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Abstract

Objective

Large articular cartilage defects are a challenge to regenerative surgery. Biomaterial scaffolds might provide valuable support for restoration of articulating surface. The performance of a composite biomaterial scaffold was evaluated in a large porcine cartilage defect.

Design

Cartilage repair capacity of a biomaterial combining recombinant human type III collagen (rhCo) and poly-(l/d)-lactide (PLA) was tested in a porcine model. A full-thickness chondral defect covering the majority of the weightbearing area was inflicted to the medial femoral condyle of the right knee. Spontaneous cartilage repair and nonoperated healthy animals served as controls. The animals were sacrificed after a 4-month follow-up. The repair tissue was evaluated with the International Cartilage Repair Society (ICRS) macroscopic score, ICRS II histological score, and with micro-computed tomography. Additionally, histopathological evaluation of lymph nodes and synovial samples were done for toxicological analyses.

Results

The lateral half of the cartilage defect in the operated groups showed better filling than the medial half. The mean overall macroscopic score for the rhCo-PLA, spontaneous, and nonoperated groups were 5.96 ± 0.33, 4.63 ± 0.42, and 10.98 ± 0.35, respectively. The overall histological appearance of the specimens was predominantly hyaline cartilage in 3 of 9 samples of the rhCo-PLA group, 2 of 8 of the spontaneous group, and 9 of 9 of the nonoperated group.

Conclusions

The use of rhCo-PLA scaffold did not differ from spontaneous healing. The repair was affected by the spatial properties within the defect, as the lateral part of the defect showed better repair than the medial part, probably due to different weightbearing conditions.

Keywords

biomaterial cartilage repair animal model

Introduction

Traumatic injuries to articular cartilage typically affect physically active, young patients.¹ Cartilage defects have a poor intrinsic repair capacity, and full-thickness cartilage defects are an independent risk factor for future total knee arthroplasty.² These defects can be repaired surgically in attempt to alleviate symptoms and improve joint function. Biomaterial scaffolds can be used for augmented cartilage repair to mimic tissue architecture and provide the regenerating tissue with structural support³ and allow for earlier weightbearing. The benefits of biomaterial scaffolds in cartilage repair were assessed by a recent consensus meeting. The consensus on the use of scaffolds in cartilage defects supports scaffold-based therapies in all chondral and osteochondral knee lesions in the absence of joint instability, malalignment, meniscal deficiency, and osteoarthritis, although chondral scaffolds should not be used for deep osteochondritis dissecans lesions.⁴ Articular cartilage consists of mainly water and functions as a shock absorber by allowing for fluid flux through the tissue. This movement of fluid together with mechanical stimulus, created by joint movement, provides nutrition to the tissue and promotes the synthesis of its extracellular matrix compounds and fibril organization.^5-7 As mechanical stimulus is crucial for cartilage tissue, early weightbearing after cartilage repair procedures has been increasingly studied. Recent clinical studies have shown that accelerated weightbearing program after a cartilage repair procedure allow for an earlier return to sports without hampering the repair result.^8,9

Our research group has developed a novel composite scaffold rhCo-PLA.^10,11 It is designed to adapt to altering weightbearing conditions in both chondral and osteochondral defects. Its recombinant human collagen (rhCo) and poly(l/d)lactide (PLA) improve both the biocompatibility and mechanical properties of the scaffold. In a preliminary study, the rhCo-PLA scaffold together with autologous chondrocytes resulted in repair tissue that was at least as good as tissue produced with autologous cells combined with a commercial collagen membrane.¹⁰

The mean defect size in recent clinical studies reporting the results of surgical cartilage repair ranges from 2.3 to 6.1 cm.^2,12-14 As the defects requiring cartilage repair are large, the performance of the rhCo-PLA scaffold needs to be tested in a large cartilage defect model. Therefore, this study was undertaken to further challenge the scaffold in a large surgically created defect in a porcine model. Aware of the soft nature of porcine subchondral bone that would increase the challenge, the used defect covered a major part of the weightbearing area of the medial femoral condyle. As the nonoperated contralateral limb in our previous study showed changes related to the surgery itself,¹⁰ a healthy nonoperated control group was included in the present study.

Materials and Methods

Pilot Defect Size Testing

To confirm the size of the largest possible lesion to the weightbearing area of medial femoral condyle, porcine femurs were obtained from an abattoir. An arthrotomy was made through a medial parapatellar approach to expose the medial condyle of the femur. The largest possible defect that would still allow suturing its outer rim was created to the condyle. The oval defect measured 11 × 17 mm (1.5 cm²). A custom-made instrument with the aforementioned measurements was then manufactured.

Scaffold Manufacturing

The rhCo-PLA scaffolds were manufactured as previously described^10,11 using recombinant human type III collagen (FibroGen Inc, San Francisco, CA, USA) and poly(l/d)lactide 96/4 (Corbion Purac, Gorinchem, The Netherlands). Briefly, the medical grade PLA was used to manufacture carded, needle punched PLA felt. The PLA felt was washed, dried, packed, and gamma-irradiated (25 kGy). The sterile PLA felt was immersed into recombinant human collagen type III (rhCo) solution and freeze-dried to form rhCo-PLA composite scaffolds. The scaffolds were further crosslinked with 14 mM EDC (N-[3-dimethylaminopropyl]-N′-ethylcarbodiimide hydrochloride, Sigma-Aldrich, Helsinki, Finland) and 6 mM NHS (N-hydroxysuccinimide; Sigma-Aldrich, Helsinki, Finland) in 95% ethanol solution, washed and freeze-dried again. The scaffolds were manufactured and packed in a laminar flow chamber in clean room environment to prevent any contamination.

