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.

Linguistic Analysis Identifies Emergent Biomaterial Fabrication Trends for Orthopaedic Applications.

The expanse of publications in tissue engineering (TE) and orthopedic TE (OTE) over the past 20 years presents an opportunity to probe emergent trends in the field to better guide future technologies that can make an impact on musculoskeletal therapies. Leveraging this trove of knowledge, a hierarchical systematic search method and trend analysis using connected network mapping of key terms is developed. Within discrete time intervals, an accelerated publication rate for anatomic orthopedic tissue engineering (AOTE) of osteochondral defects, tendons, menisci, and entheses is identified. Within these growing fields, the top-listed key terms are extracted and stratified into evident categories, such as biomaterials, delivery method, or 3D printing and biofabrication. It is then identified which categories decreased, remained constant, increased, or emerged over time, identifying the specific emergent categories currently driving innovation in orthopedic repair technologies. Together, these data demonstrate a significant convergence of material types and descriptors used across tissue types. From this convergence, design criteria to support future research of anatomic constructs that mimic both the form and function of native tissues are formulated. In summary, this review identifies large-scale trends and predicts new directions in orthopedics that will define future materials and technologies.

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.

Six-Month Outcomes of Clinically Relevant Meniscal Injury in a Large-Animal Model.

BACKGROUND: The corrective procedures for meniscal injury are dependent on tear type, severity, and location. Vertical longitudinal tears are common in young and active individuals, but their natural progression and impact on osteoarthritis (OA) development are not known. Root tears are challenging and they often indicate poor outcomes, although the timing and mechanisms of initiation of joint dysfunction are poorly understood, particularly in large-animal and human models. PURPOSE/HYPOTHESIS: In this study, vertical longitudinal and root tears were made in a large-animal model to determine the progression of joint-wide dysfunction. We hypothesized that OA onset and progression would depend on the extent of injury-based load disruption in the tissue, such that root tears would cause earlier and more severe changes to the joint. STUDY DESIGN: Controlled laboratory study. METHODS: Sham surgeries and procedures to create either vertical longitudinal or root tears were performed in juvenile Yucatan mini pigs through randomized and bilateral arthroscopic procedures. Animals were sacrificed at 1, 3, or 6 months after injury and assessed at the joint and tissue level for evidence of OA. Functional measures of joint load transfer, cartilage indentation mechanics, and meniscal tensile properties were performed, as well as histological evaluation of the cartilage, meniscus, and synovium. RESULTS: Outcomes suggested a progressive and sustained degeneration of the knee joint and meniscus after root tear, as evidenced by histological analysis of the cartilage and meniscus. This occurred in spite of spontaneous reattachment of the root, suggesting that this reattachment did not fully restore the function of the native attachment. In contrast, the vertical longitudinal tear did not cause significant changes to the joint, with only mild differences compared with sham surgery at the 6-month time point. CONCLUSION: Given that the root tear, which severs circumferential connectivity and load transfer, caused more intense OA compared with the circumferentially stable vertical longitudinal tear, our findings suggest that without timely and mechanically competent fixation, root tears may cause irreversible joint damage. CLINICAL RELEVANCE: More generally, this new model can serve as a test bed for experimental surgical, scaffold-based, and small molecule-driven interventions after injury to prevent OA progression.

Fabrication of MSC-laden composites of hyaluronic acid hydrogels reinforced with MEW scaffolds for cartilage repair.

Hydrogels are of interest in cartilage tissue engineering due to their ability to support the encapsulation and chondrogenesis of mesenchymal stromal cells (MSCs). However, features such as hydrogel crosslink density, which can influence nutrient transport, nascent matrix distribution, and the stability of constructs during and after implantation must be considered in hydrogel design. Here, we first demonstrate that more loosely crosslinked (i.e. softer, ∼2 kPa) norbornene-modified hyaluronic acid (NorHA) hydrogels support enhanced cartilage formation and maturation when compared to more densely crosslinked (i.e. stiffer, ∼6-60 kPa) hydrogels, with a >100-fold increase in compressive modulus after 56 d of culture. While soft NorHA hydrogels mature into neocartilage suitable for the repair of articular cartilage, their initial moduli are too low for handling and they do not exhibit the requisite stability needed to withstand the loading environments of articulating joints. To address this, we reinforced NorHA hydrogels with polycaprolactone (PCL) microfibers produced via melt-electrowriting (MEW). Importantly, composites fabricated with MEW meshes of 400µm spacing increased the moduli of soft NorHA hydrogels by ∼50-fold while preserving the chondrogenic potential of the hydrogels. There were minimal differences in chondrogenic gene expression and biochemical content (e.g. DNA, GAG, collagen) between hydrogels alone and composites, whereas the composites increased in compressive modulus to ∼350 kPa after 56 d of culture. Lastly, integration of composites with native tissue was assessedex vivo; MSC-laden composites implanted after 28 d of pre-culture exhibited increased integration strengths and contact areas compared to acellular composites. This approach has great potential towards the design of cell-laden implants that possess both initial mechanical integrity and the ability to support neocartilage formation and integration for cartilage repair.

