Chondrocyte deformation during the unloading phase of cyclic compression loading.

Cell volume and shape changes play a pivotal role in cellular mechanotransduction, governing cellular responses to external loading. Understanding the dynamics of cell behavior under loading conditions is essential to elucidate cell adaptation mechanisms in physiological and pathological contexts. In this study, we investigated the effects of dynamic cyclic compression loading on cell volume and shape changes, comparing them with static conditions. Using a custom-designed platform which allowed for simultaneous loading and imaging of cartilage tissue, tissues were subjected to 100 cycles of mechanical loading while measuring cell volume and shape alterations during the unloading phase at specific time points. The findings revealed a transient decrease in cell volume (13%) during the early cycles, followed by a gradual recovery to baseline levels after approximately 20 cycles, despite the cartilage tissue not being fully recovered at the unloading phase. This observed pattern indicates a temporal cell volume response that may be associated with cellular adaptation to the mechanical stimulus through mechanisms related to active cell volume regulation. Additionally, this study demonstrated that cell volume and shape responses during dynamic loading were significantly distinct from those observed under static conditions. Such findings suggest that cells in their natural tissue environment perceive and respond differently to dynamic compared to static mechanical cues, highlighting the significance of considering dynamic loading environments in studies related to cellular mechanics. Overall, this research contributes to the broader understanding of cellular behavior under mechanical stimuli, providing valuable insights into their ability to adapt to dynamic mechanical loading.

In vivo human knee varus-valgus loading apparatus for analysis of MRI-based intratissue strain and relaxometry.

The diagnosis of early-stage osteoarthritis remains as an unmet challenge in medicine and a roadblock to evaluating the efficacy of disease-modifying treatments. Recent studies demonstrate that unique patterns of intratissue cartilage deformation under cyclic loading can serve as potential biomarkers to detect early disease pathogenesis. However, a workflow to obtain deformation, strain maps, and quantitative MRI metrics due to the loading of articular cartilage in vivo has not been fully developed. In this study, we characterize and demonstrate an apparatus that is capable of applying a varus-valgus load to the human knee in vivo within an MRI environment to enable the measurement of cartilage structure and mechanical function. The apparatus was first tested in a lab environment, then the functionality and utility of the apparatus were examined during varus loading in a clinical 3T MRI system for human imaging. We found that the device enables quantitative MRI metrics for biomechanics and relaxometry data acquisition during joint loading leading to compression of the medial knee compartment. Integration with spiral DENSE MRI during cyclic loading provided time-dependent displacement and strain maps within the tibiofemoral cartilage. The results from these procedures demonstrate that the performance of this loading apparatus meets the design criteria and enables a simple and practical workflow for future studies of clinical cohorts, and the identification and validation of imaging-based biomechanical biomarkers.

Tissue engineering and future directions in regenerative medicine for knee cartilage repair: a comprehensive review.

This review evaluates the current landscape and future directions of regenerative medicine for knee cartilage repair, with a particular focus on tissue engineering strategies. In this context, scaffold-based approaches have emerged as promising solutions for cartilage regeneration. Synthetic scaffolds, while offering superior mechanical properties, often lack the biological cues necessary for effective tissue integration. Natural scaffolds, though biocompatible and biodegradable, frequently suffer from inadequate mechanical strength. Hybrid scaffolds, combining elements of both synthetic and natural materials, present a balanced approach, enhancing both mechanical support and biological functionality. Advances in decellularized extracellular matrix scaffolds have shown potential in promoting cell infiltration and integration with native tissues. Additionally, bioprinting technologies have enabled the creation of complex, bioactive scaffolds that closely mimic the zonal organization of native cartilage, providing an optimal environment for cell growth and differentiation. The review also explores the potential of gene therapy and gene editing techniques, including CRISPR-Cas9, to enhance cartilage repair by targeting specific genetic pathways involved in tissue regeneration. The integration of these advanced therapies with tissue engineering approaches holds promise for developing personalized and durable treatments for knee cartilage injuries and osteoarthritis. In conclusion, this review underscores the importance of continued multidisciplinary collaboration to advance these innovative therapies from bench to bedside and improve outcomes for patients with knee cartilage damage.

The effect of body weight on the knee joint biomechanics based on subject-specific finite element-musculoskeletal approach.