Surgical Procedures and Postoperative Care

The study was authorized by the Finnish Animal Experimentation Board (ESAVI/6113/04.10.07/2015) and conducted according to the ethical guidelines and regulations of the Finnish Act on the Protection of Animals Used for Scientific or Educational Purposes (497/2013) and Government Decree on the Protection of Animals Used for Scientific or Educational Purposes (564/2013). A total of 27 female domestic pigs (Sus scrofa domestica) were included in the study. The pigs were obtained from a local farmer at the age of 4 months. The pigs were randomized into 3 groups (n = 9 in each). The rhCo-PLA group receiving a rhCo-PLA scaffold was compared with spontaneous repair (spontaneous group) and healthy cartilage of nonoperated group. After 2 weeks of acclimatization, pigs in the 2 intervention groups were sedated with medetomidine (0.2 mg/kg, subcutaneously) and ketamine (10 mg/kg, subcutaneously). The pigs were then anesthetized with buprenorfin (0.05 mg/kg, subcutaneously), carprofen (3 mg/kg, subcutaneously), and propofol (3 mg/kg or to effect, intravenously). Preoperative antibiotics of cefuroxime (3 g, intravenously) was administered and general anesthesia was continued with isoflurane 2.5%. The animals were placed on supine position on the operating table. Through a medial parapatellar approach, the joint was opened, and the medial condyle of the femur was exposed. The outer rim of the cartilage defect was created with a custom-made punch. Then all cartilage was carefully removed with a curette. The subchondral bone was left intact so that the bone surface just started bleeding. The wound was closed in layers.

The pigs were housed separately after the surgeries. Antibiotic prophylaxis continued for 3 days postoperatively. Analgesia with carprofen 3 mg orally once a day and buprenorphine 0.01 mg/kg subcutaneously thrice a day was continued for 3 days postoperatively. The pigs were allowed free weightbearing and unrestricted movement immediately after the operations and grouped on day 3 after the surgery.

The nonoperated control group of pigs were housed in groups throughout the study period. No surgical interventions were carried out for this group.

Second Operation

Five weeks after the first surgery, the second operation was carried out. The animals were medicated and anesthetized and the joint was approached as described above. The custom-made instrument was fitted to the previously made defect and all scar and repair tissue was carefully removed with a curette. The subchondral bone was left intact so that the bone surface just started bleeding. For the rhCo-PLA group, the rhCo-PLA scaffold was tailored with the custom-made instrument to fit the lesion diameter. The scaffold was pressed to the bleeding bottom of the defect and secured with sutures. The wound was closed in layers.

For the spontaneous group, the defects were debrided but no repair procedures were carried out, and the wound was closed in layers.

Sacrifice and Sample Collection

After 4 months of follow-up, the pigs were anesthetized and sacrificed with medetomidine (0.2 mg/kg, subcutaneously), ketamine (10 mg/kg, subcutaneously), pentobarbital (60 mg/kg, intravenously), and T-61 (4-5 mL/50 kg). Both knees were opened and any possible excess synovial fluid was carefully assessed and documented. Both the femoral and tibial cartilage were assessed for any pathological changes. The distal femurs of the hind legs were then photographed, where after the medial condyle was detached with a diamond saw and stored at –20 °C in a plastic container in which a swab moistened with Ringer acetate solution was placed on the bottom.

Macroscopical Evaluation

A subjective evaluation of the overall repair covered the entire defect area. Using the International Cartilage Repair Society (ICRS) macroscopic scoring system,¹⁵ the macroscopical repair was evaluated from the lateral half of the defect area in order to match the macroscopical evaluation with the histological ICRS evaluation. The assessment was made from blinded, randomized photographs by 3 independent observers (ES, JP, TP) using a web-based survey (SurveyMonkey, San Mateo, CA, USA). The tissue of the nonoperated control group was assessed from the area corresponding the defect area in the 2 intervention groups.

Differences between the medial and the lateral half of the defects in the rhCo-PLA treatment group and the spontaneously healed group were analyzed. A MatLab (The MathWorks, Inc., Natick, MA, USA) program was developed for the defect side comparison. First, the lateral and medial defect sides were selected from the samples’ RGB (red, green, blue) photographs. Then, the photographs were converted to HSV (hue, saturation, value) color space. Comparison between the lateral and medial defect side was made with the saturation channel by calculating the mean values of the selected regions.

