A Heparan Sulfate Device for the Regeneration of Osteochondral Defects

Abstract

The regeneration of cartilage is challenging due to its low metabolic rate and avascular nature. An effective treatment for osteochondral defects remains a clinical challenge. Glycosaminoglycans such as heparan sulfate (HS) bind and enhance the activity of prochondrogenic growth factors and thus hold potential for targeted tissue regeneration without the requirement for exogenous growth factors. In this study, we examine the use of a cell- and growth factor-free HS-based technique for osteochondral repair in a rabbit model. The binding affinity between HS and several reparative proteins (TGF-β1, BMP-2, FGF-2, PDGF-BB, and VEGF₁₆₅) was studied using surface plasmon resonance. Next, an HS-impregnated gel was implanted in a large osteochondral defect in the femoral trochlea of 19 New Zealand white rabbits to study the efficacy of the treatment. Over a 12-week period, HS showed significantly enhanced subchondral bone regeneration compared with a hydrogel control. Treatment with HS also resulted in an increased presence of hyaline cartilage in the chondral region. The use of HS in osteochondral defects appears to improve both subchondral and chondral tissue repair. Our data suggest that this effect is mediated by the ability of HS to promote endogenous growth factors.

Impact Statement

Repairing damaged joint cartilage remains a significant challenge. Treatment involving microfracture, tissue grafting, or cell therapy provides some benefit, but seldom regenerates lost articular cartilage. Providing a point-of-care solution that is cell and tissue free has the potential to transform orthopedic treatment for such cases. Glycosaminoglycans such as heparan sulfate (HS) are well suited for this purpose because they provide a matrix that enhances the prochondrogenic activities of growth factors normally found at sites of articular damage. In this study, we show the potential of a novel HS device, which is free of exogenous cells or growth factors, in regenerating osteochondral defects.

Introduction

Articular cartilage is an important smooth connective tissue that reduces friction and acts to transmit loads across the joints. In traumatic events, a lesion involving both the cartilage and its subchondral bone may occur, that is, an osteochondral defect.¹ Strategies focusing on regenerating bone defects have shown promising clinical outcomes and are already commercially available, including the use of autogenous and exogenous bone, synthetic bone grafts, exogenous growth factors, and stem cells. In comparison, current treatments for the regeneration of cartilage are still challenging, due to its low metabolic rate and avascular nature.^2,3 To date, strategies to enhance cartilage repair have focused on stimulating regeneration by using endogenous or transplanted stem cells, which have been expected to differentiate into chondrocytes that secrete extracellular matrix (ECM), and thus contribute to the formation of new cartilage. For example, the current standard of care, microfracture, is routinely used in attempts to recruit endogenous stem cells and progenitors from bone marrow to the site of injured cartilage. In addition, administration of exogenous growth factors, such as transforming growth factor beta (TGF-β), vascular endothelial growth factor (VEGF), bone morphogenetic protein (BMP), and fibroblast growth factor (FGF), has also been trialed to activate endogenous stem cells and progenitors.^4–7 However, none of these treatments produces satisfactory outcomes, possibly due to the relatively short in vivo half-life of exogenous growth factors, which impairs their efficacy.

To address the issue, exogenous growth factors are often administered at supraphysiological doses to maintain an adequate threshold at the treatment site.⁸ Such approaches face significant barriers to clinical translation, as supraphysiological doses of growth factors are frequently associated with undesirable outcomes that include immune reaction,⁹ synovial inflammation,^10,11 and postoperative complications.¹² Separate to the dosing problem, there is also the need to localize the growth factor(s) to the injury site to prevent it from triggering systemic side effects, such as fibrosis and oncogenesis.^13–15 Strategies have been developed to control the release of morphogens, either through a sustained release or a lowered starting dose.^16,17 However, challenges remain in the successful delivery of growth factors in an orderly and continuous manner.^8,18

In response to these challenges, new strategies are being developed to reduce or obviate the need for exogenous growth factors. Indeed, growth factors such as VEGF and TGF-β1 are naturally present in synovial fluid and serum^19–22 and might be sufficient to drive endogenous mesenchymal stem cell (MSC) differentiation²³ if their instability can be addressed. In response, an alternate strategy that enhances the activity of local, endogenously produced growth factors at the defect site is beginning to show promise.

Our group has demonstrated the efficacious use of heparan sulfate (HS) for tissue regeneration in a number of injury models.^24,25 HS is a highly sulfated, linear polysaccharide that constitutes one of the major components of the ECM. The ability of HS to bind, stabilize, and potentiate a range of biologically relevant reparative proteins makes it well suited as a biomaterial for tissue regeneration.^26,27 Previous studies have shown that administration of HS variants at injury sites can promote both bone healing²⁸ and angiogenesis²⁹ without the use of exogenous growth factors. Therefore, we hypothesize that the application of HS to osteochondral defects may help to bind, stabilize, and amplify the activity of endogenous reparative factors and enhance osteochondral repair. In this study, we show that HS binds multiple prochondrogenic and osteogenic growth factors and, when complexed with a commercially available hyaluronic acid gel, is able to enhance the repair of large osteochondral defect model in rabbits in a single-step, point-of-care procedure, which can be readily translated to the clinic.

Materials and Methods

Surface plasmon resonance competitive binding assay

Interactions among TGF-β1 (Peprotech), BMP-2, FGF-2, PDGF-BB, VEGF₁₆₅ (R&D Systems), and porcine mucosal heparan sulfate (HS^PM; Celsus lot No. HO-10697) were measured using surface plasmon resonance (SPR)-based competitive binding assays, as previously described.³⁰ In brief, 20 mg heparin (H3149; Sigma-Aldrich) was biotinylated at free-amine groups using NHS-biotin (8.6 μM) in dimethyl sulfoxide and excess unreacted biotin removed using a 7 kDa spin column (Zeba spin; Thermo-Fisher). Heparin–biotin was immobilized on a sensor chip SA (GE Healthcare) in HBS-EP 0.05% buffer (150 mM NaCl, 10 mM HEPES, 3 mM EDTA, 0.05% [v/v] Tween-20, and pH 7.4) using the default immobilization program on a Biacore T100 until the desired 40 response units were achieved. Optimal protein concentrations for competitive inhibition experiments were determined by injecting the various proteins at a range of concentrations across the heparin-derivatized surface as previously reported.^29–31 The final concentrations used for each protein were as follows: TGF-β1—200 nM, BMP-2—25 nM, FGF-2—8 nM, VEGF₁₆₅—1.56 nM, and PDGF-BB—50 nM.