Knee osteoarthritis (OA) and obesity are major public health concerns that are closely intertwined. This intimate relationship was documented by considering obesity as the most significant preventable risk factor associated with knee OA. To date, however, the effects of obesity on the knee joint's passive-active structure and cartilage loading have been inconclusive. Hence, this study investigates the intricate relationship between obesity and knee OA, centering on the biomechanical changes in knee joint active and passive reactions during the stance phase of gait. Using a subject-specific musculoskeletal and finite element approach, muscle forces, ligament stresses, and articular cartilage contact stresses were analyzed among 60 individuals with different body mass indices (BMI) classified under healthy weight, overweight, and obese categories. Our predicted results showed that obesity significantly influenced knee joint mechanical reaction, increasing muscle activations, ligament loading, and articular cartilage contact stresses, particularly during key instances of the gait cycle-first and second peak loading instances. The study underscores the critical role of excessive body weight in exacerbating knee joint stress distribution and cartilage damage. Hence, the insights gained provide a valuable biomechanical perspective on the interaction between body weight and knee joint health, offering a clinical utility in assessing the risks associated with obesity and knee OA.

Site-specific elastic and viscoelastic biomechanical properties of healthy and osteoarthritic human knee joint articular cartilage.

Articular cartilage exhibits site-specific biomechanical properties. However, no study has comprehensively characterized site-specific cartilage properties from the same knee joints at different stages of osteoarthritis (OA). Cylindrical osteochondral explants (n = 381) were harvested from donor-matched lateral and medial tibia, lateral and medial femur, patella, and trochlea of cadaveric knees (N = 17). Indentation test was used to measure the elastic and viscoelastic mechanical properties of the samples, and Osteoarthritis Research Society International (OARSI) grading system was used to categorize the samples into normal (OARSI 0-1), early OA (OARSI 2-3), and advanced OA (OARSI 4-5) groups. OA-related changes in cartilage mechanical properties were site-specific. In the lateral and medial tibia and trochlea sites, equilibrium, instantaneous and dynamic moduli were higher (p < 0.001) in normal tissue than in early and advanced OA tissue. In lateral and medial femur, equilibrium, instantaneous and dynamic moduli were smaller in advanced OA, but not in early OA, than in normal tissue. The phase difference (0.1-0.25 Hz) between stress and strain was significantly smaller (p < 0.05) in advanced OA than in normal tissue across all sites except medial tibia. Our results indicated that in contrast to femoral and patellar cartilage, equilibrium, instantaneous and dynamic moduli of the tibia and trochlear cartilage decreased in early OA. These may suggest that the tibia and trochlear cartilage degrades faster than the femoral and patellar cartilage. The information is relevant for developing site-specific computational models and engineered cartilage constructs.

Ion-Induced Swelling Behavior of Articular Cartilage due to Non-Newtonian Flow and Its Effects on Fluid Pressure and Solid Displacement.

In this study, we examine the behavior of articular cartilage equilibrated in a salt (NaCl) solution during non-Newtonian fluid flow that follows an Ostwald-de Waele model. A linearly elastic and isotropic rectangular strip of cartilage is considered for analysis. A continuum theory of mixtures has been employed to develop a coupled system of partial differential equations for the solid displacement and the fluid pressure by considering the important factor of the ion concentration by assuming the cartilage as a deformable porous media. The coupled system of partial differential equations is solved using the numerical method named method of lines. In most cases, shear-thinning fluid is compared to the shear-thickening fluid to magnify the difference. Graphical results show that shear-thickening fluids bring more solid deformation and shows less fluid pressure in comparison to the shear-thinning fluids.

Brushing Up on Cartilage Lubrication: Polyelectrolyte-Enhanced Tribological Rehydration.