X-Ray Micro-Computed Tomography (µCT)

The frozen condyles were thawed at room temperature for 1 hour in PBSI and then imaged with Zeiss Xradia MicroXCT-400 (Zeiss, Pleasanton, CA, USA) µCT scanner. Before the actual imaging, a scout image was taken from the whole sample. Therefore, it was possible to accurately select the defect area as a field of view. The x-ray tube voltage was adjusted to 120 kV and the tube current to 83 µA. A total of 1,200 projections were taken in 360° rotation angle and 5 seconds exposure time was used. With binning 2, the resulted pixel size was 22.32 µm. Projections were reconstructed to the 3-dimensional image stack with the device manufacturer’s XMReconstructor software. Analysis volume was chosen as a cylinder with a diameter of 15 mm and a height of 6 mm. Bone volume was selected from the background by manual thresholding. The bone structural parameters bone volume fraction, trabecular thickness, and spacing were calculated as described in Palmroth et al.¹⁶ Image processing and analysis were done with the Avizo 2019.3 software (Thermo Fisher Scientific, Waltham, MA, USA) and the BoneJ plug-in¹⁷ in the Fiji software. After the imaging, the samples were refrozen at –20 °C.

Histology

The collected condyle specimens were cut around the defect area and halved from the middle of the defect with a diamond saw. The samples were fixed with 10% buffered formalin for 4 weeks, followed by decalcification in a solution containing 10% EDTA, 4% formalin, and 0.1 M phosphate buffer. The samples were halved and embedded in paraffin. The middle part of the defect was analyzed histologically by cutting the samples containing the lateral half of the defect to 5 µm sections and staining them with safranin-O. Blinded safranin-O-stained sections were evaluated by 3 independent observers (JP, ES, AV) using the ICRS II score.¹⁸

For immunohistochemical evaluation of collagen types I and II, the histological sections were prepared as previously described.^10,19 Briefly, the sections were digested with hyaluronidase (2 mg/mL, Sigma-Aldrich) and pronase (2 mg/mL, Calbiochem, Merck KGaA). Endogenous peroxidase activity was blocked with hydrogen peroxide (EnVision+ System-HRP [AEC], Dako North America Inc.) Nonspecific staining was blocked with 10% normal goat serum (Dako Denmark A/S, Glostrup, Denmark). Primary antibodies against collagen type II (ab34712, Abcam, Cambridge, UK) and collagen type I (ab34710) were diluted to 4 μg/mL in PBS supplemented with 1% bovine serum albumin (Sigma-Aldrich) and 0.1% Triton X-100. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (Dako) was applied. Antibody binding was visualized with AEC (3-amino-9-ethylcarbazole) substrate chromogen (Dako).

Histopathological Evaluation

Samples of the synovial membrane of the right and left stifle joint and popliteal lymph nodes were collected at the time of sacrifice. The samples were fixed in 10% buffered formalin, routinely processed and embedded in paraffin and cut into 5 µm tissue sections. The sections were stained with hematoxylin and eosin and evaluated under light microscope by animal pathologists (Finnish Centre for Laboratory Animal pathology, University of Helsinki, Finland).

Statistical Analyses

Differences between groups were analyzed with bootstrap type generalized estimating equations (GEE) using an exchangeable correlation structure. The weight gain of the animals was analyzed with analysis of covariance with baseline weight as covariate. P values less than 0.05 were considered statistically significant. Statistical difference between the saturation values of the medial and lateral part of the photographed defects were analyzed with t test. The statistical analyses were performed with Stata version 16.0 (StataCorp, College Station, TX, USA). The data are presented as mean ± SE unless otherwise stated.

Results

Animal Experiments

During the study, the animals gained weight from 134.1 ± 2.5 to 217.0 ± 4.5 kg. The rhCo-PLA-treated animals gained weight from 123.4 ± 1.8 to 197.4 ± 4.1 kg (Δ = 74 kg). Spontaneously healed animals gained weight from 140.5 ± 5.2 to 226.5 ± 6.7 kg (Δ = 86 kg). Weight gain for the nonoperated group was from 138.8 ± 4.7 to 228.1 ± 8.5kg (Δ = 89.3 kg). Weight gain did not differ between the groups (P = 0.31).

Between the operations, the defects had shrunken by 1 to 2 mm around the rim of the lesion and there was a deposition of soft, fibrous-like tissue macroscopically. The underlying subchondral bone seemed hardened in clinical evaluation.

One of the pigs in the spontaneous group developed a malignant hyperthermia, a condition typical for domestic pigs, and was euthanized on the second postoperative day of the second operation. Otherwise, the animals recovered well from both surgeries. One of the pigs in the rhCo-PLA treatment group showed a joint infection of unknown etiology at the time of the sacrifice but was still included in the analyses.

Macroscopic Evaluation

The repair tissue showed an uneven quality even within the same defect. It was typical for both intervention groups that the lateral edge of the large defect showed good filling but the medial half presented with poor repair ( Fig. 1 , Supplemental Figure S1). None of the defects in either intervention group showed good filling on the medial edge of the defect if the lateral side was poorly repaired. The repair tissue was in level with the surrounding cartilage in 3 of 9 samples in the rhCo-PLA group, in 1 of 8 samples in the spontaneous group, and in 9 of 9 samples in the nonoperated control group.

Figure 1.