For competitive binding assays, proteins were preincubated with 10 μg (83.3 μg/mL) HS^PM in HBS-EP 0.1% buffer before being applied to the heparin-derivatized surface at a flow rate of 30 μL/min for 2 min, followed by a 10 min dissociation period. The chip was then regenerated with two 1-min injections of 2M NaCl in HBS-EP 0.1% buffer. The response was measured as a function of time at 25°C. All data were normalized to the maximum response generated by protein alone (100% bound to chip), after which percentage of protein bound to HS^PM in solution was determined by subtracting the normalized response for each protein plus HS^PM from protein alone (% protein on chip [protein+HS^PM] minus the % protein on the chip [protein alone]. Data constitute mean ± standard deviation (SD) of three independent experiments.

Animal study design

Nineteen male New Zealand white rabbits were used for this study. All animals were healthy with an average age of 9 ± 1 months and body weight 3.9 ± 0.2 kg before the study. Bilateral osteochondral defects were surgically created in the femoral trochlear groove. Of the 38 defects created, 19 were assigned to the current study and the remainder to a parallel study to reduce the number of animals. This practice of “reduction” is in strict alignment with the three Rs (3Rs) that are guiding principles for the ethical use of animals in research. Of the 19 defects, 9 were treated with a hyaluronic acid hydrogel alone (AuxiGel^™; Termira AB, Stockholm, Sweden), and 10 were treated with the same hydrogel containing HS^PM. The two treatment groups were randomly assigned among all rabbits. Each defect received either 60 μL of the hydrogel (Gel) alone or 60 μL of hydrogel containing 10 μg of HS^PM (Gel+HS^PM).

Defect creation and gel implantation

The research protocol used for this study was approved by the Institutional Animal Care and Use Committee, Agency for Science, Technology and Research (A*STAR, Singapore, Singapore), and followed all appropriate guidelines (IACUC No. 130833). All surgical procedures were carried out aseptically under general anesthesia, consisting of a combination of ketamine (35 mg/kg) and xylazine (5 mg/kg) injections and 2% isoflurane inspiration. A skin incision was made along the anterior midline from the proximal end of the patellar to the tibial tuberosity. The knee was opened by an anteromedial para-patellar passway. The femoral trochlear groove was then exposed by shifting the patella laterally, and a single osteochondral defect (4 mm in diameter, ≈1 mm in depth) created. Microfractures (0.8 mm diameter, 2 mm depth) were made in three different places within each defect to form an inscribed triangular pattern. Treatments were given by filling the defect with Gel alone or Gel+HS^PM. In brief, 36 μL component A (hyaluronic acid solution) and 24 μL component B (PVA cross-linker solution) were mixed and allowed to set for ∼5 min before being implanted into the defect site. For the Gel+HS^PM group, 10 μg of HS^PM dissolved in 1 μL phosphate-buffered saline was added during the gelation. On-site checking was done immediately after repositioning the patella following 15 joint flexions and extensions to ensure the implant remained in the defect. Prophylactic antibiotics (Enrofloxacin, 10 mg/kg) and analgesics (Buprenorphine, 0.1 mg/kg) were administered subcutaneously for 5 days postsurgery. All rabbits were allowed to move freely in their cages and received a normal diet. At 12 weeks postsurgery, all rabbits were euthanized by lethal dose of sodium pentobarbital, and the distal end of the femurs harvested for gross evaluation, magnetic resonance imaging (MRI), and histological and immunohistochemistry (IHC) analyses.

Gross pathology observation of cartilage repair

The International Cartilage Research Society (ICRS) Visual Assessment Scale (ICRS I) was used for evaluating macroscopic parameters of cartilage repair³² that included the degree of defect repair, integration to the border zone, and macroscopic appearance. The assessment was made by trained staff unaware of the treatment groups.

Magnetic resonance observation of cartilage repair tissue

MRI data collection was done in an ultra-high-field 9.4T MRI scanner (Bruker Biospin GmbH, Ettlingen, Germany). A turbo spin echo sequence was used with echo time: 6 ms, repetition time: 1200 ms, number of averages: 2, and spatial resolution 117 × 117 × 500 μm. Magnetic resonance observation of cartilage repair tissue (MOCART) scoring^33,34 was used for evaluating the cartilage repair in nine parameters—degree of defect repair/filling, integration to border zone, surface, structure, and signal intensity of the repair tissue, subchondral lamina, subchondral bone, adhesions, and synovitis. The assessment was made by a trained staff blinded to the treatment groups.

Histology analyses

After ICRS I scoring and MRI, harvested distal ends of the femurs were fixed in 10% (v/v) neutral-buffered formalin for 1 week under vacuum and decalcified in 5% (v/v) formic acid for 1 week at room temperature. The trimmed specimens were then embedded in paraffin wax and sectioned (5 μm) horizontally from the middle of the defect. Sections were deparaffinized and stained with Masson's trichrome, Alcian blue (pH 1, counterstained with neutral red), and Safranin-O as previously reported.³⁵

IHC analyses

IHC staining was carried using a Leica Bond^™-III Autostainer (Leica Nussloch GmbH) and a Bond Refine Detection Kit (Leica Nussloch GmbH). Sections were deparaffinized with Bond Dewax Solution (Leica Nussloch GmbH) and antigen retrieval carried out by incubating with Proteinase K (20 μg/mL; Sigma-Aldrich) for 15 min at room temperature. Endogenous peroxidase activity was blocked by incubating with 3–4% (v/v) H₂O₂ for 15 min. Sections were then blocked with 10% (v/v) goat serum for 30 min before incubation with primary antibody (Collagen Type I [1:1000]; Novus Biologicals and Collagen Type II [1:2000]; and Acris Antibodies, Inc.) diluted in Bond Primary Antibody Diluent (Leica Nussloch GmbH) for 30 min at room temperature. Detection of stain was performed as described in the Bond Refine Detection Kit, and nuclei were counterstained with hematoxylin. All washes were performed with 1 × Bond Wash Solution (Leica Nussloch GmbH).