This study presents new insights into the potential role of polyelectrolyte interfaces in regulating low friction and interstitial fluid pressurization of cartilage. Polymer brushes composed of hydrophilic 3-sulfopropyl methacrylate potassium salt (SPMK) tethered to a PEEK substrate (SPMK-g-PEEK) are a compelling biomimetic solution for interfacing with cartilage, inspired by the natural lubricating biopolyelectrolyte constituents of synovial fluid. These SPMK-g-PEEK surfaces exhibit a hydrated compliant layer approximately 5 µm thick, demonstrating the ability to maintain low friction coefficients (µ ∼ 0.01) across a wide speed range (0.1-200 mm/s) under physiological loads (0.75-1.2 MPa). A novel polyelectrolyte-enhanced tribological rehydration mechanism is elucidated, capable of recovering up to ∼12% cartilage strain and subsequently facilitating cartilage interstitial fluid recovery, under loads ranging from 0.25 to 2.21 MPa. This is attributed to the combined effects of fluid confinement within the contact gap and the enhanced elastohydrodynamic behavior of polymer brushes. Contrary to conventional theories that emphasize interstitial fluid pressurization in regulating cartilage lubrication, this work demonstrates that SPMK-g-PEEK's frictional behavior with cartilage is independent of these factors and provides unabating aqueous lubrication. Polyelectrolyte-enhanced tribological rehydration can occur within a static contact area and operates independently of known mechanisms of cartilage interstitial fluid recovery established for converging or migrating cartilage contacts. These findings challenge existing paradigms, proposing a novel polyelectrolyte-cartilage tribological mechanism not exclusively reliant on interstitial fluid pressurization or cartilage contact geometry. The implications of this research extend to a broader understanding of synovial joint lubrication, offering insights into the development of joint replacement materials that more accurately replicate the natural functionality of cartilage.

Tibial and femoral articular cartilage exhibit opposite outcomes for T1ρ and T2* relaxation times in response to acute compressive loading in healthy knees.

Abnormal loading is thought to play a key role in the disease progression of cartilage, but our understanding of how cartilage compositional measurements respond to acute compressive loading in-vivo is limited. Ten healthy subjects were scanned at two timepoints (7 ± 3 days apart) with a 3 T magnetic resonance imaging (MRI) scanner. Scanning sessions included T1ρ and T2* acquisitions of each knee in two conditions: unloaded (traditional MRI setup) and loaded in compression at 40 % bodyweight as applied by an MRI-compatible loading device. T1ρ and T2* parameters were quantified for contacting cartilage (tibial and femoral) and non-contacting cartilage (posterior femoral condyle) regions. Significant effects of load were found in contacting regions for both T1ρ and T2*. The effect of load (loaded minus unloaded) in femoral contacting regions ranged from 4.1 to 6.9 ms for T1ρ, and 3.5 to 13.7 ms for T2*, whereas tibial contacting regions ranged from -5.6 to -1.7 ms for T1ρ, and -2.1 to 0.7 ms for T2*. Notably, the responses to load in the femoral and tibial cartilage revealed opposite effects. No significant differences were found in response to load between the two visits. This is the first study that analyzed the effects of acute loading on T1ρ and T2* measurements in human femoral and tibial cartilage separately. The results suggest the effect of acute compressive loading on T1ρ and T2* was: 1) opposite in the femoral and tibial cartilage; 2) larger in contacting regions than in non-contacting regions of the femoral cartilage; and 3) not different visit-to-visit.

Regeneration: AT-AMIC Technique: Limits and Indication.

Osteochondral lesion of the talus (OLT) is a commune cause of chronic ankle pain. Symptomatic lesions require surgical treatment. Currently, lesions with diameter less than 107.4 mm2 are treated with bone marrow stimulating technique with notable success rate. However, more extensive lesions show less predictable surgical results. Autologous matrix-induced chondrogenesis has proven to provide satisfactory medium and long-term results on OLTs. In the current review, we describe an all-arthroscopic technique and the Milan-Tel Aviv lesion assessment protocol.

An injectable active hydrogel based on BMSC-derived extracellular matrix for cartilage regeneration enhancement.

Articular cartilage injury impairs joint function and necessitates orthopedic intervention to restore the structure and function of the cartilage. Extracellular matrix (ECM) scaffolds derived from bone marrow mesenchymal stem cells (BMSCs) can effectively promote cell adhesion, proliferation, and chondrogenesis. However, pre-shaped ECM scaffolds have limited applicability due to their poor fit with the irregular surface of most articular cartilage defects. In this study, we fabricated an injectable active ECM hydrogel from autologous BMSCs-derived ECM by freeze-drying, liquid nitrogen milling, and enzymatic digestion. Moreover, our in vitro and in vivo results demonstrated that the prepared hydrogel enhanced chondrocyte adhesion and proliferation, chondrogenesis, cartilage regeneration, and integration with host tissue, respectively. These findings indicate that active ECM components can provide trophic support for cell proliferation and differentiation, restoring the structure and function of damaged cartilage.

Regeneration: Bone-Marrow Stimulation of the Talus-Limits and Goals.