Photographs of the medial condyles of the femur and safranin-O-stained tissue sections of the middle part of the defect showing the best, average, and the worst samples (a) in the rhCo-PLA treatment group and (b) in the spontaneously healed group, based on the macroscopic and histological scoring. rhCo, recombinant human type III collagen; PLA, poly-(l/d)-lactide.

The mean overall ICRS score for the rhCo-PLA group was 5.96 ± 0.33 (corresponding to ICRS grade III), for spontaneous group 4.63 ± 0.42 (grade III), and for healthy nonoperated group 10.98 ± 0.35 (grade II). The results showed that the healthy nonoperated group received higher scores than the 2 intervention groups. There was a statistically significant difference in the total ICRS score between the groups and the difference was located between the rhCo-PLA group and the healthy nonoperated group, and between the spontaneous group and the healthy nonoperated group (P < 0.0001) ( Fig. 2 ). Otherwise, no statistically significant differences were detected.

Figure 2.

The International Cartilage Repair Society (ICRS) macroscopic score¹⁵ showed large variation between individual animals. The subscores of (a) defect filling, (b) integration, (c) surface, and (d) the total score are shown for the study groups. Each dot represents an individual animal. Both intervention groups differed from the nonoperated healthy control group, although a statistically significant difference between the groups was only found in the total score (P < 0.0001).

The medial halves of the defect areas showed higher HSV saturation values when compared with the lateral halves ( Fig. 3 ). The mean saturation value in the rhCo-PLA group was 0.49 for the lateral part and 0.57 for the medial part (p = 0.025). The mean saturation value in the spontaneous group was 0.28 for the lateral part and 0.37 for the medial part (p = 0.003).

Figure 3.

The hue–saturation–values were higher in the medial halves of the defects in both (a) the rhCo-PLA treatment group and (b) the spontaneously healed group. rhCo, recombinant human type III collagen; PLA, poly-(l/d)-lactide.

Tissue Structure

Safranin-O staining was moderate to good in 4 of 9 specimens in the rhCo-PLA group, in 4 of 8 specimens in the spontaneous group, and in all the specimens in the nonoperated group. The same well-stained samples showed good type II collagen uptake in immunohistochemical evaluation. The type II collagen was found in the pericellular region and was more abundant in the perimeter of the defect than in the middle ( Fig. 4 ). The overall histological appearance of the specimens was predominantly hyaline cartilage in 3 of 9 samples of the rhCo-PLA group, 2 of 8 samples of the spontaneous group, and 9 of 9 samples of the nonoperated group. The rest of the samples in the 2 intervention groups showed predominantly fibrocartilaginous and fibrous repair tissue. Fibrillation and delamination of the cartilage surface was common. The tissue type in the nonoperated control group was hyaline cartilage in all animals. Typically for porcine cartilage, none of the specimens had a tidemark ( Fig. 1 , Table 1 , Supplemental Figure S1).

Figure 4.

Comparison of immunohistochemical type II collagen staining in best, average, and worst samples in the (a) rhCo-PLA treatment group and (b) spontaneously healed group. Scale bar: 2 mm. rhCo, recombinant human type III collagen; PLA, poly-(l/d)-lactide.

Table 1.

Results of the ICRS II Histological Score¹⁸ for Each Study Group.

Parameter	rhCo-PLA	Spontaneous	Nonoperated	P
Tissue morphology	33.70 ± 8.83	33.33 ± 13.39	95.00 ± 2.09	<0.0001
Matrix staining	43.70 ± 11.01	38.33 ± 14.24	96.67 ± 1.26	<0.0001
Cell morphology	37.41 ± 10.88	40.00 ± 13.80	98.33 ± 1.09	<0.0001
Chondrocyte clustering	88.15 ± 4.41	94.17 ± 4.26	75.83 ± 6.54	0.016
Surface architecture	57.78 ± 6.38	65.83 ± 8.86	90.00 ± 3.33	<0.0001
Basal integration	78.52 ± 6.55	85.83 ± 9.63	90.00 ± 7.24	NS
Formation of a tidemark	25.93 ± 10.03	20.00 ± 8.45	34.17 ± 8.21	NS
Subchondral bone abnormalities/marrow fibrosis	82.22 ± 6.38	84.17 ± 5.18	93.33 ± 3.98	NS
Inflammation	94.81 ± 4.41	100.00 ± 00.00	95.83 ± 4.17	NS
Abnormal calcification/ossification	97.78 ± 1.57	93.33 ± 5.77	100.00 ± 0.00	NS
Vascularization within the repaired tissue	79.26 ± 11.46	82.50 ± 9.92	100.00 ± 0.00	NS
Surface assessment	39.63 ± 10.16	46.67 ± 16.04	90.83 ± 2.50	<0.0001
Mid/deep zone assessment	37.78 ± 10.94	33.33 ± 13.57	99.17 ± 0.83	<0.0001
Overall assessment	38.52 ± 9.67	37.50 ± 13.39	95.83 ± 1.75	<0.0001

ICRS = International Cartilage Repair Society; rhCo = recombinant human type III collagen; PLA = poly-(l/d)-lactide; NS = nonsignificant.

The data are presented as mean ± standard error. Statistical significances between the study groups are presented for each score parameter. All the significant differences are located between the rhCo-PLA group and the nonoperated group, and between the spontaneous group and the nonoperated group with the exception of chondrocyte clustering, in which only the spontaneous group and the nonoperated group differed from each other in a statistically significant manner.

