Lawrence J. Bonassar

Abstract Differentiating mesenchymal stromal cells into healthy chondrocytes for articular cartilage (AC) requires a variety of biochemical and biomechanical stimuli. While the effects of such stimuli on chondrogenic markers are well-studied, no bioreactor has thus far collected real-time data from mRNA expression for use in closed-loop control. Here, we present the design of an automated bioreactor that applies perfusion, oscillating hydrostatic pressure (OHP), and varying concentrations of multiple biochemical growth factors to the cell cultures while measuring fluorescent markers of gene expression in real-time. Our system achieves a gradated hydrodynamic environment across the length of cell cultures with flow rates between 0.46 and 3.2 cm s −1 and shear stresses between 5.0 and 60 mPa, can apply OHP at up to 0.5 Hz and 4.83 MPa, and can dynamically control the concentrations of two biochemical growth factors. The integrated fluorescent fiberscope system can measure fluorescent dye concentrations as low as 3 nm within the cell chamber. Our LNA/DNA nano-biosensor is designed to track Sox9 mRNA, a gene critical to the chondrogenic process. The reactor is controlled via MATLAB Simulink and includes remote observation and control features to increase flexibility and minimize downtime. This novel bioreactor provides a platform for further AC research with the ultimate design goal of generating a transfer function that maps growth factor inputs to mRNA expression outputs for real-time control.

ABSTRACT Collagen type II (Col-II) and collagen type I (Col-I) are major components of articular cartilage present at different ratios at its surface. Understanding how each of these components mediates the assembly of molecular films derived from synovial fluid (SF), the native lubricant of synovial joints, is critical to explain the loss of mechanical performance in pathological conditions, guide the design of biomaterial implants meant to be in contact with SF, and develop molecular therapies to restore SF properties. This work demonstrates that Col-II articular surface model assists in scaffolding of full SF-derived films, while Col-I model lacks SF film scaffolding capabilities. However, when Col-II and Col-I are exposed to recombinant lubricin (rLub) alone, the major boundary lubricant in SF, both adsorbed and retained similar amounts. These insights, deduced from quartz crystal microbalance with dissipation, diffuse reflectance circular dichroism, and atomic force microscopy, reveal possible mechanisms underlying the loss of mechanical performance of synovial joints in pathology, where Col-I becomes the major collagenous component of the articular cartilage surface, as well as considerations for designing functional biomaterial implants. Furthermore, this work reinforces the idea of rLub as an intra-articular osteoarthritis therapy with the ability to bind to Col-II and Col-I, irrespectively. GRAPHICAL ABSTRACT

Lumbar microdiscectomy improves pain and disability but does not restore disc biology, and same-site recurrence can occur. Intradiscal autologous bone marrow aspirate concentrate (BMAC) has growing preclinical support, but human safety data are limited. To evaluate intradiscal BMAC safety and feasibility at lumbar microdiscectomy. Patients underwent lumbar microdiscectomy and 1 mL intradiscal autologous BMAC with 1-year follow-up. Primary outcomes were procedure-related complications, readmissions, reoperations, and same-site recurrence; secondary outcomes were Oswestry Disability Index (ODI) and Numeric Rating Scale (NRS) for leg and back pain; exploratory quantitative magnetic resonance imaging (MRI; T 1 -rho, T 2 ) screened for adverse compositional changes. 27 adults underwent lumbar microdiscectomy with concurrent intradiscal BMAC injections. No intraoperative or postoperative injection-related complications occurred. Same-site recurrence occurred in 1/27 (3.7%) and was managed without reoperation. Patient-reported outcome measures (PROMs) were completed by 18/27 (67%), and qMRI was obtained in 16, including 6 with complete imaging through 1 year. Among patients with completed PROMs, mean ODI, NRS-leg, and NRS-back improved at 1 year, but the uncontrolled design precludes efficacy inference. No adverse changes in T 1 -rho or T 2 were observed through 1 year. In this single-center prospective pilot, intradiscal BMAC at the time of microdiscectomy was safe and feasible, with no injection-related harm through 1 year and one same-site recurrence. Without a control group and with incomplete follow-up, findings are preliminary safety data, not evidence of efficacy or reduced recurrence risk. These results support a multicenter study with longer follow-up and a controlled comparison group.

Despite the growing prevalence of post-traumatic osteoarthritis (OA), early and effective treatment options to delay surgical intervention remain limited. Lubricin is a naturally occurring glycoprotein produced by articular chondrocytes and synovial fibroblasts that functions as the principal boundary lubricant in joints. Recently, a recombinant lubricin-like glycoprotein (rhLub) with codon optimized mucin domain was reported to have a sustained residence time (> 6 weeks) following injection into the knee joint of healthy rats. To evaluate outcomes of rhLub injection, skeletally mature Sprague-Dawley rats of both sexes (n = 18) underwent bilateral anterior cruciate ligament transection (ACLT) to initiate post-traumatic OA, followed by three intra-articular injections of 25 µL of rhLub (1.3 mg/mL) or phosphate-buffered saline (PBS) at weekly intervals beginning 1 week post-operatively. Pain sensitization was evaluated weekly throughout the 12-week study with weightbearing and pressure application measurement (PAM). Frontal plane histologic sections of the medial femorotibial joint compartment were scored using Osteoarthritis Research Society International criteria. Injections of rhLub were well-tolerated. rhLub injections protected against cartilage degeneration and improved histologic scores in male rats. Mechanical hyperalgesia and cartilage degeneration scores were greater in female rats compared to males, irrespective of treatment. This study suggests that intra-articular rhLub therapy may be most efficacious in mild-to-moderate disease.

Traumatic injury to synovial joints results in focal defects and proinflammatory changes in cartilage tissue, eventually leading to post-traumatic osteoarthritis (PTOA). Additionally, disparate incidence rates of PTOA occur between joints, particularly the knee and ankle. We hypothesize that differences in PTOA rates between knee and ankle cartilage are related to inherent differences in mechanical properties and chondrocyte sensitivity to mechanical loads. Our objective was to compare the effect of injury on the spatial patterns of cellular response between knee and ankle cartilage and relate these responses to changes in tissue mechanical properties. Cartilage explants of neonatal bovids from the talar dome of the ankle and femoral condyle of the knee were subjected to our ex vivo combined injury model which combines rapid impact injury and repetitive cartilage articulation. Explants were bisected and fluorescently stained to assess global and depth-dependent cell death, caspase activity, and mitochondrial depolarization. Explants were tested via confocal elastography to determine the local shear strain and shear modulus profile. Results showed that chondrocyte response differed between joints, with knee cartilage showing greater damage within the surface region. Additionally, ankle cartilage experienced a greater decrease in shear modulus post-injury compared to knee, causing depth-dependent shear strain to significantly increase (p < 0.0287). Furthermore, regression analyses relating cellular response to local shear strain revealed joint-specific differences in chondrocyte sensitivity, defined as the slope of cellular response versus shear strain, for cell death (knee: 873 vs ankle: 551), apoptosis (knee: 7779 vs ankle: 3577), and MT depolarization (knee: 848 vs ankle: 535).