Bone Marrow Stimulation of osteochondral lesions of the talus has been shown to be a successful way to treat cartilage injuries. Newer data suggest that Bone Marrow Stimulation is best reserved for osteochondral lesions of the talus Sizes Less Than 107.4 mm2 in area. Additionally, newer smaller and deeper techniques to perform bone marrow stimulation have resulted in less subchondral bone damage, less cancellous compaction, and superior bone marrow access with multiple trabecular access channels. Biologic adjuvants such as platelet-rich plasma (PRP), hyaluronic acid (HA), and bone marrow aspirate concentrate (BMAC) may lead to better functional outcomes when used concomitant to bone marrow stimulation.

[Effects of removing superficial layer of cartilage on the surface morphology and mechanical behavior of cartilage].

Superficial cartilage defect is an important factor that causes osteoarthritis. Therefore, it is very important to investigate the influence of superficial cartilage defects on its surface morphology and mechanical properties. In this study, the knee joint cartilage samples of adult pig were prepared, which were treated by enzymolysis with chymotrypsin and physical removal with electric friction pen, respectively. Normal cartilage and surface treated cartilage were divided into five groups: control group (normal cartilage group), chymotrypsin immersion group, chymotrypsin wiping group, removal 10% group with electric friction pen, and removal 20% group with electric friction pen. The surface morphology and structure of five groups of samples were characterized by laser spectrum confocal microscopy and environmental field scanning electron microscopy, and the mechanical properties of each group of samples were evaluated by tensile tests. The results show that the surface arithmetic mean height and fracture strength of the control group were the smallest, and the fracture strain was the largest. The surface arithmetic mean height and fracture strength of the removal 20% group with electric friction pen were the largest, and the fracture strain was the smallest. The surface arithmetic mean height, fracture strength and fracture strain values of the other three groups were all between the above two groups, but the surface arithmetic mean height and fracture strength of the removal 10% group with electric friction pen, the chymotrypsin wiping group and the chymotrypsin soaking group decreased successively, and the fracture strain increased successively. In addition, we carried out a study on the elastic modulus of different groups, and the results showed that the elastic modulus of the control group was the smallest, and the elastic modulus of the removal 20% group with electric friction pen was the largest. The above study revealed that the defect of the superficial area of cartilage changed its surface morphology and structure, and reduced its mechanical properties. The research results are of great significance for the prevention and repair of cartilage injury.

Computational modelling of articular joints with biphasic cartilage: recent advances, challenges and opportunities.

Biphasic models have been widely used to simulate the time-dependent biomechanical response of soft tissues. Modelling techniques of joints with biphasic weight-bearing soft tissues have been markedly improved over the last decade, enhancing our understanding of the function, degenerative mechanism and outcomes of interventions of joints. This paper reviews the recent advances, challenges and opportunities in computational models of joints with biphasic weight-bearing soft tissues. The review begins with an introduction of the function and degeneration of joints from a biomechanical aspect. Different constitutive models of articular cartilage, in particular biphasic materials, are illustrated in the context of the study of contact mechanics in joints. Approaches, advances and major findings of biphasic models of the hip and knee are presented, followed by a discussion of the challenges awaiting to be addressed, including the convergence issue, high computational cost and inadequate validation. Finally, opportunities and clinical insights in the areas of subject-specific modeling and tissue engineering are provided and discussed.

Regenerative capacity of human pluripotent stem cell-derived articular chondrocytes in vitro.

The ideal cell source for articular cartilage repair remains elusive. Using developmentally inspired differentiation protocols, we induced human pluripotent stem cells (hPSCs) toward articular chondrocytes capable of joint cartilage repair in rodent models, which were distinct from growth plate chondrocytes, fated to be replaced by bone in vivo. Working toward clinical translation, we demonstrated controlled differentiation into chondrocytes by comprehensive gene expression analysis at each step of the differentiation. Articular chondrocytes derived from hPSCs could be expanded several passages in vitro without losing chondrogenic potential. Furthermore, chondrocytes isolated from these articular cartilage tissues had the potential to serially regenerate new articular and growth plate cartilage tissues. Finally, the ability to cryopreserve articular chondrocytes with the desired phenotype is critical for clinical translation and here we report no loss in cell viability or regenerative potential following cryopreservation. These results support the immense potential of hPSC-derived articular chondrocytes as a cell-based therapy for cartilage repair.