The porcine accessory carpal bone as a model for biologic joint replacement for trapeziometacarpal osteoarthritis.

Given its complex shape and relatively small size, the trapezium surface at the trapeziometacarpal (TMC) joint is a particularly attractive target for anatomic biologic joint resurfacing, especially given its propensity to develop osteoarthritis, and the limited and sub-optimal treatment options available. For this to advance to clinical translation, however, an appropriate large animal model is required. In this study, we explored the porcine accessory carpal bone (ACB) as a model for the human trapezium. We characterized ACB anatomy, geometry, joint and tissue-scale mechanics, and composition across multiple donors. We showed that the ACB is similar both in size, and in the saddle shape of the main articulating surface to the human trapezium, and that loads experienced across each joint are similar. Using this information, we then devised a fabrication method and workflow to produce patient-specific tissue-engineered replicas based on CT scans, and showed that when such replicas are implanted orthotopically in an ex vivo model, normal loading is restored. Data from this study establish the porcine ACB as a model system in which to evaluate function of engineered living joint resurfacing strategies. STATEMENT OF SIGNIFICANCE: Biologic joint resurfacing, or the replacement of a joint with living tissue as opposed to metal and plastic, is the holy grail of orthopaedic tissue engineering. However, despite marked advances in engineering native-like osteochondral tissues and in matching patient-specific anatomy, these technologies have not yet reached clinical translation. Given its propensity for developing osteoarthritis, as well as its small size and complex shape, the trapezial surface of the trapeziometacarpal joint at the base of the thumb presents a unique opportunity for pursuing a biologic joint resurfacing strategy. This work establishes the porcine accessory carpal bone as an animal model for the human trapezium and presents a viable test-bed for evaluating the function of engineered living joint resurfacing strategies.

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.

Transection of the medial meniscus anterior horn results in cartilage degeneration and meniscus remodeling in a large animal model.

The meniscus plays a central load-bearing role in the knee joint. Unfortunately, meniscus injury is common and can lead to joint degeneration and osteoarthritis (OA). In small animal models, progressive degenerative changes occur with the unloading of the meniscus via destabilization of the medial meniscus (DMM). However, few large animal models of DMM exist and the joint-wide initiation of the disease has not yet been defined in these models. Thus, the goal of this study is to develop and validate a large animal model of surgically induced DMM and to use multimodal (mechanical, histological, and magnetic resonance imaging) and multiscale (joint to tissue level) quantitative measures to evaluate degeneration in both the meniscus and cartilage. DMM was achieved using an arthroscopic approach in 13 Yucatan minipigs. One month after DMM, joint contact area decreased and peak pressure increased, indicating altered load transmission as a result of meniscus destabilization. By 3 months, the joint had adapted to the injury and load transmission patterns were restored to baseline, likely due to the formation and maturation of a fibrovascular scar at the anterior aspect of the meniscus. Despite this, we found a decrease in the indentation modulus of the tibial cartilage and an increase in cartilage histopathology scores at 1 month compared to sham-operated animals; these deleterious changes persisted through 3 months. Over this same time course, meniscus remodeling was evident through decreased proteoglycan staining in DMM compared to sham menisci at both 1 and 3 months. These findings support that arthroscopic DMM results in joint degeneration in the Yucatan minipig and provide a new large animal testbed in which to evaluate therapeutics and interventions to treat post-traumatic OA that originates from a meniscal injury.

Measuring clinically relevant knee motion with a self-calibrated wearable sensor.

Low-cost sensors provide a unique opportunity to continuously monitor patient progress during rehabilitation; however, these sensors have yet to demonstrate the fidelity and lack the calibration paradigms necessary to be viable tools for clinical research. The purpose of this study was to validate a low-cost wearable sensor that accurately measured peak knee extension during clinical exercises and needed no additional equipment for calibration. Sagittal plane knee motion was quantified using a 9-axis motion sensor and directly compared to motion capture data. The motion sensor measured the field strength of a strong earth magnet secured to the distal femur, which was correlated with knee angle during a simple calibration process. Peak knee motions and kinematic patterns were compared with motion capture data using paired t-tests and cross correlation, respectively. Peak extension values during seated knee extensions were accurate within 5 degrees across all subjects (root mean square error: 2.6 degrees, P = 0.29). Knee flexion during gait strongly correlated (0.84 ≤ rxy ≤ 0.99) with motion capture measurements but demonstrated peak flexion errors of 10 degrees. In this study, we present a low-cost sensor (≈$ 35 US) that accurately determines knee extension angle following a calibration procedure that did not require any other equipment. Our findings demonstrate that this sensor paradigm is a feasible tool to monitor patient progress throughout physical therapy. However, dynamic motions that are associated with soft-tissue artifact may limit the accuracy of this type of wearable sensor.

A Wearable Magnet-Based System to Assess Activity and Joint Flexion in Humans and Large Animals.