Histological scoring

ICRS II scoring was used to evaluate tissue filling and neotissue phenotype.³⁶ Qualitative analysis of the neotissue was based on cell morphology and ECM content. Hyaline cartilage was characterized by the presence of rounded cells in lacunae that express glycosaminoglycan (GAG) and Type II collagen. Fibrocartilage was defined by cells that produce GAG and Type I collagen. Hybrid cartilage exhibits positive staining for both of the Type I and Type II collagen and GAG, while fibrous tissue is positive for Type I collagen alone. Analysis of the percentage of tissue filling was performed using a grid superimposed over the tissue slide. Assessment was made by a trained staff blinded to the treatment groups.

Statistical analysis methods

Values are expressed as mean ± SD. MOCART variables, ICRS I, and ICRS II parameters were analyzed using the multiple t-test function (one unpaired t-test per variable) in GraphPad Prism 7.03 software. Differences in repair cartilage phenotype and tissue filling percentages were determined using a one-way analysis of variance using Tukey's test. Statistical significance was set at p-value <0.05.

Results

HS^PM binds growth factors with varying affinity

To assess the growth factor-binding affinity of HS^PM, various proteins that are important for wound repair (TGF-β1, VEGF₁₆₅, BMP-2, PDGF-BB, and FGF-2) were combined with HS^PM and the complex applied to a heparin-coated sensor chip. Protein:HS^PM complex formation was indicated by a reduction in the amount of protein binding to the heparin-coated chip surface. A greater reduction in binding suggested that the protein has a higher affinity for HS^PM. Because proteins produce variable responses due to variations in binding affinity,³⁷ different concentrations of each protein were used. Once optimized, each protein was incubated with 10 μg HS^PM (83.3 μg/mL) and the complex applied to the sensor chip. Reduced protein binding to the senor chip was observed for all proteins when combined with HS^PM (Fig. 1A–E). When normalized to protein alone (no complexation with HS^PM), the relative level of protein sequestered into solution versus control was determined (Fig. 1F). HS^PM showed varying affinities toward TGF-β1, VEGF_165, BMP-2, PDGF-BB, and FGF-2.

FIG. 1.

Binding of growth factors important to wound healing. Growth factors (A) TGF-β1; (B) VEGF-A₁₆₅; (C) BMP-2; (D) PDGF-BB; and (E) FGF-2 were precomplexed with HS^PM before being applied to a heparin-derivatized SPR sensor chip. Growth factors sequestered into solution results in a reduction in R.U. on the sensor chip. (F) Data are represented as the percentage of protein bound to HS^PM in solution. Data are representative of the mean ± SD of three independent experiments. BMP, bone morphogenetic protein; FGF, fibroblast growth factor; R.U. response units; SD, standard deviation; SPR, surface plasmon resonance; PDGF-BB, platelet-derived growth factor composed of two B subunits; TGF-β1, transforming growth factor beta 1; VEGF, vascular endothelial growth factor.

HS^PM improves the macro- and microscopic healing of rabbit osteochondral defects

All animals survived until the end point, and no adverse events were seen. All the 19 retrieved samples were evaluated macroscopically using ICRS I scoring and were imaged by MRI, but 2 samples were not included for MOCART scoring due to poor positioning of the samples. Post-MRI, one sample in the Gel group was damaged during histological processing and removed from the study. The remaining samples were processed for ICRS II scoring. Scattergrams of data from microscopic evaluations highlighted that one sample in the Gel+HS^PM group (ID# Y781L-R) was probably a biological outlier (data not shown). Therefore, a Grubbs' outlier test,³⁸ with an alpha level set at 0.05, was used to assess the data set (GraphPad Prism, version 7.03; GraphPad Software, San Diego, CA). Examination of the histological sections on this particular sample suggested there were problems during sectioning, and therefore the data point was removed. Therefore, 19 samples were used for ICRS I scoring, 17 samples for MOCART scoring, and 17 samples for ICRS II scoring and additional histomorphometry. Detailed animal usage data are listed in Supplementary Table S1.

The harvested explants were photographed using a Nikon digital camera D3300 and representative images showing the best, and the worst performers of the healed defects from the two groups are presented in Figure 2A. Gross morphological evaluation showed that surfaces of defects treated with Gel+HS^PM had a higher degree of integration with surrounding host cartilage and were more intact and smoother in appearance (Fig. 2A). In contrast, scattered fissures and poor tissue healing was more evident for defects treated with Gel. Despite the variation between the best and the worst performers within each group, treatment with Gel+HS^PM scored higher in all of the ICRS I parameters, while also having the least coefficient of variation (CV) in treatment outcomes (Fig. 2B). Treatment with Gel+HS^PM resulted in an average ICRS I score of 10.40 ± 2.07 and median score of 11 compared with defects treated with Gel that averaged 9.11 ± 2.42 and had a median score of 8 (Fig. 2C). Moreover, treatment with Gel+HS^PM resulted in cartilage of “normal” appearance (ICRS I score = 12, Grade I) in 40% (4 out of 10) of defects, compared with 22% (2 out of 9) in the Gel group. A similar result was observed for cartilage of “normal or nearly normal” appearance (score ≥8, Grade II and above) in 78% of Gel-treated defects (7 out of 9) compared with 90% in Gel+HS^PM-treated defects (9 out of 10). The difference is, however, not statistically significant (p = 0.23).

FIG. 2.

ICRS I Scores 12 weeks posttreatment. (A) Representative images showing the macroscopic appearance of osteochondral defects treated with hyaluronic acid (Gel) or hyaluronic acid+porcine mucosal heparan sulfate (Gel+HS^PM) 12 weeks postsurgery (N = 19). Left: representative images of the best performers from both groups; right: representative images of the worst performers from both groups. Ruler markings indicate 1 mm. (B) Heat map of ICRS I scores. Three parameters were assessed: degree of defect repair, integration to border zone, and macroscopic appearance. Treatment with Gel+HS^PM resulted in higher ICRS I scores and less variability compared with Gel in all three parameters. (C) Box plot of the overall ICRS I scores for Gel+HS^PM and Gel 12 weeks postsurgery. ICRS, International Cartilage Research Society. Color images are available online.