The PLA fibers of the rhCo-PLA scaffold were detected in histological evaluation of all the rhCo-PLA-treated animals, confirming that the scaffolds stayed on place. Histologically, only one sample in the rhCo-PLA group showed a small cyst-like defect in the subchondral bone. Otherwise, no subchondral bone defects were seen in the histological sections of any of the studied groups.

The ICRS II histological score revealed that the healthy nonoperated group received higher score than the 2 intervention groups in all subscores, except for cell clustering and inflammation ( Table 1 ). The rhCo-PLA group and the spontaneous group differed in a statistically significant manner from the nonoperated group in tissue morphology, matrix staining, cell morphology, surface architecture, superficial assessment, middle/deep zone assessment, and overall score (P < 0.0001 for each parameter). Chondrocyte clustering showed a difference between the spontaneous group and the healthy nonoperated group (P = 0.016).

Histopathological Evaluation

The synovium samples exhibited minimal or mild synovitis with multifocal infiltration of lymphocytes and plasma cells in the subintima in 9 of 9 animals in the rhCo-PLA group, in 4 of 9 animals in the spontaneous group, and in 0 of 9 animals in the nonoperated group. Foreign material resembling suture material was observed in three pigs in the rhCo-PLA group. Additionally, foreign material particles were observed in 1 pig in the spontaneous group and 1 in the nonoperated group. One pig in the nonoperated control group had a cyst consisting of organized hemorrhage in the meniscus, likely originating from trauma. No other pathological findings related to the rhCo-PLA scaffolds were observed in the lymph nodes or synovium of these animals.

Minimal or mild reactive follicular hyperplasia of the lymph nodes, which is a frequent and usual finding in young animals, was seen in all samples with the exception of 2 animals in the nonoperated group. In addition, all samples exhibited a mild to moderate infiltration of eosinophils and occasional yellow to brown pigmented granular material (hemosiderin) in the medullary sinuses.

Subchondral Bone

The average size of the subchondral bone defects was slightly larger in the spontaneously healed group (74.9 ± 30.1 mm³) than in the rhCo-PLA group (60.8 ± 51.8 mm³), although the difference was not statistically significant ( Fig. 5 ). The 3 study groups did not show statistically significant differences in the bone structural parameters of trabecular thickness, trabecular separation of bone volume fraction.

Figure 5.

Results of the x-ray micro-computed tomography (µCT) showing (a) bone volume fraction in %, (b) trabecular thickness in µm, and (c) trabecular spacing in µm in each study group. (d) A comparison of the defect size in mm³ in the rhCo-PLA and spontaneous groups. Each dot represents an individual animal. There were no statistically significant differences between the groups. rhCo = recombinant human type III collagen; PLA = poly-(l/d)-lactide.

Discussion

The results of this study show that the lateral part of the medial condyle of the femur has a better healing capacity than the medial part of the condyle. This is probably due to the weightbearing conditions. In both intervention groups, the lateral part of the defect within the medial condyle of the femur showed statistically better repair than the medial part, according to macroscopic analyses. The mechanical stimulus from weight bearing is considered an important factor in cartilage anabolism. Mechanical stimulus improves chondrocyte viability²⁰ and cartilage extracellular matrix deposition²¹ in vivo and ex vivo. The lateral part of the medial condyle bears weight²² and might therefore have an improved cartilage repair capacity. The rhCo-PLA scaffold has shown weight-adaptive properties,³ which provides the regenerating tissue with mechanical cues and potentially improves its performance in comparison with other biomaterial scaffolds.

The use of an animal model allows for standardizing the test group as well as the location and the size of the defect, enabling an effective comparison of the 2 intervention groups. The porcine model was chosen as it has, together with the equine model, thick cartilage giving the closest approximation to human cartilage.²³ As seen in our previous study, the nonoperated contralateral limb, which is often used as a control, is also affected by the surgeries.¹⁰ Therefore, it does not represent a normal, healthy porcine cartilage. In this study, a healthy nonoperated group to which no interventions were done was used as a control. Interestingly, not even the healthy group received grade I ICRS macroscopic score, indicative of normal cartilage, suggesting that spontaneous cartilage defects in domestic pigs are common. These animals are bred to gain muscle mass quickly, and they are typically slaughtered at the age of 4 months. The animals used in this study were of 8 months of age at the time of sacrifice, weighing on average 217 kg, which puts a heavy strain on their joints. The healthy, nonoperated group gave valuable information on porcine cartilage and subchondral bone. This study benchmarks normal tissue in porcine cartilage repair studies and can thus later be used as a reference in evaluating repair results in later experiments.

Previous experiments have shown subchondral bone pathologies when repairing porcine cartilage with biomaterial scaffolds.^10,24,25 Due to the soft nature of the subchondral bone of domestic pigs, osteochondral—and even chondral—repair in this model is difficult, and sinking of the scaffolds common. In this study, despite the small subchondral bone voids in the 2 intervention groups, the trabecular thickness and spacing of the subchondral bone did not show statistically significant differences from normal porcine subchondral bone. The rhCo-PLA implants did not show signs of detachment or sinking into the bone.