Quantification of Cartilage Poroelastic Material Properties Via Analysis of Loading-Induced Cell Death.

Articular cartilage (AC) is a load-bearing tissue that covers long bones in synovial joints. The biphasic/poroelastic mechanical properties of AC help it to protect joints by distributing loads, absorbing impact forces, and reducing friction. Unfortunately, alterations in these mechanical properties adversely impact cartilage function and precede joint degeneration in the form of osteoarthritis (OA). Thus, understanding what factors regulate the poroelastic mechanical properties of cartilage is of great scientific and clinical interest. Transgenic mouse models provide a valuable platform to delineate how specific genes contribute to cartilage mechanical properties. However, the poroelastic mechanical properties of murine articular cartilage are challenging to measure due to its small size (thickness ∼ 50 microns). In the current study, our objective was to test whether the poroelastic mechanical properties of murine articular cartilage can be determined based solely on time-dependent cell death measurements under constant loading conditions. We hypothesized that in murine articular cartilage subjected to constant, sub-impact loading from an incongruent surface, cell death area and tissue strain are closely correlated. We further hypothesized that the relationship between cell death area and tissue strain can be used-in combination with inverse finite element modeling-to compute poroelastic mechanical properties. To test these hypotheses, murine cartilage-on-bone explants from different anatomical locations were subjected to constant loading conditions by an incongruent surface in a custom device. Cell death area increased over time and scaled linearly with strain, which rose in magnitude over time due to poroelastic creep. Thus, we were able to infer tissue strain from cell death area measurements. Moreover, using tissue strain values inferred from cell death area measurements, we applied an inverse finite element modeling procedure to compute poroelastic material properties and acquired data consistent with previous studies. Collectively, our findings demonstrate in the key role poroelastic creep plays in mediating cell survival in mechanically loaded cartilage and verify that cell death area can be used as a surrogate measure of tissue strain that enables determination of murine cartilage mechanical properties.

Ex vivo storage of human osteochondral allografts: Long-term analysis over 300 days using a Ringer-based solution.

Large osteochondral defects are a major challenge in orthopedics, for which osteochondral allograft (OCA) transplantation is nowadays considered as an option, especially in young patients. However, a major issue with OCA is the need for graft storage, which ensures adequate cartilage integrity over time. The aim of this study was to test how long a Ringer-based storage solution can provide good graft quality after explantation and thus meet the requirements for OCA. For this purpose, human osteochondral allografts of the knee and ankle were analyzed. Live/Dead analysis was performed and glycosaminoglycan, as well as hydroxyproline content, were measured as crucial chondrocyte integrity factors. Furthermore, biomechanical tests focusing on stress relaxation and elastic compression modulus were performed. The critical value of 70% living chondrocytes, which corresponds to a number of 300 cells/mm², was reached after an average of 16 weeks of storage. In addition, a constant cell shrinkage was observed over time. The amount of glycosaminoglycan and hydroxyroline showed a slight and constant decrease over time, but no significant differences when compared from Day 0 to the values at Weeks 40-43. Biomechanical testing also revealed no significant differences at the different time points. Therefore, the results show that the Ringer-based storage solution at 4°C is able to provide a chondrocyte survival of 70% until Week 16. This is comparable to previously published storage solutions. Therefore, the study contributes to the establishment of a Ringer-based osteochondral allograft transplantation system for countries where medium-based storage solution cannot be approved.

Biomechanical properties of articular cartilage in different regions and sites of the knee joint: acquisition of osteochondral allografts.

Osteochondral allograft (OCA) transplantation involves grafting of natural hyaline cartilage and supporting subchondral bone into the cartilage defect area to restore its biomechanical and tissue structure. However, differences in biomechanical properties and donor-host matching may impair the integration of articular cartilage (AC). This study analyzed the biomechanical properties of the AC in different regions of different sites of the knee joint and provided a novel approach to OCA transplantation. Intact stifle joints from skeletally mature pigs were collected from a local abattoir less than 8 h after slaughter. OCAs were collected from different regions of the joints. The patella and the tibial plateau were divided into medial and lateral regions, while the trochlea and femoral condyle were divided into six regions. The OCAs were analyzed and compared for Young's modulus, the compressive modulus, and cartilage thickness. Young's modulus, cartilage thickness, and compressive modulus of OCA were significantly different in different regions of the joints. A negative correlation was observed between Young's modulus and the proportion of the subchondral bone (r = - 0.4241, P < 0.0001). Cartilage thickness was positively correlated with Young's modulus (r = 0.4473, P < 0.0001) and the compressive modulus (r = 0.3678, P < 0.0001). During OCA transplantation, OCAs should be transplanted in the same regions, or at the closest possible regions to maintain consistency of the biomechanical properties and cartilage thickness of the donor and recipient, to ensure smooth integration with the surrounding tissue. A 7 mm depth achieved a higher Young's modulus, and may represent the ideal length.