MOCART scoring using MRI images (Fig. 3A) showed that the repair tissue in defects treated with HS^PM had higher scores in all nine parameters (Fig. 3B, C). Specifically, HS^PM treatment significantly improved the intactness of the surface of repair tissue (p = 0.016) and subchondral bone (p = 0.048) as well as the signal intensity of the repair tissue (p = 0.0342) (Fig. 3B). As a result, the overall MOCART score was significantly higher for treatment with Gel+HS^PM (12.25 ± 1.91) compared with Gel alone (8.78 ± 3.15), suggesting a more complete and homogeneous restoration of repair tissue in defects treated with Gel+HS^PM (p = 0.0165) (Fig. 3B, C).

FIG. 3.

MRI and MOCART scores. (A) Representative images showing the MRI images of osteochondral defects treated with hyaluronic acid (Gel) or hyaluronic acid+porcine mucosal heparan sulfate (Gel+HS^PM) 12 weeks postsurgery (N = 19). The MOCART score of the representative images are given for reference. Scale bar = 5 mm. White arrows indicate the edge of the defect. (B) MOCART scores (N = 17). (C) Heat map of MOCART scores (N = 17). Treatment with Gel+HS^PM resulted in higher MOCART scores especially in parameters of the surface of the repair tissue, signal intensity of the repair tissue, and subchondral bone compared with Gel. Asterisks in (B) p < 0.05. MOCART, magnetic resonance observation of cartilage repair tissue; MRI, magnetic resonance imaging. Color images are available online.

Decalcified sections were stained with Masson's Trichrome, Safranin-O, Alcian blue, or immunostained for the presence of Type I and Type II collagen (Fig. 4). Most of the cartilage formed was hybrid cartilage, characterized by the expression of GAG and both Type I and Type II collagen (Fig. 4). When defects were treated with Gel+HS^PM, an abundance of bone filling the defect to the level of the tidemark in the uninjured host tissue was observed in most of the samples. This was not observed in the Gel-alone-treated defects; instead, Safranin O-positive staining was observed below the height of the host tidemark, indicating on-going endochondral bone formation (or remnant hyaluronic acid gel) in the subchondral region. Overall, this suggests inadequate growth of subchondral bone when Gel+HS^PM was omitted (Fig. 5A, B). In Gel-alone-treated defects, we observed cartilage deposited on regenerated subchondral bone, but the cells in the chondral layer were unordered and had an elongated, spindle-shaped morphology (Fig. 5C). In contrast, defects treated with Gel+HS^PM contained cells with a clear chondrocyte-like morphology that were oval shaped and stacked within well-organized lacuna in the regenerated chondral layer (Fig. 5D). The ICRS II scores also reflected this difference, with Gel+HS^PM-treated defects scoring higher than Gel-treated defects in all 13 parameters (Fig. 6A), with significantly higher scores for cell morphology (p = 0.0415) and subchondral bone regeneration (p = 0.0159) compared with Gel (Fig. 6B). Also, Gel+HS^PM treatment produced more consistent ICRS II scores (a lower CV) for all parameters (Fig. 6A), indicating a more reliable outcome. The overall ICRS II assessment (Fig. 6C) showed significantly higher scores for the Gel+HS^PM (89.15 ± 6.03) group compared with the Gel group (78.26 ± 8.46), suggesting that the use of HS^PM leads to better repair outcomes in osteochondral defects (p = 0.0075).

FIG. 4.

Representative histology of a normal trochlea before surgery (left column), the defect postsurgery (second column), and 12 weeks posttreatment with Gel (animal ID: BLK516L-R) or Gel+HS^PM (animal ID: BLK785L_L; N = 17). Sections were stained with Masson's Trichrome to detect collagen (blue), cell cytoplasm (red) and nuclei (black), and Safranin-O and Alcian blue to detect GAGs (red and blue, respectively). Immunohistochemistry was used to assess the distribution of Collagen type I and II. Scale bar: 1 mm. Boxed areas in Masson's trichrome-stained images are shown at a higher magnification in Figure 5A and B. GAGs, glycosaminoglycans. Color images are available online.

FIG. 5.

Representative histology at the margins of defects. (A) Higher magnification of boxed area in Figure 4 (Gel treatment). Treatment with Gel showing poor subchondral bone regeneration. (C) Boxed area in (A) at higher magnification showing an abundance of spindle shaped, elongated cells (arrows). (B) Treatment with Gel+HS^PM results in subchondral bone regeneration up to the level of the tidemark in the host bone that is well integrated with surrounding tissue. (D) Boxed area in (B) at higher magnification highlighting the oval-shaped chondrocyte-like morphology of cells (white arrows) stacked in the newly formed chondral tissue (NC). Masson's Trichrome staining, scale bars: 250 μm in (A, B), 50 μm in (C, D). B, bone; HC, hyaline cartilage; NC, new cartilage; TM, tidemark. Color images are available online.

FIG. 6.

ICRS II Scores 12 weeks posttreatment (N = 17). (A) Heatmap of individual scores for ICRS II parameters and the variability in scores (coefficient of variation). Treatment with Gel+HS^PM resulted in higher scores for the various ICRS II parameters with less variability compared with Gel. (B) Cell morphology and subchondral bone were significantly improved following treatment with Gel+HS^PM. (C) Box plot showing increased overall ICRS II score following treatment with Gel+HS^PM compared with Gel. *p < 0.05, **p < 0.01. Color images are available online.