Young animals typically have the capacity of spontaneous cartilage healing,²⁶ but it has been stated that porcine cartilage does not regenerate in large defects.²³ Although neither of the intervention groups showed repair tissue completely similar to healthy cartilage, the relatively short follow-up period of 4 months produced partial repair in both study groups. The use of the rhCo-PLA scaffold did not hamper the repair capacity of the young porcine cartilage. The rhCo-PLA and the spontaneous groups did not differ from one another when the repair tissue was evaluated with ICRS macroscopic score or ICRS II histological score.

Foreign material, resembling suture material, was found in the synovium of 3 animals in the rhCo-PLA group. Foreign material was also found in 1 pig in the spontaneous group and 1 in the nonoperated group. Suture material used in the 2 intervention groups could explain the foreign material found in their synovium. One animal in the spontaneously healed group showed presence of foreign material surrounded or phagocytized by macrophages in the synovium and joint capsule. Surprisingly, 1 animal in the nonoperated group also exhibited foreign material in the synovium, without a cellular reaction. Although foreign bodies in the knee joint are uncommon, there are case reports that describe foreign bodies migrating into the knee joint after a trauma.^27,28 This might be the case with this one animal, although the origin of the particle remains unknown. No other pathological findings related to the rhCo-PLA scaffolds were observed in the lymph nodes or synovium of these animals. The lymph nodes of the animals in all groups showed minimal or mild follicular hyperplasia. As this was seen also in the healthy, nonoperated group, this finding lacks clinical significance. However, the synovium of the operated animals exhibited signs of inflammation. The mild synovitis was seen in all the rhCo-PLA-treated animals and in 4 of 9 of the spontaneously healed animals. The inflammation might be associated with the operation itself, the materials used in the operation, or developing osteoarthritis.^29,30

Bioabsorbable aliphatic polyesters, such as poly(lactic-co-glycolic acid) (PLGA) and PLA, are widely used in medicine due to their favorable properties. Their applications include suture materials, drug delivery, and bone screws, as well as biomaterial scaffolds intended for cartilage repair. The wounds of both groups were closed with polyglactin 910 sutures, which are made of a copolymer composed of 90% glycolide and 10% l-lactide (Ethicon Wound Closure Manual, 2007, Ethicon, Inc.). The suture material and the PLA used in the rhCo-PLA scaffold degrade by hydrolysis. This lowers the pH of the surroundings and may elicit inflammatory responses.^31,32 A foreign body immune response is triggered by any polymeric implant.³³ In a clinical setting, local inflammatory reactions due to these materials are rare, and seen mostly with bulky products containing large quantities of the polymers, however. In clinical data covering 2,528 patients, the incidence of local inflammation elicited by polyglycolic acid was 5.3% and by polylactic acid 0.2%.³⁴ One of the pigs in the rhCo-PLA-treated group developed a purulent joint infection of the operated joint. The etiology of the infection is unclear but microbiological testing in the enrichment culture showed growth of coagulase-negative Staphylococcus bacteria that was sensitive to all analyzed antibiotics.

The heterogeneity of the repair tissue between individual animals and even in the same defect creates a challenge for the evaluation of the defects. This issue was managed by focusing the assessment to the middle part of the defect both in macroscopical and histological scoring. There was large variation in the repair results between individual animals but mostly, the variation seen in the results was due to the spatial variations in the repair capacity within each defect. Further studies are needed to confirm whether this is due to different weightbearing conditions.

Conclusions

In this study of a large full-thickness articular cartilage defect in a porcine model, the use of rhCo-PLA scaffold did not differ from spontaneous healing. Both intervention groups showed inferior cartilage quality compared with healthy nonoperated porcine cartilage. The lateral side of the medial condyle of the femur showed good cartilage repair in both spontaneously healed and rhCo-PLA-augmented groups. Thus, the weightbearing conditions may be of high importance when repairing articular cartilage.

Supplemental Material

sj-tif-1-car-10.1177_19476035211030988 – Supplemental material for Cartilage Repair Capacity within a Single Full-Thickness Chondral Defect in a Porcine Autologous Matrix-Induced Chondrogenesis Model Is Affected by the Location within the Defect

Supplemental material, sj-tif-1-car-10.1177_19476035211030988 for Cartilage Repair Capacity within a Single Full-Thickness Chondral Defect in a Porcine Autologous Matrix-Induced Chondrogenesis Model Is Affected by the Location within the Defect by E. Salonius, A. Meller, T. Paatela, A. Vasara, J. Puhakka, M. Hannula, A.-M. Haaparanta, I. Kiviranta and V. Muhonen in CARTILAGE

Footnotes

Acknowledgements

The authors wish to thank Hannu Kautiainen (Medcare Oy) for his expertise in the statistical analyses and prof. emer. Satu Sankari) at the Finnish Centre for Laboratory Animal Pathology (FCLAP) for her histopathological expertise. Histological images were generated using 3DHISTECH Pannoramic 250 FLASH II digital slide scanner at Genome Biology Unit supported by HiLIFE and the Faculty of Medicine, University of Helsinki, and Biocenter Finland.