Articular cartilage is a load-bearing, hierarchically organized tissue composed of a network of type II collagen embedded in an aggrecan-rich polyelectrolyte gel. Its ability to resist deformation and dissipate energy arises from spatially varying matrix composition and architecture. Here, we review experimental and theoretical advances that elucidate the mechanistic basis of cartilage shear mechanics. Recent studies have shown that the tissue operates near a rigidity transition, in which small changes in collagen density, cross-linking, or osmotic stress can produce large, nonlinear changes in shear stiffness. We discuss how this behavior is captured by models rooted in rigidity percolation, continuum elasticity, and micromechanics, and how these frameworks connect depth-dependent composition to macroscale mechanical response. Throughout, we emphasize physical principles that describe observations across native, degraded, and engineered tissues, and we highlight emerging strategies for designing cartilage-inspired materials with tunable, anisotropic mechanics, with applications in soft robotics, synthetic gels, and load-bearing biomaterials.

The incorporation of robust meniscus-to-bone interfaces into tissue-engineered menisci is critical for their clinical translation. Generating gradients in collagen fiber organization and mineral content for tissue-engineered entheses is essential for achieving native tissue-like mechanics; however, engineering such gradients remains challenging. This study presents a tissue-engineered enthesis model consisting of a fibrochondrocyte-seeded cylinder of type I collagen gel with trabecular bone plugs on both ends. Using a tri-chamber bioreactor, spatially controlled biochemical (e.g., TGF-β1 and glucose) and biomechanical stimuli were applied, generating native-like collagen fiber structure and mechanics within tissue-engineered enthesis constructs. Confocal elastography revealed a more uniform local strain distribution and reduced peak strain in the enthesis constructs cultured in a tri-chamber bioreactor compared to those in a single chamber bioreactor, likely attributed to the enhanced collagen fiber organization. To further improve the integration at the collagen-bone plug interface, we introduced partially demineralized bone plugs to the constructs. Partial demineralization improved the mechanical performance of enthesis constructs, decreasing peak strain by > 30% and strain gradients by 50%, while increasing toughness and strain at failure by 50% and 40%, respectively. Overall, these findings highlight the importance of zone-specific biochemical and biomechanical stimuli and biomimetic scaffold materials to improve tissue-engineered implants.

Viscosupplementation is one of the most common forms of osteoarthritis (OA) therapy. It involves injecting a lubricant, typically hyaluronic acid (HA), into the joint with the goal of restoring the synovial fluid viscosity. Interestingly, the clinical performance of viscosupplements is more strongly correlated with their effective viscosity than their bulk viscosity. Effective viscosity is a mathematical construct used to reconcile a viscosupplement's material properties with its lubrication performance. Previous findings have hinted that effective viscosity is more than a theoretical concept, and may reflect a physical phenomenon mediated by lubricin at the articular surface. However, this had not yet been experimentally demonstrated. Therefore, this study: a) used microrheology to measure localized increases in the viscosity of HA near the articular surface; and b) assessed the role of lubricin in mediating this effect. To investigate this, nanoscale fluorescent beads were suspended in HA solutions, and bead Brownian motion was analyzed at various distances from the articular surface of lubricin-intact and lubricin-removed cartilage explants. This study found that HA exhibits 38 % increased viscosity at the articular surface of lubricin-intact cartilage compared to lubricin-removed cartilage, and overnight incubation in synovial fluid restored the native viscosity distribution. Elevated viscosity persisted up to 50 μm from the surface, a length-scale consistent with earlier studies. This is the first study to directly visualize localized changes in the viscosity of HA near the articular surface of cartilage resulting from lubricin-HA interactions, providing compelling evidence that effective viscosity reflects a physical phenomenon that can be measured and observed.

cell construct holds promise for advancing cartilage repair efforts. Our approach involves the development of a multichambered perfusion tissue bioreactor that regulates fluid shear stress levels similar to the gradated hydrodynamic environment in articular cartilage. COMSOL modeling reveals our tapered cell chamber design will produce three different shear levels, high in the 22-41 mPa range, medium in the 4.5-8.4 mPa range, and low in the 2.2-3.8 mPa range and distributed across the surface of our mesenchymal stromal cell (MSC) encapsulated construct. In a 14-day bioreactor culture, we assess how the fluid shear magnitude and cell vertical location within a 3D construct influence cell chondrogenesis. Notably, Sox9 expression for MSCs cultivated in our reactor shows spatially patterned gene upregulations that encode key chondrogenic marker proteins. Beginning with the high shear stress region, lubricin and type II collagen gene increases of 410- and 370-fold indicate cell movement toward a superficial zone archetype, which is further supported by histological and immunohistochemical stains illustrating the formation of a dense proteoglycan matrix enriched with lubricin, versican, and collagen types I and II molecules. For the medium shear stress region, high aggrecan and type II collagen gene expressions of 2.3- and 400-fold, respectively, along with high proteoglycan analysis, show movement toward a superficial/midzone cartilage archetype. For low shear stress regions, higher collagen type II and X gene upregulations of 550- and 8300-fold, the latter being 2× of that for the high shear regime, indicate cell movement toward deep zone characteristics. Collectively, biochemical analysis, histology, and gene expression data demonstrate that our fluid shear bioreactor induces formation of a stratified structure within tissue-engineered constructs, demonstrating the feasibility of using this approach to recapitulate the structure of native articular cartilage.

The use of bioprinters as depositional tools for bioinks and cells has expanded greatly over the past two decades. Bioprinting combines hydrogels with cells to produce customized constructs for personalized medicine. However, several challenges hinder the clinical use of these constructs. Quality control metrics for bioprinting involve the assessment of critical quality attributes at every stage of production. Currently, bioprinted constructs are manually assessed using destructive methods that occur post-production, requiring the creation of multiple products per patient. Reproducing printed constructs is difficult due to time-sensitive bioink properties that require lengthy optimization processes to print with accuracy. In addition, the collection, processing, and testing of cell bioactivity for each printed construct greatly increases production costs. To address these challenges, non-destructive, real-time monitoring can be integrated into the bioprinting process. The goal of this review paper is to reimagine the function of a bioprinter from a simple tool of production to one capable of evaluating constructs in real-time. This review features recent advances in the field for real-time monitoring with a focus on time-sensitive bioink properties, print accuracy, and cell health. Automated assessment and quantification of time-sensitive bioink qualities such as mixing, pH, temperature, and viscosity will enhance construct quality by enabling the rapid optimization of printing parameters. Meanwhile, real-time monitoring of cell health through concentration, viability, and type serves as an indicator for bioactivity. Construct accuracy and reproducibility are also improved through the identification, prediction, and correction of defects during printing. Incorporating real-time monitoring into the bioprinting process using closed-loop feedback would improve the reproducibility, quality, and translation of constructs into the clinic.