Further analysis of the amount of tissue filling the defect (total tissue filling %) showed no significant difference between the two treatments (p = 0.09), irrespective of the tissue layer (Fig. 7A). However, rather than simply assessing the amount of tissue, it is more important to determine the relative amounts of bone tissue in the subchondral layer (Fig. 7B) and cartilage in the chondral layer (Fig. 7C). The data show that when defects were treated with Gel+HS^PM, significantly more bone tissue filled the subchondral layer (79.77% ± 4.94%, p = 0.0393) compared with the Gel group (61.19% ± 6.75%) (Fig. 7B). However, the amount of newly formed cartilage in the chondral region was similar for both treatments (87.50% ± 35.36% for Gel versus 85.71% ± 33.50% for Gel+HS^PM) (Fig. 7C). The data also show that most of the cartilage formed was hybrid cartilage, with no fibrocartilage being observed (Fig. 7D). Interestingly, hyaline cartilage was observed in two out of nine (22%) Gel+HS^PM-treated defects, but not in any Gel-treated defects (Fig. 7D).

FIG. 7.

Tissue filling regenerated osteochondral defects 12 weeks posttreatment (N = 17). (A) Defects are nearly completely filled with tissue, irrespective of treatment. (B) New bone forms a significantly (p = 0.0393) greater proportion of tissue deposited in the subchondral region when defects are treated with Gel+HS^PM. (C) Similar amounts of regenerated cartilage fill the chondral region when treated with either Gel+HS^PM or Gel. (D) Hybrid cartilage is the predominant tissue type deposited in the chondral region, irrespective of treatment and increased incidence of hyaline cartilage fill the chondral region when defects are treated with Gel+HS^PM. Hyaline cartilage was only present in defects treated with Gel+HS^PM. (*p < 0.05).

Discussion

Autologous chondrocyte implantation for the treatment of large, full-thickness osteochondral defects is gaining clinical momentum.^39–43 However, significant donor site morbidity and a high percentage of fibrocartilage at the repair site continue to plague its therapeutic efficacy.⁴⁴ As an alternate strategy, we chose to examine a cell-free matrix that does not require surgical harvesting of donor tissue, which therefore provides a point-of-care treatment option for patients. Key to this strategy is the development of an implantable device with the ability to integrate with the host and fully restore the structure and function of the lost tissue. GAGs such as HS are well-suited for this purpose because they provide a matrix that enhances the prochondrogenic activities of endogenous growth factors found at sites of articular damage. In this study, we evaluated the ability of an HS-impregnated gel to repair large osteochondral defects in the femoral trochlea of rabbits over a 12-week period.

The use of SPR enables an assessment of the interactions between two components, such as HS and a HS-binding protein, in a label-free system. In the context of cartilage repair, SPR allows HS to be screened for binding against many proregenerative HS-binding proteins, with higher binding representing higher affinity toward a protein. When HS displays a high affinity or ability to bind large quantities of proregenerative proteins in SPR assays, it is likely that this HS can retain and stabilize these factors within a wound site or defect, enhancing the endogenous healing response. Using SPR-based analytics, we show here that high-affinity complexes are formed between the rich mixture that is HS^PM and many notably reparative proteins such as TGF-β1, BMP-2, FGF-2, PDGF-BB, and VEGF₁₆₅. We observed considerable binding variability that highlights the heterogeneity of HS—while some HS chains may contain an enrichment of binding sites for one protein, they may lack binding sites for others. Despite the varied affinity profile, this study showed that HS^PM was able to bind the pro-osteogenic factor BMP-2⁴⁵ as well as the prochondrogenic factor TGF-β1.⁴⁶ Also notable was the binding of HS^PM to the progenitor mitogen FGF-2⁴⁷ as well as the provascular factors VEGF₁₆₅^48,49 and PDGF-BB.⁵⁰ This mixed binding profile is important because the repair of osteochondral injuries involve the restoration of both subchondral bone and the overlying cartilage, together with blood vessels and other tissues at the repair site. Although HS^PM containing scaffolds were not preloaded with protein growth factors, the addition of HS^PM may bind endogenous growth factors,⁵¹ enhancing the biological signals that help to mediate the repair cascade.^52,53

We have previously shown that HS stimulates the proliferation of MSCs in an FGF-2-dependent manner^54–56 and that heparin/HS preparations enhance TGF-β1-induced SMAD2/3 signaling, an important initiator of the chondrogenic signaling cascade.³⁰ We have also shown that affinity-selected HS prolongs BMP-2-mediated SMAD1/5/8 signaling²⁸ and osteogenic differentiation, as well as the formation of VEGF₁₆₅-mediated vascular networks.²⁹ This suggests that HS-containing scaffolds may favor the proliferation and differentiation of stem cells toward those desired cellular responses needed for osteochondral repair, thus highlighting the potential of HS preparations as reparative agents in a rabbit femoral trochlear grove defect model.

In our animal study, osteochondral defects treated with Gel+HS^PM showed higher MOCART and ICRS I scores compared to the Gel alone group, indicating improved filling of the defects and integration with surrounding host tissue. MRI analyses showed more intact surface and subchondral bone in defects treated with Gel+HS^PM. The majority of defects treated with Gel+HS^PM (90%) had a surface morphology that appeared normal or nearly normal (ICRS I scores ≥8, Grade II and above) compared with those treated with Gel alone (78%). Detailed histological assessment using ICRS II scoring indicated abundant new subchondral bone reaching the level of the tidemark in adjacent host bone was formed following Gel+HS^PM treatment, but not with Gel alone. The reduced amount of new bone formation may be due to less absorption of the HA gel, or insufficient calcification of newly formed ECM in the absence of HS^PM. Importantly, regenerated hyaline cartilage in the chondral layer was only observed following treatment with Gel+HS^PM. Although hybrid cartilage was found in nearly all defects, more oval-shaped chondrocytes were observed with Gel+HS^PM treatment. Such improvement in the cartilage layer following treatment with Gel+HS^PM can be attributed to enhanced subchondral bone regeneration because articular cartilage growth relies on a sufficiency of subchondral bone for mechanical support and nutrition.^57,58 Furthermore, there is growing evidence that biochemical and molecular crosstalk occurs between cells in cartilage and subchondral bone.^59,60 It is plausible that HS^PM helps to mediate the interdependence between the two compartments by stabilizing and enhancing the actions of reparative factors at the injury site.⁶¹ Also, by virtue of its anti-inflammatory qualities,^62,63 HS may help swing the balance in favor of regeneration rather than degeneration that typically dominates this type of injury. Future efforts will focus on improving the quality rather than the amount of repaired tissue, including the restoration of aggrecan levels and the mechanical properties of the repaired cartilage tissue.

