Purpose

Current cell based cartilage regeneration strategies are based on porous scaffolds requiring high amount of de-novo synthesis of extracellular matrix from the implanted cells. Insufficient clinical outcome suggests that the generated repair tissue does not fulfill the required properties and is frequently not hyaline. Therefore replacing the missing cartilage matrix with a functional biomaterial could improve the outcome. Decellularized allogenic cartilage, as homologous tissue, is a promising biomaterial to support chondrogenic matrix formation. Due to the complex fine structure we aimed at preserving its integrity and use it as bulk material. In order to achieve repopulation of this exceptionally dense matrix with cells, we developed a new strategy based on laser engraving.

Methods and Materials

Articular cartilage biopsies were engraved with different types of lasers and patterns, were then decellularized and partially glycosaminoglycan (GAG) depleted. The materials were then seeded with adipose-derived stromal cells (ASC) or chondrocytes to evaluate cell adhesion, chondrogenic differentiation and in vivo performance in a nude mouse model. Mechanical testing, nanoCT and (immuno-)histochemistry were used to analyze the samples.

Results

Engraving of crossed line incisions was the optimal pattern for a suitable cell-matrix ratio still retaining the stability but providing enough space for cells. The material was several times stiffer than commercial scaffolds and filled the defect space to more than a half. Cells adhered well to the matrix surface and neo-tissue integrated with it. Collagen fibrils were oriented along the incisions and, notably, the incisions exerted a chondrogenic effect on chondrocytes as well as ASC, which was apparently strongest in the incision tip.

Conclusion

Laser-engraved decellularized articular cartilage provides a chondrogenic environment and higher load-bearing than commercial scaffolds but also serves as homologous tissue replacement taking load off the cells to fill out the defect completely by newly synthesized matrix.

Purpose

The objective of this multicenter, randomized and controlled trial was to compare the outcome of patients affected by joint surface lesions (JSLs), with or without concurrent OA, treated with an aragonite-based osteochondral implant (Agili-C™, CartiHeal Ltd, Israel) to a control group, treated with arthroscopic debridement/microfracture.

Methods and Materials

251 subjects were enrolled in 26 medical centers, according to the following criteria : 1) age 21–75 years; 2) up to three JSLs, ICRS Grade IIIa or above, located on the femoral condyles and/or trochlea; 3) total treatable area from 1 to 7 cm² ; 4) bony defect depth less than 8mm; 5) knee OA grade 0-3 according to Kellgren-Lawrence score. Subjects were randomized to the implant or debridement/microfracture in a 2:1 ratio. Evaluation was performed at 6,12,18 and 24 months, based on: KOOS (primary endpoint), IKDC-subjective, Tegner and SF-12 questionnaires. Subjects also underwent MRI evaluation at 12 and 24 months to assess defect fill. Failures (i.e. need for any secondary treatment) and adverse events were recorded.

Results

Both groups presented comparable demographic characteristics and baseline values. The implant group showed statistically superior outcome in the primary endpoint and all secondary endpoints at all follow-up visits. The magnitude of improvement in the implant group was twice as large than the control group in terms of mean KOOS improvement. Similar results were documented in all other scores recorded. Responder rate (defined a priori as at least 30-points improvement in KOOS) was 77.8% in the implant group compared to only 33.6% in the control (p<0.0001). At 24 months, 88.5% of the implanted subjects had at least 75% defect fill on MRI compared to 30.9% of subjects treated with debridement/microfracture (p<0.0001). Failure rate was 7.2% for the implant group vs 21.4% for control.

Conclusion

The aragonite-based implant provided superior clinical and radiographic outcome compared to debridement/microfractures at 24 month evaluation.

Purpose

Purpose A key aspect for the success of autologous osteochondral transplantations is the fit of the subchondral bone plate of the transplant to the recipient area. (Baumbach et al., Draenert et al.). Hence, the aim of this study is to find a way to create precise milling templates and to check whether an experienced surgeon can improve his results by using templates in an animal cadaver study.

Methods and Materials

Methods We performed autologous osteochondral transplants of the posterior lateral condyle to the femoral head of the same bone on 50 femoral pig bones. For 25 bones milling templates were created and used in the transplant process. Cone beam CT scans of the bones were taken. The articular surface of the condyle was matched with the femoral head in the Mimics Inprint 3D Software (Materialise, Belgium) (Figure 1). Templates (Figure 2) were printed using a photopolymer jetting system (Formlabs, USA). For transplantation Surgical Diamond Instruments (BoneArtis, Switzerland) were used. Using CT scans, the fit of the subchondral bone plate of the transplant to the surrounding subchondral bone plate was measured at 0°, 90°, 180°, 270° of the transplant cylinder.

Results

Results The average gap between the subchondral bone plate of the transplant and the implant site was 0.27mm in the group with milling-templates and 0.73mm in the group without. We performed a t-test, showing that the milling template transplants outperformed the transplant without a template at a 1% significance level (p<0.01). Furthermore, milling templates displayed a 72% lower variance (σ² = 0.086) versus the transplants without a template (σ² = 0.312).

