Evaluation of surgical fixation methods for the implantation of melt electrowriting-reinforced hyaluronic acid hydrogel composites in porcine cartilage defects.

The surgical repair of articular cartilage remains an ongoing challenge in orthopedics. Tissue engineering is a promising approach to treat cartilage defects; however, scaffolds must (i) possess the requisite material properties to support neocartilage formation, (ii) exhibit sufficient mechanical integrity for handling during implantation, and (iii) be reliably fixed within cartilage defects during surgery. In this study, we demonstrate the reinforcement of soft norbornene-modified hyaluronic acid (NorHA) hydrogels via the melt electrowriting (MEW) of polycaprolactone to fabricate composite scaffolds that support encapsulated porcine mesenchymal stromal cell (pMSC, three donors) chondrogenesis and cartilage formation and exhibit mechanical properties suitable for handling during implantation. Thereafter, acellular MEW-NorHA composites or MEW-NorHA composites with encapsulated pMSCs and precultured for 28 days were implanted in full-thickness cartilage defects in porcine knees using either bioresorbable pins or fibrin glue to assess surgical fixation methods. Fixation of composites with either biodegradable pins or fibrin glue ensured implant retention in most cases (80%); however, defects treated with pinned composites exhibited more subchondral bone remodeling and inferior cartilage repair, as evidenced by micro-computed tomography (micro-CT) and safranin O/fast green staining, respectively, when compared to defects treated with glued composites. Interestingly, no differences in repair tissue were observed between acellular and cellularized implants. Additional work is required to assess the full potential of these scaffolds for cartilage repair. However, these results suggest that future approaches for cartilage repair with MEW-reinforced hydrogels should be carefully evaluated with regard to their fixation approach for construct retention and surrounding cartilage tissue damage.

In Situ Assessment of Porcine Osteochondral Repair Tissue in the Visible-Near Infrared Spectral Region.

Standard assessment of cartilage repair progression by visual arthroscopy can be subjective and may result in suboptimal evaluation. Visible-near infrared (Vis-NIR) fiber optic spectroscopy of joint tissues, including articular cartilage and subchondral bone, provides an objective approach for quantitative assessment of tissue composition. Here, we applied this technique in the 350-2,500 nm spectral region to identify spectral markers of osteochondral tissue during repair with the overarching goal of developing a new approach to monitor repair of cartilage defects in vivo. Full thickness chondral defects were created in Yucatan minipigs using a 5-mm biopsy punch, and microfracture (MFx) was performed as a standard technique to facilitate repair. Tissues were evaluated at 1 month (in adult pigs) and 3 months (in juvenile pigs) post-surgery by spectroscopy and histology. After euthanasia, Vis-NIR spectra were collected in situ from the defect region. Additional spectroscopy experiments were carried out in vitro to aid in spectral interpretation. Osteochondral tissues were dissected from the joint and evaluated using the conventional International Cartilage Repair Society (ICRS) II histological scoring system, which showed lower scores for the 1-month than the 3-month repair tissues. In the visible spectral region, hemoglobin absorbances at 540 and 570 nm were significantly higher in spectra from 1-month repair tissue than 3-month repair tissue, indicating a reduction of blood in the more mature repair tissue. In the NIR region, we observed qualitative differences between the two groups in spectra taken from the defect, but differences did not reach significance. Furthermore, spectral data also indicated that the hydrated environment of the joint tissue may interfere with evaluation of tissue water absorbances in the NIR region. Together, these data provide support for further investigation of the visible spectral region for assessment of longitudinal repair of cartilage defects, which would enable assessment during routine arthroscopy, particularly in a hydrated environment.

Gravity-based patterning of osteogenic factors to preserve bone structure after osteochondral injury in a large animal model.

Chondral and osteochondral repair strategies are limited by adverse bony changes that occur after injury. Bone resorption can cause entire scaffolds, engineered tissues, or even endogenous repair tissues to subside below the cartilage surface. To address this translational issue, we fabricated thick-shelled poly(D,L-lactide-co-glycolide) microcapsules containing the pro-osteogenic agents triiodothyronine andß-glycerophosphate, and delivered these microcapsules in a large animal model of osteochondral injury to preserve bone structure. We demonstrate that the developed microcapsules rupturedin vitrounder increasing mechanical loads, and readily sink within a liquid solution, enabling gravity-based patterning along the osteochondral surface. In a large animal, these mechanically-activated microcapsules (MAMCs) were assessed through two different delivery strategies. Intra-articular injection of control MAMCs enabled fluorescent quantification of MAMC rupture and cargo release in a synovial joint setting over timein vivo. This joint-wide injection also confirmed that the MAMCs do not elicit an inflammatory response. In the contralateral hindlimbs, chondral defects were created, MAMCs were patternedin situ, and nanofracture (Nfx), a clinically utilized method to promote cartilage repair, was performed. The Nfx holes enabled marrow-derived stromal cells to enter the defect area and served as repeatable bone injury sites to monitor over time. Animals were evaluated one and two weeks after injection and surgery. Analysis of injected MAMCs showed that bioactive cargo was released in a controlled fashion over two weeks. A bone fluorochrome label injected at the time of surgery displayed maintenance of mineral labeling in the therapeutic group, but resorption in both control groups. Alkaline phosphatase (AP) staining at the osteochondral interface revealed higher AP activity in defects treated with therapeutic MAMCs. Overall, this study develops a gravity-based approach to pattern bioactive factors along the osteochondral interface, and applies this novel biofabrication strategy to preserve bone structure after osteochondral injury.