In the present study, the biodistribution of HS^PM and the presence of augmented growth factor(s) were not assessed. Therefore, the observed HS^PM-enhanced tissue regeneration may have resulted from an improved microenvironment during the early stages of wound healing, or through medium/long-term changes in the available pool of reparative factors and cells. Also, the biological variation in the animal study⁶⁴ and the numbers of animals used may have an impact on the outcome. Also, clinical variation is common in medical practice and may cause unexpected results.⁶⁵ In assessing such variability, we noted that the Gel+HS^PM treatment group not only promoted tissue repair but also reduced the CV for all 3 parameters of the ICRS I score and all 14 parameters of the ICRS II scoring system. It is therefore possible that improved outcomes with HS^PM had a major contribution toward reducing the variable healing responses between animals, a finding that has important implications for future clinical studies.

Our results highlight that the restoration of subchondral bone is an important pillar for cartilage regeneration. Moreover, full restoration of osteochondral defects demands a multiphasic engineering approach. A variety of biphasic scaffolds mimicking the structure of osteochondral tissues have been studied in the presence or absence of exogenous growth factors.⁶⁶ Future investigations focusing on biphasic scaffolds loaded with HS variants to manipulate the biodistribution of endogenous growth factors are needed to further fine-tune osteochondral responses. The data presented here emphasize the considerable therapeutic potential of medical devices containing HS at articular sites, warranting their further development as biomaterials that support tissue restoration.

Conclusion

We have shown that HS^PM materials can enhance osteochondral tissue regeneration, possibly by helping to orchestrate the activities of endogenous growth factors and cells, highlighting their therapeutic value in harnessing endogenous repair cascades. We believe that the development of such an HS-based strategy for osteochondral repair may spare the need for exogenous applications of growth factors or cell transplantation.

Footnotes

Acknowledgments

The authors acknowledge the funding support of Singapore's Agency for Science, Technology and Research (A*STAR) and Institute of Medical Biology (IMB). Funding was also provided by the National Medical Research Council (NMRC), Singapore (No. CIRG16may052).

Authors' Contributions

V.N., J.H.H., and S.M.C. obtained the funding. J.H.L., V.N., J.H.H., K.S., and S.M.C. designed the study. J.H.L. and R.A.A.S. performed the in vitro studies. J.H.L., X.R., and T.C.T. conducted the surgeries. J.H.L., X.L., X.R., T.C.T., R.A.A.S., K.S., S.S., K.B., and S.M.C. analyzed and interpreted the data. J.H.L., R.A.A.S., and X.L. assembled the data and drafted the article. All authors revised the article and gave final approval of the article.

Disclosure Statement

No competing financial interests exist.

References

van Dijk

C.N.

, Reilingh

M.L.

, Zengerink

, and van Bergen

C.J.A.

Osteochondral defects in the ankle: why painful?. Knee Surg Sport Traumatol Arthrosc, 18, 570, 2010.

Sobol

, Shekhter

, Guller

, Baum

, and Baskov

Laser-induced regeneration of cartilage. J Biomed Opt, 16, 080902, 2011.

Zhang

, Hu

, and Athanasiou

K.A.

The role of tissue engineering in articular cartilage repair and regeneration. Crit Rev Biomed Eng, 37, 1, 2009.

Ying

, Chen

, Hu

, et al. Effect of different doses of transforming growth factor-β1 on cartilage and subchondral bone in osteoarthritic temporomandibular joints. Br J Oral Maxillofac Surg, 51, 241, 2013.

Murata

, Yudoh

, and Masuko

The potential role of vascular endothelial growth factor (VEGF) in cartilage. Osteoarthritis Cartilage, 16, 279, 2008.

Chubinskaya

, Hurtig

, and Rueger

D.C.

OP-1/BMP-7 in cartilage repair. Int Orthop, 31, 773, 2007.

Moore

E.E.

, Bendele

A.M.

, Thompson

D.L.

, et al. Fibroblast growth factor-18 stimulates chondrogenesis and cartilage repair in a rat model of injury-induced osteoarthritis. Osteoarthritis Cartilage, 13, 623, 2005.

James

A.W.

, LaChaud

, Shen

, et al. A review of the clinical side effects of bone morphogenetic protein-2. Tissue Eng Part B Rev, 22, 284, 2016.

Baker

, Reynolds

H.M.

, Lumicisi

, and Bryson

C.J.

Immunogenicity of protein therapeutics: the key causes, consequences and challenges. Self Nonself, 1, 314, 2010.

10.

Leah

Osteoarthritis: TGF-β overload at bones of cartilage degeneration. Nat Rev Rheumatol, 9, 382, 2013.

11.

Allen

J.B.

, Manthey

C.L.

, Hand

A.R.

, Ohura

, Ellingsworth

, and Wahl

S.M.

Rapid onset synovial inflammation and hyperplasia induced by transforming growth factor beta. J Exp Med, 171, 231, 1990.

12.

Cole

, Veeravagu

, Jiang

, and Ratliff

J.K.

Usage of recombinant human bone morphogenetic protein in cervical spine procedures: analysis of the MarketScan longitudinal database. J Bone Joint Surg Am., 96, 1409, 2014.

13.

Jakowlew

S.B.

Transforming growth factor-beta in cancer and metastasis. Cancer Metastasis Rev, 25, 435, 2006.

14.

Bakker

A.C.

, van de Loo

F.A.J.

, van Beuningen

H.M.

, et al. Overexpression of active TGF-beta-1 in the murine knee joint: evidence for synovial-layer-dependent chondro-osteophyte formation. Osteoarthritis Cartilage, 9, 128, 2001.

15.

Yang

Y.-A.

, Dukhanina

, Tang

, et al. Lifetime exposure to a soluble TGF-beta antagonist protects mice against metastasis without adverse side effects. J Clin Invest, 109, 1607, 2002.

16.

Mao

A.S.

, and Mooney

D.J.

Regenerative medicine: current therapies and future directions. Proc Natl Acad Sci U S A, 112, 14452, 2015.

17.

Liu

, Zhou

, Xu

, Zhang

, Liu

C.-H.