Functional outcomes, such as joint flexion and gait, are important indicators of efficacy in musculoskeletal research. Current technologies that objectively assess these parameters, including visual tracking systems and force plates, are challenging to deploy in long-term translational and clinical studies. To that end, we developed a wearable device that measures both physical activity and joint flexion using a single integrated sensor and magnet system, and hypothesized that it could evaluate post-operative functional recovery in an unsupervised setting. To demonstrate the feasibility of measuring joint flexion, we first compared knee motion from the wearable device to that acquired from a motion capture system to confirm that knee flexion measurements during normal human gait, predicted via changes in magnetic field strength, closely correlated with data acquired by motion capture. Using this system, we then monitored a porcine cohort after bilateral stifle arthrotomy to investigate longitudinal changes in physical activity and joint flexion. We found that unsupervised activity declined immediately after surgery, with a return to pre-operative activity occurring over a period of 2 weeks. By providing objective, individualized data on locomotion and joint function, this magnet-based system will facilitate the in vivo assessment of novel therapeutics in translational orthopaedic research.

Biphasic Finite Element Modeling Reconciles Mechanical Properties of Tissue-Engineered Cartilage Constructs Across Testing Platforms.

Cartilage tissue engineering is emerging as a promising treatment for osteoarthritis, and the field has progressed toward utilizing large animal models for proof of concept and preclinical studies. Mechanical testing of the regenerative tissue is an essential outcome for functional evaluation. However, testing modalities and constitutive frameworks used to evaluate in vitro grown samples differ substantially from those used to evaluate in vivo derived samples. To address this, we developed finite element (FE) models (using FEBio) of unconfined compression and indentation testing, modalities commonly used for such samples. We determined the model sensitivity to tissue radius and subchondral bone modulus, as well as its ability to estimate material parameters using the built-in parameter optimization tool in FEBio. We then sequentially tested agarose gels of 4%, 6%, 8%, and 10% weight/weight using a custom indentation platform, followed by unconfined compression. Similarly, we evaluated the ability of the model to generate material parameters for living constructs by evaluating engineered cartilage. Juvenile bovine mesenchymal stem cells were seeded (2 × 107 cells/mL) in 1% weight/volume hyaluronic acid hydrogels and cultured in a chondrogenic medium for 3, 6, and 9 weeks. Samples were planed and tested sequentially in indentation and unconfined compression. The model successfully completed parameter optimization routines for each testing modality for both acellular and cell-based constructs. Traditional outcome measures and the FE-derived outcomes showed significant changes in material properties during the maturation of engineered cartilage tissue, capturing dynamic changes in functional tissue mechanics. These outcomes were significantly correlated with one another, establishing this FE modeling approach as a singular method for the evaluation of functional engineered and native tissue regeneration, both in vitro and in vivo.

Anatomic Mesenchymal Stem Cell-Based Engineered Cartilage Constructs for Biologic Total Joint Replacement.

Cartilage has a poor healing response, and few viable options exist for repair of extensive damage. Hyaluronic acid (HA) hydrogels seeded with mesenchymal stem cells (MSCs) polymerized through UV crosslinking can generate functional tissue, but this crosslinking is not compatible with indirect rapid prototyping utilizing opaque anatomic molds. Methacrylate-modified polymers can also be chemically crosslinked in a cytocompatible manner using ammonium persulfate (APS) and N,N,N',N'-tetramethylethylenediamine (TEMED). The objectives of this study were to (1) compare APS/TEMED crosslinking with UV crosslinking in terms of functional maturation of MSC-seeded HA hydrogels; (2) generate an anatomic mold of a complex joint surface through rapid prototyping; and (3) grow anatomic MSC-seeded HA hydrogel constructs using this alternative crosslinking method. Juvenile bovine MSCs were suspended in methacrylated HA (MeHA) and crosslinked either through UV polymerization or chemically with APS/TEMED to generate cylindrical constructs. Minipig porcine femoral heads were imaged using microCT, and anatomic negative molds were generated by three-dimensional printing using fused deposition modeling. Molded HA constructs were produced using the APS/TEMED method. All constructs were cultured for up to 12 weeks in a chemically defined medium supplemented with TGF-ß3 and characterized by mechanical testing, biochemical assays, and histologic analysis. Both UV- and APS/TEMED-polymerized constructs showed increasing mechanical properties and robust proteoglycan and collagen deposition over time. At 12 weeks, APS/TEMED-polymerized constructs had higher equilibrium and dynamic moduli than UV-polymerized constructs, with no differences in proteoglycan or collagen content. Molded HA constructs retained their hemispherical shape in culture and demonstrated increasing mechanical properties and proteoglycan and collagen deposition, especially at the edges compared to the center of these larger constructs. Immunohistochemistry showed abundant collagen type II staining and little collagen type I staining. APS/TEMED crosslinking can be used to produce MSC-seeded HA-based neocartilage and can be used in combination with rapid prototyping techniques to generate anatomic MSC-seeded HA constructs for use in filling large and anatomically complex chondral defects or for biologic joint replacement.