Optimized Media Volumes Enable Homogeneous Growth of Mesenchymal Stem Cell-Based Engineered Cartilage Constructs.

Despite marked advances in the field of cartilage tissue engineering, it remains a challenge to engineer cartilage constructs with homogeneous properties. Moreover, for engineered cartilage to make it to the clinic, this homogeneous growth must occur in a time-efficient manner. In this study we investigated the potential of increased media volume to expedite the homogeneous maturation of mesenchymal stem cell (MSC) laden engineered constructs over time in vitro. We assessed the MSC-laden constructs after 4 and 8 weeks of chondrogenic culture using bulk mechanical, histological, and biochemical measures. These assays were performed on both the intact total constructs and the construct cores to elucidate region-dependent differences. In addition, local strain transfer was assessed to quantify depth-dependent mechanical properties throughout the constructs. Our findings suggest that increased media volume enhances matrix deposition early in culture and ameliorates unwanted regional heterogeneities at later time points. Taken together, these data support the use of higher media volumes during in vitro culture to hasten tissue maturation and increase the core strength of tissue constructs. These findings will forward the field of cartilage tissue engineering and the translation of tissue engineered constructs.

Fabrication and maturation of integrated biphasic anatomic mesenchymal stromal cell-laden composite scaffolds for osteochondral repair and joint resurfacing.

Articular cartilage injury can lead to joint-wide erosion and the early onset of osteoarthritis. To address this, we recently developed a rapid fabrication method to produce patient-specific engineered cartilage tissues to replace an entire articular surface. Here, we extended that work by coupling a mesenchymal stromal cell-laden hydrogel (methacrylated hyaluronic acid) with the porous polycaprolactone (PCL) bone integrating phase and assessed the composition and mechanical performance of these constructs over time. To improve initial construct stability, PCL/hydrogel interface parameters were first optimized by varying PCL pretreatment (with sodium hydroxide before ethanol) before hydrogel infusion. Next, cylindrical osteochondral constructs were formed and cultured in media containing transforming growth factor ß3 for up to 8 weeks, with constructs evaluated for viability, histological features, and biochemical content. Mechanical properties were also assessed in axial compression and via an interface shear strength assay. Results showed that the fabrication process was compatible with cell viability, and that construct biochemical content and mechanical properties increased with time. Interestingly, compressive properties peaked at 5 weeks, while interfacial shear properties continued to improve beyond this time point. Finally, these fabrication methods were combined with a custom mold developed from limb-specific computed tomography imaging data to create an anatomic implantable cell-seeded biologic joint surface, which showedmaturation similar to the osteochondral cylinders. Future work will apply these advances in large animal models of critically sized osteochondral defects to study repair and whole joint resurfacing.

Emerging therapies for cartilage regeneration in currently excluded 'red knee' populations.

The field of articular cartilage repair has made significant advances in recent decades; yet current therapies are generally not evaluated or tested, at the time of pivotal trial, in patients with a variety of common comorbidities. To that end, we systematically reviewed cartilage repair clinical trials to identify common exclusion criteria and reviewed the literature to identify emerging regenerative approaches that are poised to overcome these current exclusion criteria. The term "knee cartilage repair" was searched on clinicaltrials.gov. Of the 60 trials identified on initial search, 33 were further examined to extract exclusion criteria. Criteria excluded by more than half of the trials were identified in order to focus discussion on emerging regenerative strategies that might address these concerns. These criteria included age (<18 or >55 years old), small defects (<1 cm2), large defects (>8 cm2), multiple defect (>2 lesions), BMI >35, meniscectomy (>50%), bilateral knee pathology, ligamentous instability, arthritis, malalignment, prior repair, kissing lesions, neurologic disease of lower extremities, inflammation, infection, endocrine or metabolic disease, drug or alcohol abuse, pregnancy, and history of cancer. Finally, we describe emerging tissue engineering and regenerative approaches that might foster cartilage repair in these challenging environments. The identified criteria exclude a majority of the affected population from treatment, and thus greater focus must be placed on these emerging cartilage regeneration techniques to treat patients with the challenging "red knee".