, and Wang

Controlled release of growth factors for regenerative medicine. Curr Pharm Des, 21, 1627, 2015.

18.

Wang

, Wang

, Lu

W.W.

, Zhen

, Yang

, and Peng

Novel biomaterial strategies for controlled growth factor delivery for biomedical applications. NPG Asia Mater, 9, e435, 2017.

19.

Vignola

, Picco

, Falcini

, Sabatini

, Buoncompagni

, and Gattorno

Serum and synovial fluid concentration of vascular endothelial growth factor in juvenile idiopathic arthritides. Rheumatology (Oxford), 41, 691, 2002.

20.

Jiang

, Qiu

Y.-T.

, Chen

M.-J.

, Zhang

Z.-Y.

, and Yang

Synovial TGF-β1 and MMP-3 levels and their correlation with the progression of temporomandibular joint osteoarthritis combined with disc displacement: a preliminary study. Biomed Rep, 1, 218, 2013.

21.

Wahl

S.M.

, Allen

J.B.

, Wong

H.L.

, Dougherty

S.F.

, and Ellingsworth

L.R.

Antagonistic and agonistic effects of transforming growth factor-beta and IL-1 in rheumatoid synovium. J Immunol, 145, 2514, 1990.

22.

Lettesjö

, Nordström

, Ström

, et al. Synovial fluid cytokines in patients with rheumatoid arthritis or other arthritic lesions. Scand J Immunol, 48, 286, 1998.

23.

Coupes

B.M.

, Williams

, Roberts

I.S.

, Short

C.D.

, and Brenchley

P.E.

Plasma transforming growth factor beta(1) and platelet activation: implications for studies in transplant recipients. Nephrol Dial Transplant, 16, 361, 2001.

24.

Bramono

D.S.

, Murali

, Rai

, et al. Bone marrow-derived heparan sulfate potentiates the osteogenic activity of bone morphogenetic protein-2 (BMP-2). Bone, 50, 954, 2012.

25.

Bramono

D.S.

, Rider

D.A.

, Murali

, Nurcombe

, and Cool

S.M.

The effect of human bone marrow stroma-derived heparan sulfate on the ex vivo expansion of human cord blood hematopoietic stem cells. Pharm Res, 28, 1385, 2011.

26.

Melrose

Glycosaminoglycans in wound healing. Bone Tissue Regen Insights, 7, 29, 2016.

27.

Lee

S.S.

, Fyrner

, Chen

, et al.. Sulfated glycopeptide nanostructures for multipotent protein activation. Nat Nanotechnol, 12, 821, 2017.

28.

Murali

, Rai

, Dombrowski

, et al. Affinity-selected heparan sulfate for bone repair. Biomaterials, 34, 5594, 2013.

29.

Wang

, Poon

, Murali

, et al. Engineering a vascular endothelial growth factor 165-binding heparan sulfate for vascular therapy. Biomaterials, 35, 6776, 2014.

30.

Lee

, Wee

, Gunaratne

, et al. Structural determinants of heparin-transforming growth factor-β1 interactions and their effects on signaling. Glycobiology, 25, 1491, 2015.

31.

Smith

R.A.A.

, Chua

R.J.E.

, Carnachan

S.M

, et al. Retention of the structure and function of heparan sulfate biomaterials after gamma irradiation. Tissue Eng Part A, 24, 729, 2017.

32.

van den Borne

M.P.J.

, Raijmakers

N.J.H.

, Vanlauwe

, et al. International Cartilage Repair Society (ICRS) and Oswestry macroscopic cartilage evaluation scores validated for use in Autologous Chondrocyte Implantation (ACI) and microfracture. Osteoarthritis Cartilage, 15, 1397, 2007.

33.

Marlovits

, Striessnig

, Resinger

C.T.

, et al. Definition of pertinent parameters for the evaluation of articular cartilage repair tissue with high-resolution magnetic resonance imaging. Eur J Radiol, 52, 310, 2004.

34.

Marlovits

, Singer

, Zeller

, Mandl

, Haller

, and Trattnig

Magnetic resonance observation of cartilage repair tissue (MOCART) for the evaluation of autologous chondrocyte transplantation: determination of interobserver variability and correlation to clinical outcome after 2 years. Eur J Radiol, 57, 16, 2006.

35.

Brehm

, Aklin

, Yamashita

, et al. Repair of superficial osteochondral defects with an autologous scaffold-free cartilage construct in a caprine model: implantation method and short-term results. Osteoarthritis Cartilage, 14, 1214, 2006.

36.

Mainil-Varlet

, Van Damme

, Nesic

, Knutsen

, Kandel

, and Roberts

A new histology scoring system for the assessment of the quality of human cartilage repair: ICRS II. Am J Sports Med, 38, 880, 2010.

37.

, and Esko

J.D.

Demystifying heparan sulfate–protein interactions. Annu Rev Biochem, 83, 129, 2014.

38.

Grubbs

F.E.

Procedures for detecting outlying observations in samples. Technometrics, 11, 1, 1969.

39.

Barlow

, Downham

, and Griffin

Arthroscopy in knee osteoarthritis: a systematic review of the literature. Acta Orthop Belg, 81, 1, 2015.

40.

Chawla

, Twycross-Lewis

, and Maffulli

Microfracture produces inferior outcomes to other cartilage repair techniques in chondral injuries in the paediatric knee. Br Med Bull, 116, 93, 2015.

41.

Oussedik

, Tsitskaris

, and Parker

Treatment of articular cartilage lesions of the knee by microfracture or autologous chondrocyte implantation: a systematic review. Arthroscopy, 31, 732, 2015.

42.

Richter

D.L.

, Schenck

R.C.

, Wascher

D.C.

, and Treme

Knee articular cartilage repair and restoration techniques: a review of the literature. Sports Health, 8, 153, 2016.

43.

Carver

S.E.

, and Heath

C.A.

Influence of intermittent pressure, fluid flow, and mixing on the regenerative properties of articular chondrocytes. Biotechnol Bioeng, 65, 274, 1999.

44.

Lee

C.R.

, Grodzinsky

A.J.

, Hsu

H.P.

, Martin

S.D.