Conclusion

Conclusion The use of 3D printed milling-templates can improve the fit of the subchondral bone plate in autologous osteochondral transplantation and create a more replicable result throughout the series of surgeries, as displayed by the difference in variance.

Purpose

The overall goal of this study was to develop an ‘off-the-shelf’, bilayered scaffold for osteochondral defect repair that combines tissue-specific extracellular matrix (ECM) derived biomaterials to drive zonal repair with a tailored pore architecture to recapitulate the hierarchical collagen structure of articular cartilage (AC).

Methods and Materials

Freeze-dried anisotropic scaffolds were produced from solubilized ECM using a novel methodology through (i) modifying freeze-drying kinetics and (ii) controlling direction of heat transfer. 6x6mm osteochondral defects were created in the trochlear ridge of goats. Joints were assigned to one of the two groups: Empty control or AC-Bone ECM-derived bilayered scaffold. Tissue repair was evaluated at 6 months.

Results

In this study we developed a novel freeze-drying method which concurrently increased the mean pore size and aligned the pore orientation within the scaffolds. This fabrication method resulted in significant increases in glycosaminoglycan deposition by MSCs and enhanced collagen fiber alignment in the scaffolds (Fig 1A). We established that ECM biomaterials derived from solubilized bone were optimal for bone repair applications when compared to scaffolds derived from other musculoskeletal tissues in a subcutaneous mouse model (Fig 1B). Finally, we observed that implantation of an “off-the-shelf” bilayered ECM scaffold improved tissue repair outcomes in a caprine model (Fig 1C). The scaffold supported the development of a cartilage repair tissue that possessed high levels of GAG and type II collagen. Furthermore, the scaffold was found to support the development of a native AC-like collagen alignment through the depth of the repair tissue and to significantly reduce the collagen fibre dispersion in the superficial layer of AC to levels comparable with native tissue controls.

Conclusion

This scaffold technology represents a promising clinical option for the repair of osteochondral defects which could be used as a standalone scaffold or could be used in combination with autologous cells or growth factors to enhance repair.

Purpose

Focal knee resurfacing implants (FKRIs) are typically intended to treat cartilage defects in middle-aged patients. Most FKRIs are metal-based, implying potential stress-shielding, hampering follow-up using MR imaging and potentially leading to degeneration of the opposing articulating cartilage due to the large mismatch in elastic modulus. To overcome these drawbacks, a bi-layered non-degradable thermoplastic polycarbonate-urethane (TPU)-based FKRI is proposed. We hypothesized that by approaching the elastic modulus of cancellous bone and articular cartilage with a bi-layerd design, the implants would osseointegrate and induce less damage to the articulating cartilage when compared to metal-based implants. The purpose of this study was to evaluate the performance of this implant in a caprine model.

Methods and Materials

TPU-based, mushroom-shaped implants composed of a Bionate® II 80A top-layer and a BCP-coated Bionate® 75D/zirconium oxide (40/60 wt%) composite stem were injection moulded. Bi-layered metal implants (cobalt-chromium and titanium) and sham-operated knees served as control (n=8 per group). Surgery was performed bilaterally in the stifle joints of Dutch milk goats. After a follow-up of six months, the opposing cartilage was evaluated histologically using the Modified Mankin Score (MMS; 0-25). Bone histomorphometry was performed to assess osseointegration. Implant positioning was assed using laser scanning.

Results

The tibial cartilage articulating with the metal and TPU-based implants had a mean MMS of 14.79±3.45 and 10.31±1.89 respectively (p<0.01). The MMS of the metal implant group was significantly higher than the sham-operated group (p<0.001), while no significant difference was observed between the TPU-based implants and sham-operated knees (p=0.94). No significant difference in the bone-to-implant-contact was observed between the metal and TPU implant groups (p=0.72). No significant difference in implant positioning was observed.

Conclusion

TPU-based implants show promise as joint preserving implants with satisfactory osseointegration and less articulating cartilage damage when compared to metal implants. Longer follow-up studies are warranted to evaluate the long-term effects of TPU-based FKRIs.

Purpose

A major challenge in cartilage tissue engineering (TE) is to develop scaffolds capable of providing an instructive biomimetic environment to effectively drive mesenchymal stromal cells (MSCs) differentiation. Hydrogels have emerged as promising biomaterials for this purpose, due to their biocompatibility and ability to mimic the tissue extracellular matrix. Recently, graphene oxide (GO) emerged as a promising nanomaterial for cartilage TE due to chondroinductive properties when embedded into polymeric formulations. It has been also shown that piezoelectric nanomaterials, like barium titanate (BaTiO3) nanoparticles, can be exploited as nanoscale transducers capable of inducing cell growth/differentiation. Ultrasound waves are an interesting tool to facilitate chondrogenesis. In particular, it has been demonstrated that low-intensity pulsed ultrasound (LIPUS) regulates the differentiation of ASCs.