Osteochondral lesions (OCL) are common among young patients and often require surgical interventions since cartilage has a poor capacity for self-repair. Bone marrow stimulation (BMS) has been used clinically for decades to treat OCLs, however a persisting challenge with BMS and other cartilage repair strategies is the inferior quality of the resulting fibrocartilaginous repair tissue. Lubrication-based therapies have the potential to improve the quality of cartilage repair tissue as joint lubrication is linked to local cartilage tissue strains and subsequent cellular responses including death and apoptosis. Recently, a full length recombinant human lubricin (rhLubricin) was developed and has been shown to lower friction in cartilage. This study investigated the effect of a single delayed injection of rhLubricin on cartilage repair in an in vivo rabbit OCL model using gross macroscopic evaluation, surface profilometry, histology, and tribology. Moderate improvement in macroscopic scores for cartilage repair were observed. Notably, quantitative analysis of Safranin-O histology showed that rhLubricin treated joints had significantly higher glycosaminoglycan content compared to saline treated joints, and there were no differences in repair integration between groups. Furthermore, rhLubricin treated joints had significantly lower friction coefficients tested across three sliding speeds compared to saline treated joints (rhLubricin: 0.15 ± 0.03 at 0.1 mm/s to 0.12 ± 0.03 at 10 mm/s, Saline: 0.22 ± 0.06 at 0.1 mm/s to 0.19 ± 0.05 at 10 mm/s). Overall, a single delayed injection of rhLubricin improved the quality and lubricating ability of the repair cartilage tissue without inhibiting repair tissue integration.

Patients with cartilage defects often experience increased meniscal degeneration. It remains unclear whether meniscal damage occurs concurrently with cartilage injury or due to later joint pathology. Limited data exists on how isolated cartilage injuries affect meniscal structure and degeneration. In osteoarthritis models, alterations to the structure and composition of meniscal ECM components have been observed, including meniscus hypertrophy characterized by excessive glycosaminoglycan deposition and fibrochondrocyte rounding. Although proteoglycan deposition increases in early OA, the timing of GAG changes relative to collagen disruption remains unclear. This study examined the correlation between changes in local proteoglycan deposition, cell morphology, and the collagen network in the meniscus following cartilage damage using an in vivo rabbit model. A medial osteochondral defect was created on the femoral condyle of New Zealand white male rabbits, and menisci were harvested 12 weeks later. Our results indicate that a medial osteochondral defect drives pathology in the underlying meniscus, likely due to altered loading conditions. The medial menisci of defect joints exhibited increased proteoglycan deposition and hypertrophy, with increased cell roundness and area in regions of elevated GAGs. Local collagen architecture showed increased fiber diameter in the medial menisci of defect joints, which positively correlated with increased GAG coverage. Abnormal collagen structures were observed, including wider variations in fiber diameters and areas of small fibers with low second harmonic generation signals, indicating poorly organized collagen. A deeper understanding of GAG regulation and fibrochondrocyte pathology in injured meniscus tissue could aid in the development of therapeutics and inform disease progression.

In the late stage of osteoarthritis (OA), few options are available to improve patient quality of life beyond joint arthroplasty. Exposed bone and direct bone-on-bone contact in late-stage OA lead to severe pain and joint stiffness. These symptoms could potentially be relieved through sufficient lubrication of the bone surface. Herein, we report a diblock copolymer that significantly reduces the coefficient of friction at bone interfaces. One block of the copolymer contains positively charged quaternary amines that are designed to electrostatically interact with the negatively charged bone mineral composite, and the other block resists compression to lubricate the interface by a bottle-brush structure consisting of poly(acrylic acid) backbones and polyethylene glycol side chains. The diblock copolymer effectively lubricated the bone surface to a level that is superior (trabecular bone) or comparable (subchondral bone) to the coefficient of friction of cartilage in phosphate buffered saline under boundary mode conditions. To our knowledge, this is the first study showing effective bone lubrication via a synthetic lubricant and thus could serve as a potential treatment for late-stage OA when bone surfaces are exposed.

New Zealand white rabbits are a prevalent model species used to study preclinical articular cartilage repair therapies. The composition and structure of rabbit articular cartilage have been extensively characterized, yet the local shear properties of the tissue are unknown. Characterizing the local shear properties is essential for understanding the structure–function relationship in the tissue and relating the rabbit preclinical model to human disease. Therefore, the objectives of this study were to (1) characterize the local shear properties of articular cartilage from the femoral condyles of New Zealand white rabbits, (2) determine if local protein content or matrix structure correlated with local shear properties, and (3) compare microscale shear moduli values of rabbit cartilage to those previously reported for human, equine, and bovine tissues. Local shear strains and moduli varied with rabbit cartilage tissue depth; shear modulus was highest ~ 50 μm below the tissue surface and decreased to plateau values around 150 μm, mirroring the trend with shear strains. Local shear strains showed significant correlations with local protein content but not matrix organization. Rabbit cartilage shear properties followed similar spatial trends as bovine, equine, and human tissue in the first ~ 100 um of the tissue depth. However, rabbit tissue then differentiated from the larger animals as shear modulus values plateaued and did not increase by an order of magnitude like that seen in the larger species. Local shear properties of rabbit articular cartilage capture the surface properties of human, equine, and bovine cartilage but mechanically lack the deep zone region.

Collagen is a key extracellular matrix protein found in connective tissues. The structure and organization of collagen fibers play a crucial role in determining tissue function and how tissues respond to mechanical loads. Small leucine-rich proteoglycans (SLRPs) are well-known facilitators of collagen fibrillogenesis in connective tissues. While the role of SLRPs has been extensively documented in tissues such as tendon and skin, their functions are primarily inferred from changes observed in knockout models. Additionally, their specific roles and influences of their addition to a system, particularly in collagen gel-based materials, remain underexplored. Previous in vitro studies of SLRPs have been partly limited by the challenges associated with obtaining pure SLRPs in sufficient quantities and with appropriate glycosylation. Therefore, novel methods to reliably produce SLRPs at the required quality and scale are needed. In this study, we first evaluated the feasibility of producing recombinant decorin, biglycan, and fibromodulin using HEK293-F cells. Subsequently, we investigated the effect of SLRP supplementation on high-density collagen gels using scanning electron microscopy and assessed the impact on tensile properties. Our findings demonstrated that each SLRP uniquely influenced collagen structure at both the fibril and fiber levels, consequently modifying the tissues' mechanical response to load. Decorin, in particular, exhibited significant differences in tensile properties compared to biglycan and fibromodulin, underscoring its distinct role in promoting a structurally and mechanically robust response under tensile load.

