmacological agents.