Declaration of Conflicting Interests

The author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: A-MH and VM are cofounders, board members, and major shareholders in Askel Healthcare Ltd, a spin-off from Tampere University of Technology and University of Helsinki, aiming to commercialize COPLA cartilage repair technology based on rhCol-PLA scaffold studied in this article. AV is a shareholder in Askel Healthcare Ltd. The other authors have no conflicts of interest to declare.

Ethical Approval

The study was authorized by the Finnish Animal Experimentation Board (ESAVI/6113/04.10.07/2015).

Animal Welfare

This study was conducted according to the ethical guidelines and regulations of the Finnish Act on the Protection of Animals Used for Scientific or Educational Purposes (497/2013) and Government Decree on the Protection of Animals Used for Scientific or Educational Purposes (564/2013).

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The study was conducted with the financial support of Tekes (grant 40171/14), Helsinki University Hospital (grant TYH2015207), and Novo Nordisk Foundation Exploratory pre-seed grant No. 17196. The sponsors of this study had no role in the study design, collection, analysis, or interpretation of the data; in the writing of the manucript; or in the decision to submit the manuscript for publication.

ORCID iDs

E. Salonius

T. Paatela

Supplemental Material

Supplementary material for this article is available on the Cartilage website at .

References

Gelber

Hochberg

Mead

Wang

Wigley

Klag

. Joint injury in young adults and risk for subsequent knee and hip osteoarthritis. Ann Intern Med. 2000;133(5):321-8. doi:10.7326/0003-4819-133-5-200009050-00007

Everhart

Abouljoud

Kirven

Flanigan

. Full-thickness cartilage defects are important independent predictive factors for progression to total knee arthroplasty in older adults with minimal to moderate osteoarthritis: data from the Osteoarthritis Initiative. J Bone Joint Surg Am. 2019;101(1):56-63. doi:10.2106/JBJS.17.01657

Gasik

Zühlke

Haaparanta

Muhonen

Laine

Bilotsky

, et al The importance of controlled mismatch of biomechanical compliances of implantable scaffolds and native tissue for articular cartilage regeneration. Front Bioeng Biotechnol. 2018;6:187. doi:10.3389/fbioe.2018.00187

Filardo

Andriolo

Angele

Berruto

Brittberg

Condello

, et al Scaffolds for knee chondral and osteochondral defects: indications for different clinical scenarios. a consensus statement. Cartilage. Published online January 15, 2020. doi:10.1177/1947603519894729

Mow

Holmes

Lai

. Fluid transport and mechanical properties of articular cartilage: a review. J Biomech. 1984;17(5):377-94. doi:10.1016/0021-9290(84)90031-9

Bernhard

Vunjak-Novakovic

. Should we use cells, biomaterials, or tissue engineering for cartilage regeneration? Stem Cell Res Ther. 2016;7(1):56. doi:10.1186/s13287-016-0314-3

Zhang

Qiu

Gao

Zhang

. Advances in application of mechanical stimuli in bioreactors for cartilage tissue engineering. Tissue Eng Part B Rev. 2017;23(4):399-411. doi:10.1089/ten.TEB.2016.0427

Ebert

Edwards

Fallon

Ackland

Janes

Wood

. Two-year outcomes of a randomized trial investigating a 6-week return to full weightbearing after matrix-induced autologous chondrocyte implantation. Am J Sports Med. 2017;45(4):838-48. doi:10.1177/0363546516673837

Wei

Liu

. Effects of early weightbearing on microfracture treatment of osteochondral lesions of talus with subchondral bone defects. Curr Med Sci. 2019;39(1):88-93. doi:10.1007/s11596-019-2004-8

10.

Muhonen

Salonius

Haaparanta

Järvinen

Paatela

Meller

, et al Articular cartilage repair with recombinant human type II collagen/polylactide scaffold in a preliminary porcine study. J Orthop Res. 2016;34(5):745-53. doi:10.1002/jor.23099

11.

Salonius

Kontturi

Laitinen

Haaparanta

Korhonen

Nystedt

, et al Chondrogenic differentiation of human bone marrow-derived mesenchymal stromal cells in a three-dimensional environment. J Cell Physiol. 2020;235(4):3497-507. doi:10.1002/jcp.29238

12.

Riboh

Cvetanovich

Cole

Yanke

. Comparative efficacy of cartilage repair procedures in the knee: a network meta-analysis. Knee Surg Sports Traumatol Arthrosc. 2017;25(12):3786-99. doi:10.1007/s00167-016-4300-1

13.

Jones

Kelley

Arshi

McAllister

Fabricant

. Comparative effectiveness of cartilage repair with respect to the minimal clinically important difference. Am J Sports Med. 2019;47(13):3284-93. doi:10.1177/0363546518824552

14.

Gou

Tseng

Wang

Chen

Shyu

Weng

, et al Autologous chondrocyte implantation versus microfracture in the knee: a meta-analysis and systematic review. Arthroscopy. 2020;36(1):289-303. doi:10.1016/j.arthro.2019.06.033

15.