Intervertebral disc (IVD) degeneration is a common source of back pain. The IVD is a complex structure that consists of an outer annular ring, an inner nucleus pulposus, and flanking cartilaginous endplates, which together allow for daily mobility by distributing loads and acting as a flexible segment within the spine. Both age and mechanical overload can drive the development of a pathologic disc microenvironment that includes alterations in mechanics, solute transport, and inflammation. Such changes in the disc have negative consequences on resident cells that promote their senescence, apoptosis, and contribution to furthering disc degeneration through mitochondrial dysfunction and the release of reactive oxygen species, proteases, and cytokines. This crosstalk between IVD cells and their microenvironment creates a feedback loop that eventually manifests into such clinical conditions as disc height loss, herniations, and total IVD collapse. Developing a holistic understanding of how this feedback loop is initiated and may be halted will enable the development of novel therapeutics that not only provide analgesic benefit but also help rebuild the deteriorated disc.

Degenerative disc disease is a significant cause of chronic low back pain, often leading to disability and high health care costs. Current treatments, including physical therapy, pain management, and surgical interventions such as spinal fusion and total disc replacement, do not reverse degeneration. Bioactive therapies offer a potential alternative by targeting the underlying degenerative process. Cell-based therapies, including the use of mesenchymal stem cells and platelet-rich plasma, aim to restore disc structure and function by promoting extracellular matrix production and reducing inflammation. Early studies show potential benefits in pain relief and disc regeneration, but long-term efficacy remains unclear. Nucleus pulposus augmentation and replacement strategies, such as the use of hydrogel implants and in situ curing polymers, are aimed at restoring disc height and biomechanical function. While these strategies are promising, issues such as implant durability and migration require further study. Total disc replacement preserves motion and avoids adjacent-segment disease, but outcomes depend on patient selection and implant design. Despite encouraging results, bioactive therapies still require research to establish long-term safety and effectiveness. Advancements in biomaterials, patient selection criteria, and clinical trials will determine their role in the future management of degenerative disc disease.

Development of non-surgical disease-modifying interventions for knee osteoarthritis (OA) remains a persistent challenge despite decades of efforts. Therapeutic transport to cartilage in synovial joints is hindered by the dense, negatively charged cartilage matrix, and further challenged by rapid synovial fluid clearance within hours to days. In this study, we investigated ultrasmall (dh ~ 6 nm) fluorescent core-shell silica nanoparticles (Cornell Prime Dots, or C’ Dots), which have received FDA-investigational new drug approval for multiple human clinical trials in oncology, as cartilage-penetrating delivery vehicles for applications in knee OA. Across multiple length and time scales, we examined the relationship between C’ Dot tissue and cellular transport kinetics and whole joint clearance. In vitro, C’ Dots penetrated cartilage explants within 30 min (D ~ 2 μm2/s). C’ Dots were internalized by chondrocytes within 24 h and were retained in vesicular structures for up to 5 days. In vivo, C’ Dot clearance following intra-articular knee injection was well described by two distinct time constants (τ1 ~ 18 hours, τ2 ~ 3 weeks), consistent with mechanisms of synovial- and tissue-mediated clearance. C’ Dot clearance rates were not affected by surgically-induced cruciate ligament transection. Notably, C’ Dots remained in the knee longer than 3 months after a single injection and were localized to cartilage, meniscus, ligaments, and synovium. Collectively, these results illustrate the potential of C’ Dots for long-term delivery of conjugated therapeutics in the knee.

Background: The voluntary recall and ban of several textured breast implant models worldwide, secondary to their association with Breast Implant-Associated Anaplastic Large Cell Lymphoma, has limited the key benefit of a textured surface─positional stability. We have engineered a Positionally Stable Smooth Implant (PSSI) containing millimeter-scaled cylindrical wells on the implant surface for capsule ingrowth and device stabilization. Objectives: To evaluate the long-term positional stability of PSSI designs in vivo and characterize capsule formation. Methods: Miniature breast implants were manufactured using poly(dimethylsiloxane). PSSI were designed with various dimensions of well width, depth, and number. Comparison groups consisted of smooth and textured implants. Six sterilized implants per group were implanted subcutaneously into the bilateral dorsa of Sprague–Dawley rats. Implant rotation was measured with MicroCT every 2 weeks. Implant-capsule units were explanted at 3 months for histological analysis. Results: All PSSI groups exhibited significantly less cumulative positional rotation than smooth implants ( p < 0.05), with stability comparable to that of textured implants. Upon explantation, microCT and gross examination revealed notable capsule ingrowth within the PSSI wells. Histological evaluation of foreign body response showed significantly fewer pro-inflammatory M1 macrophages in the PSSI capsules compared to the textured control. Additionally, myofibroblast expression, which is implicated in capsular contracture, was significantly lower in both the PSSI and textured groups compared to smooth implants. Conclusions: This novel smooth-surface breast implant design provided equivalent positional stability and reduced pro-inflammatory M1 macrophage expression compared to textured implants. These results suggest a promising, safer alternative to textured implants for inducing positional stability.

Abstract Bioprinting produces personalized, cell-laden constructs for tissue regeneration through the additive layering of bio-ink, an injectable hydrogel infused with cells. Currently, bioprinted constructs are assessed for quality by measuring cellular properties post-production using destructive techniques, necessitating the creation of multiple constructs and increasing the production costs of bioprinting. To reduce this burden, cell properties in bio-ink can be monitored in real-time during printing. We incorporated dielectric impedance spectroscopy (DIS) onto a syringe for real-time measurement of primary chondrocytes suspended in phosphate buffered saline (PBS) using impedance (| Z |) and phase angle ( θ ) from 0.1 to 25 000 kHz. Cell concentration and viability ranged from 0.1 × 10 6 cells ml −1 to 125 × 10 6 cells ml −1 and from 0%to 94%, respectively. Samples with constant or with changing cell concentration were exposed to various flow conditions from 0.5 to 4 ml min −1 . The background PBS signal was subtracted from the sample, allowing for comparisons across devices and providing insight into the dielectric properties of the cells, and was labeled as | Z cells | and θ cells . | Z cells | shared a linear correlation with cell concentration and viability. Flow rate had minimal effect on our results, and | Z cells | responded on the order of seconds as cell concentration was altered over time. Notably, sensitivity to cell concentration and viability were dependent on frequency and were highest for | Z cells | when θ cells was minimized. Cell concentration and viability showed an additive effect on | Z cells | that was modeled across multiple frequencies, and deconvolution of these signals could result in real-time predictions of cell properties in the future. Overall, DIS was found to be a suitable technique for real-time sensing of cell concentration and viability during bioprinting.