, and Spector

Effects of harvest and selected cartilage repair procedures on the physical and biochemical properties of articular cartilage in the canine knee. J Orthop Res, 18, 790, 2000.

45.

Luu

H.H.

, Song

W.-X.

, Luo

, et al. Distinct roles of bone morphogenetic proteins in osteogenic differentiation of mesenchymal stem cells. J Orthop Res, 25, 665, 2007.

46.

Worster

A.A.

, Nixon

A.J.

, Brower-Toland

B.D.

, and Williams

Effect of transforming growth factor beta1 on chondrogenic differentiation of cultured equine mesenchymal stem cells. Am J Vet Res, 61, 1003, 2000.

47.

Solchaga

L.A.

, Penick

, Goldberg

V.M.

, Caplan

A.I.

, and Welter

J.F.

Fibroblast growth factor-2 enhances proliferation and delays loss of chondrogenic potential in human adult bone-marrow-derived mesenchymal stem cells. Tissue Eng Part A, 16, 1009, 2010.

48.

Duyndam

M.C.A.

, Hilhorst

M.C.G.W.

, Schlüper

H.M.M.

, et al. Vascular endothelial growth factor-165 overexpression stimulates angiogenesis and induces cyst formation and macrophage infiltration in human ovarian cancer xenografts. Am J Pathol, 160, 537, 2002.

49.

Yue

, and Tomanek

R.J.

Effects of VEGF(165) and VEGF(121) on vasculogenesis and angiogenesis in cultured embryonic quail hearts. Am J Physiol Heart Circ Physiol, 280, H2240, 2001.

50.

Battegay

E.J.

, Rupp

, Iruela-Arispe

, Sage

E.H.

, and Pech

PDGF-BB modulates endothelial proliferation and angiogenesis in vitro via PDGF beta-receptors. J Cell Biol, 125, 917, 1994.

51.

McCaffrey

T.A.

, Falcone

D.J.

, Vicente

, Du

, Consigli

, and Borth

Protection of transforming growth factor-beta 1 activity by heparin and fucoidan. J Cell Physiol, 159, 51, 1994.

52.

Lyon

, Rushton

, and Gallagher

J.T.

The interaction of the transforming growth factor-betas with heparin/heparan sulfate is isoform-specific. J Biol Chem, 272, 18000, 1997.

53.

Zehe

, Engling

, Wegehingel

, Schafer

, and Nickel

Cell-surface heparan sulfate proteoglycans are essential components of the unconventional export machinery of FGF-2. Proc Natl Acad Sci U S A, 103, 15479, 2006.

54.

Dombrowski

, Song

S.J.

, Chuan

, et al. Heparan sulfate mediates the proliferation and differentiation of rat mesenchymal stem cells. Stem Cells Dev, 18, 661, 2009.

55.

Titmarsh

D.M.

, Tan

C.L.L.

, Glass

N.R.

, Nurcombe

, Cooper-White

J.J.

, and Cool

S.M.

Microfluidic screening reveals heparan sulfate enhances human mesenchymal stem cell growth by modulating fibroblast growth factor-2 transport. Stem Cells Transl Med, 6, 1178, 2017.

56.

Wijesinghe

S.J.

, Ling

, Murali

, et al. Affinity selection of FGF2-binding heparan sulfates for ex vivo expansion of human mesenchymal stem cells. J Cell Physiol, 232, 566, 2017.

57.

Wen

, Lu

W.W.

, and Chiu

K.Y.

Importance of subchondral bone in the pathogenesis and management of osteoarthritis from bench to bed. J Orthop Transl, 2, 16, 2014.

58.

Imhof

, Breitenseher

, Kainberger

, Rand

, and Trattnig

Importance of subchondral bone to articular cartilage in health and disease. Top Magn Reson Imaging, 10, 180, 1999.

59.

Findlay

D.M.

, and Atkins

G.J.

Osteoblast-chondrocyte interactions in osteoarthritis. Curr Osteoporos Rep, 12, 127, 2014.

60.

Yuan

X.L.

, Meng

H.Y.

, Wang

Y.C.

, et al. Bone–cartilage interface cross talk in osteoarthritis: potential pathways and future therapeutic strategies. Osteoarthritis Cartilage, 22, 1077, 2014.

61.

Fisher

M.C.

, Li

, Seghatoleslami

M.R.

, Dealy

C.N.

, and Kosher

R.A.

Heparan sulfate proteoglycans including syndecan-3 modulate BMP activity during limb cartilage differentiation. Matrix Biol, 25, 27, 2006.

62.

J.-P.

, and Vlodavsky

Heparin, heparan sulfate and heparanase in inflammatory reactions. Thromb Haemost, 102, 823, 2009.

63.

Ferro

Heparan sulfate inhibitors and their therapeutic implications in inflammatory illnesses. Expert Opin Ther Targets, 17, 965, 2013.

64.

Howard

B.R.

Control of variability. ILAR J, 43, 194, 2002.

65.

Kennedy

P.J.

, Leathley

C.M.

, and Hughes

C.F.

Clinical practice variation. Med J Aust, 193(8 Suppl), S97, 2010.

66.

, Ding

, Wang

, Zhuang

, and Chen

Biomimetic biphasic scaffolds for osteochondral defect repair. Regen Biomater, 2, 221, 2015.

Supplementary Material

Please find the following supplemental material available below.

For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.

For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.

0.03 MB

0.00 MB

A Heparan Sulfate Device for the Regeneration of Osteochondral Defects

Abstract

Impact Statement

Introduction

Materials and Methods

Surface plasmon resonance competitive binding assay

Animal study design

Defect creation and gel implantation

Gross pathology observation of cartilage repair

Magnetic resonance observation of cartilage repair tissue

Histology analyses

IHC analyses

Histological scoring

Statistical analysis methods

Results

HSPM binds growth factors with varying affinity

HSPM improves the macro- and microscopic healing of rabbit osteochondral defects

Discussion

Conclusion

Footnotes

Acknowledgments

Authors' Contributions

Disclosure Statement

References

Supplementary Material

HS^PM binds growth factors with varying affinity

HS^PM improves the macro- and microscopic healing of rabbit osteochondral defects