Peterson

Minas

Brittberg

Nilsson

Sjögren-Jansson

Lindahl

. Two- to 9-year outcome after autologous chondrocyte transplantation of the knee. Clin Orthop Relat Res. 2000;(374):212-34. doi:10.1097/00003086-200005000-00020

16.

Palmroth

Pitkänen

Hannula

Paakinaho

Hyttinen

Miettinen

, et al Evaluation of scaffold microstructure and comparison of cell seeding methods using micro-computed tomography-based tools. J R Soc Interface. 2020; 17(165):20200102. doi:10.1098/rsif.2020.0102

17.

Doube

Kłosowski

Arganda-Carreras

Cordelières

Dougherty

Jackson

, et al BoneJ: free and extensible bone image analysis in ImageJ. Bone. 2010;47(6):1076-9. doi:10.1016/j.bone.2010.08.023

18.

Mainil-Varlet

Van Damme

Nesic

Knutsen

Kandel

Roberts

. A new histology scoring system for the assessment of the quality of human cartilage repair: ICRS II. Am J Sports Med. 2010;38(5):880-90. doi:10.1177/0363546509359068

19.

Salonius

Rieppo

Nissi

Pulkkinen

Brommer

Brünott

, et al Critical-sized cartilage defects in the equine carpus. Connect Tissue Res. 2019;60(2_suppl):95-106. doi:10.1080/03008207.2018.1455670

20.

Bhardwaj

Devi

Mandal

. Tissue-engineered cartilage: the crossroads of biomaterials, cells and stimulating factors. Macromol Biosci. 2015;15(2_suppl):153-82. doi:10.1002/mabi.201400335

21.

Mauck

Soltz

Wang

Wong

Chao

Valhmu

, et al Functional tissue engineering of articular cartilage through dynamic loading of chondrocyte-seeded agarose gels. J Biomech Eng. 2000;122(3):252-60. doi:10.1115/1.429656

22.

Koo

Gold

Andriacchi

. Considerations in measuring cartilage thickness using MRI: factors influencing reproducibility and accuracy. Osteoarthritis Cartilage. 2005;13(9):782-9. doi:10.1016/j.joca.2005.04.013

23.

Moran

Ramesh

Brama

O’Byrne

O’Brien

Levingstone

. The benefits and limitations of animal models for translational research in cartilage repair. J Exp Orthop. 2016;3(1):1. doi:10.1186/s40634-015-0037-x

24.

Vasara

Hyttinen

Pulliainen

Lammi

Jurvelin

Peterson

, et al Immature porcine knee cartilage lesions show good healing with or without autologous chondrocyte transplantation. Osteoarthritis Cartilage. 2006;14(10):1066-74. doi:10.1016/j.joca.2006.04.003

25.

Pulliainen

Vasara

Hyttinen

Tiitu

Valonen

Kellomäki

, et al Poly-L-D-lactic acid scaffold in the repair of porcine knee cartilage lesions. Tissue Eng. 2007;13(6):1347-55. doi:10.1089/ten.2006.0347

26.

Ahern

Parvizi

Boston

Schaer

. Preclinical animal models in single site cartilage defect testing: a systematic review. Osteoarthritis Cartilage. 2009;17(6):705-13. doi:10.1016/j.joca.2008.11.008

27.

Devgan

Mudgal

. An unusual case of foreign body knee that spontaneously migrated inside and out of the joint: arthroscopic removal. Knee Surg Sports Traumatol Arthrosc. 2007;15(6):758-60. doi:10.1007/s00167-006-0197-4

28.

Palmers

Dierickx

Peene

Bijnens

. An unusual metallic foreign body in the lateral tibiofemoral compartment. Arthroscopy. 2002;18(3):325-8. doi:10.1053/jars.2002.30060

29.

Sellam

Berenbaum

. The role of synovitis in pathophysiology and clinical symptoms of osteoarthritis. Nat Rev Rheumatol. 2010;6(11):625-35. doi:10.1038/nrrheum.2010.159

30.

Scanzello

Goldring

. The role of synovitis in osteoarthritis pathogenesis. Bone. 2012;51(2_suppl):249-57. doi:10.1016/j.bone.2012.02.012

31.

Gentile

Chiono

Carmagnola

Hatton

. An overview of poly(lactic-co-glycolic) acid (PLGA)-based biomaterials for bone tissue engineering. Int J Mol Sci. 2014;15(3):3640-59. doi:10.3390/ijms15033640

32.

Haaparanta

Uppstu

Hannula

Ellä

Rosling

Kellomäki

. Improved dimensional stability with bioactive glass fibre skeleton in poly(lactide-co-glycolide) porous scaffolds for tissue engineering. Mater Sci Eng C Mater Biol Appl. 2015;56:457-66. doi:10.1016/j.msec.2015.07.013

33.

da Silva

Kaduri

Poley

Adir

Krinsky

Shainsky-Roitman

, et al. Biocompatibility, biodegradation and excretion of polylactic acid (PLA) in medical implants and theranostic systems. Chem Eng J. 2018;340:9-14. doi:10.1016/j.cej.2018.01.010

34.

Böstman

Pihlajamäki

. Adverse tissue reactions to bioabsorbable fixation devices. Clin Orthop Relat Res. 2000;(371):216-27.

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