Abstract Osteochondral lesions (OCL) are common among young patients and often require surgical interventions since cartilage has a poor capacity for self-repair. Bone marrow stimulation (BMS) has been used clinically for decades to treat OCLs, however a persisting challenge with BMS and other cartilage repair strategies is the inferior quality of the resulting fibrocartilaginous repair tissue. Lubrication-based therapies have the potential to improve the quality of cartilage repair tissue as joint lubrication is linked to local cartilage tissue strains and subsequent cellular responses including death and apoptosis. Recently, a full length recombinant human lubricin (rhLubricin) was developed and has been shown to lower friction in cartilage. This study investigated the effect of a single delayed injection of rhLubricin on cartilage repair in an in vivo rabbit OCL model using gross macroscopic evaluation, surface profilometry, histology, and tribology. Moderate improvement in macroscopic scores for cartilage repair were observed. Notably, quantitative analysis of Safranin-O histology showed that rhLubricin treated joints had significantly higher glycosaminoglycan content compared to saline treated joints, and there were no differences in repair integration between groups. Furthermore, rhLubricin treated joints had significantly lower friction coefficients tested across three sliding speeds compared to saline treated joints (rhLubricin: 0.15 ± 0.03 at 0.1 mm/s to 0.12 ± 0.03 at 10 mm/s, Saline: 0.22 ± 0.06 at 0.1 mm/s to 0.19 ± 0.05 at 10 mm/s). Overall, a single delayed injection of rhLubricin improved the quality and lubricating ability of the repair cartilage tissue without inhibiting repair tissue integration.

The complex collagen network of the native meniscus and the gradient of the density and alignment of this network through the meniscal enthesis is essential for the proper mechanical function of these tissues. This architecture is difficult to recapitulate in tissue-engineered replacement strategies. Prenatally, the organization of the collagen fiber network is established and aggrecan content is minimal. In vitro, fibrochondrocytes (FCCs) produce proteoglycans and associated glycosaminoglycan (GAG) chains early in culture, which can inhibit collagen fiber formation during the maturation of tissue-engineered menisci. Thus, it would be beneficial to both specifically and temporarily block deposition of proteoglycans early in culture. In this study, we transiently inhibited aggrecan production by meniscal fibrochondrocytes using siRNA in collagen gel-based tissue-engineered constructs. We evaluated the effect of siRNA treatment on the formation of collagen fibrils and bulk and microscale tensile properties. Specific inhibition of aggrecan production by fibrochondrocytes via siRNA was successful both in 2D monolayer cell culture and 3D tissue culture. This inhibition during early maturation of these in vitro constructs increased collagen fibril diameter by more than 2-fold. This increase in fibril diameter allowed these tissues to distribute strains more effectively at the local level, particularly at the interface of the bone and soft tissue. These data show that siRNA can be used to modulate the ECM to improve collagen fiber formation and mechanical properties in tissue-engineered constructs, and that a transient decrease in aggrecan promotes the formation of a more robust fiber network.

The low friction nature of articular cartilage has been attributed to the synergistic interaction between lubricin and hyaluronic acid in the synovial fluid (SF). Lubricin is a mucinous glycoprotein that lowers the boundary mode coefficient of friction of articular cartilage in a dose-dependent manner. While there have been multiple attempts to produce recombinant lubricin and lubricin mimetic cartilage lubricants over the last two decades, these materials have not found clinical use due to challenges associated with large scale production, manufacturing, and purification. Recently, a novel method using codon scrambling was developed to produce a stable, full-length bioengineered equine lubricin (eLub) in large reproducible quantities. While preliminary frictional analysis of eLub and other recombinantly produced forms revealed they can lubricate cartilage, a complete tribological characterization is lacking, with previous studies evaluating the friction coefficient only at a single dose or a single speed. The objective of this study was to analyze the dose-dependent tribological properties of eLub using the Stribeck framework of tribological analysis. Recombinantly produced eLub at doses greater than 1.5 mg/mL exhibits friction coefficients on par with healthy bovine SF, and a maximal 5 mg/mL dose exhibits a nearly 50% lower friction coefficient than healthy SF. eLub also modulates the shift in lubrication mode of the cartilage from the high friction boundary mode to the low friction minimum mode at high concentrations.

Post-traumatic osteoarthritis develops following an inciting injury to a joint and results in cartilage degeneration. Mechanical loading, including articulation, drives anabolic responses in cartilage clinically, in vivo, and in vitro. Tribological articulation, or sliding of cartilage on a glass counterface, has long been used as an in vitro tool to study cartilage tissue behavior. However, it is unclear if tribological articulation affects chondrocyte fate following injury, and if the timing of articulation impacts the resultant effect. The goal of this study was to investigate the effect of tribological articulation on injured cartilage tissue at two time points: (i) performed immediately after injury and (ii) 24 h after injury. Neonatal bovine femoral cartilage explants were injured using a rapid spring-loaded impactor and subsequently subjected to tribological articulation. Cell death due to impact injury was highest near the articular surface, suggesting a strain-dependent mechanism. Immediate articulation following injury mitigated cell death compared to injury alone or delayed articulation; markers for both general cell death and early-stage apoptosis were markedly decreased in the explants that were immediately slid. Interestingly, mitigation of cell death due to sliding was most predominant at the cartilage surface. Tribological articulation is known to create fluid flow within the tissue, predominantly at the articular surface, which could drive the protective response seen here. Altogether, this work shows that perturbations to the cellular environment immediately following cartilage injury significantly impact chondrocyte fate.

Progressive cartilage degradation, synovial inflammation, and joint lubrication dysfunction are key markers of osteoarthritis. The composition of synovial fluid (SF) is altered in OA, with changes to both hyaluronic acid and lubricin, the primary lubricating molecules in SF. Lubricin's distinct bottlebrush mucin domain has been speculated to contribute to its lubricating ability, but the relationship between its structure and mechanical function in SF is not well understood. Here, we demonstrate the application of a novel mucinase (StcE) to selectively degrade lubricin's mucin domain in SF to measure its impact on joint lubrication and friction. Notably, StcE effectively degraded the lubricating ability of SF in a dose-dependent manner starting at nanogram concentrations (1-3.2 ng/mL). Further, the highest StcE doses effectively degraded lubrication to levels on par with trypsin, suggesting that cleavage at the mucin domain of lubricin is sufficient to completely inhibit the lubrication mechanism of the collective protein component in SF. These findings demonstrate the value of mucin-specific experimental approaches to characterize the lubricating properties of SF and reveal key trends in joint lubrication that help us better understand cartilage function in lubrication-deficient joints.