The aim of this study was to investigate whether dose-controlled LIPUS is able to influence chondrogenic differentiation of ASCs embedded in a 3D piezoelectric hydrogel.

Methods and Materials

ASCs at 2*10⁶ cells/mL were embedded in a 3D VitroGel RGD^® hydrogel with or without nanoparticles (GO, 25 µg/ml, BaTiO₃, 50 µg/ml) and subjected to LIPUS stimulation every 2 days, until day 10 of culture.

Hydrogels were cultured and chondrogenically differentiated for 2, 7, 10 and 28 days. At each time point cell viability, cytotoxicity, gene expression and immunohistochemistry (COL2, aggrecan, SOX9, and COL1) were evaluated.

Results

In both 3D hydrogels we evidenced that LIPUS treatment did not affect negatively the viability of the embedded cells. LIPUS boosted the chondrogenic differentiation of ASCs laden in 3D piezoelectric hydrogel: the chondrogenic genes and proteins markers (COL2,aggrecan and SOX9) were increased while the fibrotic marker COL1 was decreased compared to control samples (non piezoelectric hydrogels and piezoelectric hydrogels not stimulated with LIPUS).

Conclusion

These results suggest that the combination of LIPUS and 3D piezoelectric hydrogels push the differentiation of ASCs and represent a promising tool in the field of cartilage TE.

Purpose

Functional cartilage repair is hindered by the lack of integration between the cartilage graft and the surrounding cartilage. To improve integration, we designed a polylactide-co-glycolide (PLGA) and poly-ε-caprolactone (PCL) electrospun scaffold, which was previously shown to promote cell migration to the graft-tissue interface [1]. This study tests the hypothesis that IGF-1 delivery and electroactive graphite nanoparticle incorporation in the scaffold will enhance deposition of a cartilage-like matrix and improve graft-host integration.

Methods and Materials

Polymer meshes were electrospun [1] with or without graphite and/or IGF-1. In vitro: Primary bovine chondrocytes were cultured on the mesh and their response over 21 days was evaluated histologically (n=3) and biochemically (n=5). In vivo: A cartilage defect was created in a bovine osteochondral plug, and the extracted cartilage core was wrapped in a mesh and press-fit back into the defect. Samples were implanted subcutaneously in athymic rats for six weeks to assess cartilage repair through histology (n=3) and mechanical testing (n=8).

Results

In vitro results suggest IGF-1 and graphite increased neocartilaginous matrix deposition (Fig. 1). Preliminary results also support graphite’s immunomodulatory properties (data not shown). Moreover, all experimental groups promoted hyaline cartilage-like tissue formation with higher GAG and collagen deposition and increased type II collagen, compared to control (Fig. 2). Histologically, there was near-seamless integration in several of the samples treated with IGF-1 or graphite, with push-out testing confirming a significant improvement in integration strength.

Conclusion

IGF-1 is a known chondrocyte chemoattractant and enriches cell presence at the graft-host cartilage interface. The electroactive nature of graphite readily attracts negatively-charged adhesive proteins [2]; thus, these nanoparticles likely promote GAG retention and regulate processes of cartilage repair. These findings demonstrate the potential of this novel bioactive scaffold to regulate chondrocyte response at the nanoscale and improve graft-host cartilage integration.

References: [1] Boushell et al., 2019. [2] Hsiao et al., 2013.

Purpose

Intra-articular injection with biologicals like pro-catabolic cytokine neutralizing antibodies to treat osteoarthritis (OA) is challenging due to the short retention time of these biologicals in the joint space. To address this issue, we first attach the antibody fragments to polymer conjugates that can form an hydrogel by in situ cross linking upon injection in the intra-articular space. To test this concept we have employed the variable domain of heavy chain of single chain only antibody (VHHs) neutralizing TNFα for cytokine capture. The aim of this study is to provide a proof of this strategy aimed at creating a cytokine sink in the joint space.

Methods and Materials

In this study, we have selected a VHH that effectively neutralizes TNFα. Recombinant DNA technology was used to introduce an unpaired cysteine in the C-terminus of the VHH. Hyaluronic acid was functionalized with maleimide, needed for coupling of VHH, and tyramine residues, needed for hydrogel formation. Hydrogels functionalized with the VHH were prepared by mixing the hyaluronic acid-VHH-tyramine conjugates with the enzyme peroxidase and H2O2. Biological activity of the functionalized hydrogels was measured using an NFκb responsive luciferase reporter cell line after stimulation with TNFα.

Results

We show successful conjugation of the VHH to the polymer backbone via thiol-maleimide chemistry, while maintaining the binding affinity of the VHH against TNFα. The VHH functionalized polymer could be used for making stable hydrogels after tyramine mediated cross linking. Using an NF-κB luciferase reporter cell line we demonstrated that hydrogels functionalized with the VHH efficiently inactivated TNFα.

Conclusion

We successfully developed a biocompatible and efficient way to couple VHH to hyaluronic acid. These conjugates could be used for generation of cytokine sinks capable of capturing different pro-catabolic cytokines involved in OA after an intra-articular injection.