Mechanobiology of Hyaluronan: Connecting Biomechanics and Bioactivity in Musculoskeletal Tissues.

Hyaluronan (HA) plays well-recognized mechanical and biological roles in articular cartilage and synovial fluid, where it contributes to tissue structure and lubrication. An understanding of how HA contributes to the structure of other musculoskeletal tissues, including muscle, bone, tendon, and intervertebral discs, is growing. In addition, the use of HA-based therapies to restore damaged tissue is becoming more prevalent. Nevertheless, the relationship between biomechanical stimuli and HA synthesis, degradation, and signaling in musculoskeletal tissues remains understudied, limiting the utility of HA in regenerative medicine. In this review, we discuss the various roles and significance of endogenous HA in musculoskeletal tissues. We use what is known and unknown to motivate new lines of inquiry into HA biology within musculoskeletal tissues and in the mechanobiology governing HA metabolism by suggesting questions that remain regarding the relationship and interaction between biological and mechanical roles of HA in musculoskeletal health and disease.

Regular Running Is Related to the Knee Joint Cartilage Structure in Healthy Adults.

PURPOSE: The purpose of this study was to determine whether regular running distance and biomechanics are related to medial central femur cartilage (MCFC) structure. METHODS: The cross-sectional study sample consisted of 1164 runners and nonrunners aged 18-65 yr. Participants completed questionnaires on physical activity and their running history. We performed quantitative magnetic resonance imaging of knee cartilage-T2 relaxation time (T2) mapping (high T2 indicates cartilage degeneration)-and a running biomechanical analysis using a three-dimensional motion capture system. A 14-d monitoring of the physical activity was conducted. RESULTS: Those aged 35-49 yr were at 84% higher odds of having MCFC T2 in the highest level (85th percentile, P < 0.05) compared with youngest adults indicating that MCFC structures may be altered with aging. Being male was associated with 34% lower odds of having T2 at the highest level ( P < 0.05) compared with females. Nonrunners and runners with the highest weekly running distance were more likely to have a high T2 compared with runners with running distance of 6-20 km·wk -1 ( P < 0.05). In addition, the maximal knee internal adduction moment was associated with a 19% lower odds of having T2 at the highest level ( P < 0.05). CONCLUSIONS: Females compared with males and a middle-aged cohort compared with the younger cohort seemed to be associated with the degeneration of MCFC structures. Runners who ran 6-20 km·wk -1 were associated with a higher quality of their MCFC compared with highly active individuals and nonrunners. Knee frontal plane biomechanics was related to MCFC structure indicating a possibility of modifying the medial knee collagen fibril network through regular running.

Pericellular Matrix Formation and Atomic Force Microscopy of Single Primary Human Chondrocytes Cultured in Alginate Microgels.

One of the main components of articular cartilage is the chondrocyte's pericellular matrix (PCM), which is critical for regulating mechanotransduction, biochemical cues, and healthy cartilage development. Here, individual primary human chondrocytes (PHC) are encapsulated and cultured in 50 µm diameter alginate microgels using drop-based microfluidics. This unique culturing method enables PCM formation and manipulation of individual cells. Over ten days, matrix formation is observed using autofluorescence imaging, and the elastic moduli of isolated cells are measured using AFM. Matrix production and elastic modulus increase are observed for the chondrons cultured in microgels. Furthermore, the elastic modulus of cells grown in microgels increases ≈ten-fold over ten days, nearly reaching the elastic modulus of in vivo PCM. The AFM data is further analyzed using a Gaussian mixture model and shows that the population of PHCs grown in microgels exhibit two distinct populations with elastic moduli averaging 9.0 and 38.0 kPa. Overall, this work shows that microgels provide an excellent culture platform for the growth and isolation of PHCs, enabling PCM formation that is mechanically similar to native PCM. The microgel culture platform presented here has the potential to revolutionize cartilage regeneration procedures through the inclusion of in vitro developed PCM.