Articular joints facilitate motion and transfer loads to underlying bone through a combination of cartilage tissue and synovial fluid, which together generate a low-friction contact surface. Traumatic injury delivered to cartilage and the surrounding joint capsule causes secretion of proinflammatory cytokines by chondrocytes and the synovium, triggering cartilage matrix breakdown and impairing the ability of synovial fluid to lubricate the joint. Once these inflammatory processes become chronic, posttraumatic osteoarthritis (PTOA) development begins. However, the exact mechanism by which negative alterations to synovial fluid leads to PTOA pathogenesis is not fully understood. We hypothesize that removing the lubricating macromolecules from synovial fluid alters the relationship between mechanical loads and subsequent chondrocyte behavior in injured cartilage. To test this hypothesis, we utilized an ex vivo model of PTOA that involves subjecting cartilage explants to a single rapid impact followed by continuous articulation within a lubricating bath of either healthy synovial fluid, phosphate-buffered saline (PBS), synovial fluid treated with hyaluronidase, or synovial fluid treated with trypsin. These treatments degrade the main macromolecules attributed with providing synovial fluid with its lubricating properties; hyaluronic acid and lubricin. Explants were then bisected and fluorescently stained to assess global and depth-dependent cell death, caspase activity, and mitochondrial depolarization. Explants were tested via confocal elastography to determine the local shear strain profile generated in each lubricant. These results show that degrading hyaluronic acid or lubricin in synovial fluid significantly increases middle zone chondrocyte damage and shear strain loading magnitudes, while also altering chondrocyte sensitivity to loading.

Background: Mechanical augmentation upon implantation is essential for the long-term success of tissue-engineered intervertebral discs (TE-IVDs). Previous studies utilized stiffer materials to fabricate TE-IVD support structures. However, these materials undergo various failure modes in the mechanically challenging IVD microenvironment. FlexiFil (FPLA) is an elastomeric 3D printing filament that is amenable to the fabrication of support structures. However, no present study has evaluated the efficacy of a flexible support material to preserve disc height and support the formation of hydrated tissues in a large animal model. Methods: We leveraged results from our previously developed FE model of the minipig spine to design and test TE-IVD support cages comprised of FPLA and PLA. Specifically, we performed indentation to assess implant mechanical response and scanning electron microscopy to visualize microscale damage. We then implanted FPLA and PLA support cages for 6 weeks in the minipig cervical spine and monitored disc height via weekly x-rays. TE-IVDs cultured in FPLA were also implanted for 6 weeks with weekly x-rays and terminal T2 MRIs to quantify tissue hydration at study endpoint. Results: Results demonstrated that FPLA cages withstood nearly twice the deformation of PLA without detrimental changes in mechanical performance and minimal damage. In vivo, FPLA cages and stably implanted TE-IVDs restored native disc height and supported the formation of hydrated tissues in the minipig spine. Displaced TE-IVDs yielded disc heights that were superior to PLA or discectomy-treated levels. Conclusions: FPLA holds great promise as a flexible and bioresorbable material for enhancing the long-term success of TE-IVD implants.

Quercetin, recognized for its antioxidant, anti-inflammatory, and antibacterial properties, faces limited biomedical application due to its low solubility. Cotton, a preferred wound dressing material over synthetic ones, lacks inherent antibacterial and wound-healing attributes and can benefit from quercetin features. This study explores the potential of overcoming these challenges through the inclusion complexation of quercetin with cyclodextrins (CDs) and the development of a nanofibrous coating on a cotton nonwoven textile. Hydroxypropyl-beta-cyclodextrin (HP-β-CD) and hydroxypropyl-gamma-cyclodextrin (HP-γ-CD) formed inclusion complexes of quercetin, with chitosan added to enhance antibacterial properties. Phase solubility results showed that inclusion complexation can enhance quercetin solubility up to 20 times, with HP-γ-CD forming a more stable inclusion complexation compared with HP-β-CD. Electrospinning of the nanofibers from HP-β-CD/Quercetin and HP-γ-CD/Quercetin aqueous solutions without the use of a polymeric matrix yielded a uniform, smooth fiber morphology. The structural and thermal analyses of the HP-β-CD/Quercetin and HP-γ-CD/Quercetin nanofibers confirmed the presence of inclusion complexes between quercetin and each of the CDs (HP-β-CD and HP-γ-CD). Moreover, HP-β-CD/Quercetin and HP-γ-CD/Quercetin nanofibers showed a near-complete loading efficiency of quercetin and followed a fast-releasing profile of quercetin. Both HP-β-CD/Quercetin and HP-γ-CD/Quercetin nanofibers showed significantly higher antioxidant activity compared to pristine quercetin. The HP-β-CD/Quercetin and HP-γ-CD/Quercetin nanofibers also showed antibacterial activity, and with the addition of chitosan in the HP-γ-CD/Quercetin system, the Chitosan/HP-γ-CD/Quercetin nanofibers completely eliminated the investigated bacteria species. The nanofibers were nontoxic and well-tolerated by cells, and exploiting the quercetin and chitosan anti-inflammatory activities resulted in the downregulation of IL-6 and NO secretion in both immune as well as regenerative cells. Overall, CD inclusion complexation markedly enhances quercetin solubility, resulting in a biofunctional antioxidant, antibacterial, and anti-inflammatory wound dressing through a nanofibrous coating on cotton textiles.

Abstract Intra‐articular injections of hyaluronic acid (HA) are the cornerstone of osteoarthritis (OA) treatments. However, the mechanism of action and efficacy of HA viscosupplementation are debated. As such, there has been recent interest in developing synthetic viscosupplements. Recently, a synthetic 4 wt% polyacrylamide (pAAm) hydrogel was shown to effectively lubricate and bind to the surface of cartilage in vitro. However, its ability to localize to cartilage and alter the tribological properties of the tissue in a live articulating large animal joint is not known. The goal of this study was to quantify the distribution and extent of localization of pAAm in the equine metacarpophalangeal or metatarsophalangeal joint (fetlock joint), and determine whether preferential localization of pAAm influences the tribological properties of the tissue. An established planar fluorescence imaging technique was used to visualize and quantify the distribution of fluorescently labeled pAAm within the joint. While the pAAm hydrogel was present on all surfaces, it was not uniformly distributed, with more material present near the site of the injection. The lubricating ability of the cartilage in the joint was then assessed using a custom tribometer across two orders of magnitude of sliding speed in healthy synovial fluid. Cartilage regions with a greater coverage of pAAm, that is, higher fluorescent intensities, exhibited friction coefficients nearly 2‐fold lower than regions with lesser pAAm ( R rm = −0.59, p &lt; 0.001). Collectively, the findings from this study indicate that intra‐articular viscosupplement injections are not evenly distributed inside a joint, and the tribological outcomes of these materials is strongly determined by the ability of the material to localize to the articulating surfaces in the joint.

Collagen scaffolds seeded with human chondrocytes have shown great potential for cartilage repair and regeneration. However, these porous scaffolds buckle under low compressive forces, creating regions of highly localized deformations that can cause cell death and deteriorate the integrity of the engineered tissue. We perform three-dimensional (3D) tomography-based characterization to track the evolution of collagen scaffolds’ microstructure under large deformation. The results illustrate how instabilities produce a spatially varying compaction across the specimens, with more pronounced collapse near the free boundaries. We discover that, independent of differences in pore-size distributions, all collagen scaffolds examined displayed strong auxetic behavior i.e., their transverse area contracts under compression, as a result of the instability cascade. This feature, typically characteristic of engineered metamaterials, is of critical importance for the performance of collagen scaffolds in tissue engineering, especially regarding the persistent challenge of lateral integration in cartilage constructs.

Augmentation of the nasal dorsum often requires implantation of structural material. Existing methods include autologous, cadaveric or alloplastic materials and injectable hydrogels. Each of these options is associated with considerable limitations. There is an ongoing need for precise and versatile implants that produce long-lasting craniofacial augmentation. Four separate polylactic acid (PLA) dorsal nasal implant designs were 3D-printed. Two implants had internal PLA rebar of differing porosities and two were designed as "shells" of differing porosities. Shell designs were implanted without infill or with either minced or zested processed decellularized ovine cartilage infill to serve as a "biologic rebar", yielding eight total treatment groups. Scaffolds were implanted heterotopically on rat dorsa (N = 4 implants per rat) for explant after 3, 6, and 12 months followed by volumetric, histopathologic, and biomechanical analysis. Low porosity implants with either minced cartilage or PLA rebar infill had superior volume retention across all timepoints. Overall, histopathologic and immunohistochemical analysis showed a resolving inflammatory response with an M1/M2 ratio consistently favoring tissue regeneration over the study course. However, xenograft cartilage showed areas of degradation and pro-inflammatory infiltrate contributing to volume and contour loss over time. Biomechanical analysis revealed all constructs had equilibrium and instantaneous moduli higher than human septal cartilage controls. Biocompatible, degradable polymer implants can induce healthy neotissue ingrowth resulting in guided soft tissue augmentation and offer a simple, customizable and clinically-translatable alternative to existing craniofacial soft tissue augmentation materials. PLA-only implants may be superior to combination PLA and xenograft implants due to contour irregularities associated with cartilage degradation.

In various biological systems, analyzing how cell behaviors are coordinated over time would enable a deeper understanding of tissue-scale response to physiologic or superphysiologic stimuli. Such data is necessary for establishing both normal tissue function and the sequence of events after injury that lead to chronic disease. However, collecting and analyzing these large datasets presents a challenge-such systems are time-consuming to process, and the overwhelming scale of data makes it difficult to parse overall behaviors. This problem calls for an analysis technique that can quickly provide an overview of the groups present in the entire system and also produce meaningful categorization of cell behaviors. Here, we demonstrate the application of an unsupervised method-the Variational Autoencoder (VAE)-to learn the features of cells in cartilage tissue after impact-induced injury and identify meaningful clusters of chondrocyte behavior. This technique quickly generated new insights into the spatial distribution of specific cell behavior phenotypes and connected specific peracute calcium signaling timeseries with long term cellular outcomes, demonstrating the value of the VAE technique.

ABSTRACT Lubricin, a lubricating glycoprotein abundant in synovial fluid, forms a low-friction brush polymer interface in tissues exposed to sliding motion including joints, tendon sheaths, and the surface of the eye. Despite its therapeutic potential in diseases such as osteoarthritis and dry eye disease, there are few sources available. Through rational design, we developed a series of recombinant lubricin analogs that utilize the species-specific tissue-binding domains at the N- and C-termini to increase biocompatibility while replacing the central mucin domain with an engineered variant that retains the lubricating properties of native lubricin. In this study, we demonstrate the tissue binding capacity of our engineered lubricin product and its retention in the joint space of rats. Next, we present a new bioprocess chain that utilizes a human-derived cell line to produce O -glycosylation consistent with that of native lubricin and a purification strategy that capitalizes on the positively charged, hydrophobic N- and C-terminal domains. The bioprocess chain is demonstrated at 10 L scale in industry-standard equipment utilizing commonly available ion exchange, hydrophobic interaction and size exclusion chromatography resins. Finally, we confirmed the purity and lubricating properties of the recombinant biolubricant. The biomolecular engineering and bioprocessing strategies presented here are an effective means of lubricin production and could have broad applications to the study of mucins in general.

Reconstruction of the human auricle remains a formidable challenge for plastic surgeons. Autologous costal cartilage grafts and alloplastic implants are technically challenging, and aesthetic and/or tactile outcomes are frequently suboptimal. Using a small animal “bioreactor”, we have bioengineered full-scale ears utilizing decellularized cartilage xenograft placed within a 3D-printed external auricular scaffold that mimics the size, shape, and biomechanical properties of the native human auricle. The full-scale polylactic acid ear scaffolds were 3D-printed based upon data acquired from 3D photogrammetry of an adult ear. Ovine costal cartilage was processed either through mincing (1 mm3) or zesting (< 0.5 mm3), and then fully decellularized and sterilized. At explantation, both the minced and zested neo-ears maintained the size and contour complexities of the scaffold topography with steady tissue ingrowth through 6 months in vivo. A mild inflammatory infiltrate at 3 months was replaced by homogenous fibrovascular tissue ingrowth enveloping individual cartilage pieces at 6 months. All ear constructs were pliable, and the elasticity was confirmed by biomechanical analysis. Longer-term studies of the neo-ears with faster degrading biomaterials will be warranted for future clinical application.

Abstract Inflammation of the synovium, known as synovitis, plays an important role in the pathogenesis of osteoarthritis (OA). Synovitis involves the release of a wide variety of pro‐inflammatory mediators in synovial fluid (SF) that damage the articular cartilage extracellular matrix and induce death and apoptosis in chondrocytes. The composition of synovial fluid is dramatically altered by inflammation in OA, with changes to both hyaluronic acid and lubricin, the primary lubricating molecules in SF. However, the relationship between key biochemical markers of joint inflammation and mechanical function of SF is not well understood. Here, we demonstrate the application of a novel analytical framework to measure the effective viscosity for SF lubrication of cartilage, which is distinct from conventional rheological viscosity. Notably, in a well‐established equine model of synovitis, this effective lubricating viscosity decreased by up to 10,000‐fold for synovitis SF compared to a ~4 fold change in conventional viscosity measurements. Further, the effective lubricating viscosity was strongly inversely correlated ( r = −0.6 to −0.8) to multiple established biochemical markers of SF inflammation, including white blood cell count, prostaglandin E 2 (PGE 2 ), and chemokine ligand (CCLs) concentrations, while conventional measurements of viscosity were poorly correlated to these markers. These findings demonstrate the importance of experimental and analytical approaches to characterize functional lubricating properties of synovial fluid and their relationships to soluble biomarkers to better understand the progression of OA.

This is the dataset for our paper titled " Application of a Variational Autoencoder for Clustering and Analyzing in situ Articular Cartilage Cellular Response to Mechanical Stimuli". Corresponding author information: Email: ht452@cornell.edu (Han Kheng Teoh); ic64@cornell.edu (Itai Cohen) This dataset is shared under a Creative Commons Attribution 4.0 International license (CC BY 4.0); the data will be openly available to share and adapt, but appropriate credit to the original data creators is required upon reuse. When using this dataset, please cite: The dataset: Jingyang Zheng, Han Kheng Teoh, Michelle L. Delco, Lawrence J. Bonassar , and Itai Cohen. (2024) Data from: Application of a Variational Autoencoder for Clustering and Analyzing in situ Articular Cartilage Cellular Response to Mechanical Stimuli [dataset]. Zenodo. https://doi.org/10.5281/zenodo.10565588 AND the paper: Jingyang Zheng, Han Kheng Teoh, Michelle L. Delco, Lawrence J. Bonassar , and Itai Cohen. (2024) Application of a Variational Autoencoder for Clustering and Analyzing in situ Articular Cartilage Cellular Response to Mechanical Stimuli. PLOS One https://doi.org/10.1371/journal.pone.0297947 The work was supported by the NIH National Institute of Arthritis and Musculoskeletal and Skin Diseases, Contract: K08AR068470, R03AR075929, and The Harry M. Zweig Fund for Equine Research. This work was also supported by the NIH National Institute of Neurological Disorders and Stroke. Contract: R01NS116595. Additionally, this work was supported by the National Science Foundation grants DMR-1807602, CMMI 1927197, and BMMB-1536463. Lastly, this work made use of the Cornell Center for Materials Research Shared Facilities, which are supported through the NSF MRSEC program (DMR-1719875). DATA & FILE OVERVIEW ------------------------------------------------- The dataset contains two folders : Data and Code. In the Data folder, the experimental data is organized into three subfolders, specifying the date when the experiment was performed. Each subfolder contains the following files: all_locs.mat - contains the cells (x,y) position. The data is organized as a Nx2 array, where N is the number of cells in the sample. blue_all.mat - contains the post-impact NMP (cell death) intensity for each cell. The data is organized as a TxN array, where T is the number of time points the NMP (cell death) intensity was measured. green_all.mat - contains the post impact Ca^{2+} intensity for each cell. The data is organized as a TxN array, where T is the number of time points the Ca^{2+} intensity was measured. red_all.mat - contains the post impact TMRM (mitochrondrial polarity) intensity for each cell. The data is organized as a TxN array, where T is the number of time points the TMRM (mitochondrial polarity) intensity was measured. impact_intensity.mat - contains the Ca^{2+} intensity during impact for each cell. The data is organized as a TxN array, where T is the number of time points the Ca^{2+} intensity was measured. impact_locs.mat - contains the cells' (x,y) position within the impact site. The data is organized as a Nx2 array, where N is the number of cells in the sample. D_skl_dd_mm_yy.p - contains the symmetrized KL divergence between cells' latent representation. The data is organized as a N by N array, where N is the number of cells. In addition, the Data folder also contains : model_weights.p file - contains the weights and biases for the trained VAE network used in this study. The Code folder contains: decoders.py - contains a class function for the VAE decoder. encoders.py - contains a class function for the VAE encoder. loaders.py - contains a function that partitions the cell data into a training set and a test set. wrapper.py - contains a class function that trains a VAE. Cartilage VAE - Part I.ipynb - contains the code necessary to generate Figures 1 to 5 in the manuscript. Cartilage VAE - Part II.ipynb - contains the code necessary to generate Figures 5 to 9 in the manuscript.

This is the dataset for our paper titled " Application of a Variational Autoencoder for Clustering and Analyzing in situ Articular Cartilage Cellular Response to Mechanical Stimuli". Corresponding author information: Email: ht452@cornell.edu (Han Kheng Teoh); ic64@cornell.edu (Itai Cohen) This dataset is shared under a Creative Commons Attribution 4.0 International license (CC BY 4.0); the data will be openly available to share and adapt, but appropriate credit to the original data creators is required upon reuse. When using this dataset, please cite: The dataset: Jingyang Zheng, Han Kheng Teoh, Michelle L. Delco, Lawrence J. Bonassar , and Itai Cohen. (2024) Data from: Application of a Variational Autoencoder for Clustering and Analyzing in situ Articular Cartilage Cellular Response to Mechanical Stimuli [dataset]. Zenodo. https://doi.org/10.5281/zenodo.10565588 AND the paper: Jingyang Zheng, Han Kheng Teoh, Michelle L. Delco, Lawrence J. Bonassar , and Itai Cohen. (2024) Application of a Variational Autoencoder for Clustering and Analyzing in situ Articular Cartilage Cellular Response to Mechanical Stimuli. PLOS One https://doi.org/10.1371/journal.pone.0297947 The work was supported by the NIH National Institute of Arthritis and Musculoskeletal and Skin Diseases, Contract: K08AR068470, R03AR075929, and The Harry M. Zweig Fund for Equine Research. This work was also supported by the NIH National Institute of Neurological Disorders and Stroke. Contract: R01NS116595. Additionally, this work was supported by the National Science Foundation grants DMR-1807602, CMMI 1927197, and BMMB-1536463. Lastly, this work made use of the Cornell Center for Materials Research Shared Facilities, which are supported through the NSF MRSEC program (DMR-1719875). DATA & FILE OVERVIEW ------------------------------------------------- The dataset contains two folders : Data and Code. In the Data folder, the experimental data is organized into three subfolders, specifying the date when the experiment was performed. Each subfolder contains the following files: all_locs.mat - contains the cells (x,y) position. The data is organized as a Nx2 array, where N is the number of cells in the sample. blue_all.mat - contains the post-impact NMP (cell death) intensity for each cell. The data is organized as a TxN array, where T is the number of time points the NMP (cell death) intensity was measured. green_all.mat - contains the post impact Ca^{2+} intensity for each cell. The data is organized as a TxN array, where T is the number of time points the Ca^{2+} intensity was measured. red_all.mat - contains the post impact TMRM (mitochrondrial polarity) intensity for each cell. The data is organized as a TxN array, where T is the number of time points the TMRM (mitochondrial polarity) intensity was measured. impact_intensity.mat - contains the Ca^{2+} intensity during impact for each cell. The data is organized as a TxN array, where T is the number of time points the Ca^{2+} intensity was measured. impact_locs.mat - contains the cells' (x,y) position within the impact site. The data is organized as a Nx2 array, where N is the number of cells in the sample. D_skl_dd_mm_yy.p - contains the symmetrized KL divergence between cells' latent representation. The data is organized as a N by N array, where N is the number of cells. In addition, the Data folder also contains : model_weights.p file - contains the weights and biases for the trained VAE network used in this study. The Code folder contains: decoders.py - contains a class function for the VAE decoder. encoders.py - contains a class function for the VAE encoder. loaders.py - contains a function that partitions the cell data into a training set and a test set. wrapper.py - contains a class function that trains a VAE. Cartilage VAE - Part I.ipynb - contains the code necessary to generate Figures 1 to 5 in the manuscript. Cartilage VAE - Part II.ipynb - contains the code necessary to generate Figures 5 to 9 in the manuscript.