Mesenchymal stromal cells modulate YAP by verteporfin to mimic cartilage development and construct cartilage organoids based on decellularized matrix scaffolds.

The mechanical elasticity or stiffness of the ECM modulates YAP activity to regulate the differentiation of stem cells during the development and defect regeneration of cartilage tissue. However, the understanding of the scaffold-associated mechanobiology during the initiation of chondrogenesis and hyaline cartilaginous phenotype maintenance remains unclear. In order to elucidate such mechanisms to promote articular cartilage repair by producing more hyaline cartilage, we identify the relationship between YAP subcellular localization and variation of the cartilage structure and organization during the early postnatal explosive growth in incipient articular cartilage. Next, we prepared a decellularized cartilage scaffold with different stiffness (2-33 kPa) to investigate the effect of scaffold stiffness on the formation of hyaline cartilage by mesenchymal stem cells and the change of YAP activity. Furthermore, we simulated the decrease of cellular YAP activity during postnatal cartilage development by inhibiting YAP activity with verteporfin, and realized that the timing of drug incorporation was critical to regulate the differentiation of MSCs to hyaline chondrocytes and inhibit their hypertrophy and fibrosis. On this basis, we constructed hyaline cartilage organoids by decellularized matrix scaffolds. Collectively, the results herein demonstrate that YAP plays a critical role during in vitro chondrogenic differentiation which is tightly regulated by biochemical and mechanical regulation.

Dipolar Relaxation of Water Protons in the Vicinity of a Collagen-like Peptide.

Quantitative magnetic resonance imaging is one of the few available methods for noninvasive diagnosis of degenerative changes in articular cartilage. The clinical use of the imaging data is limited by the lack of a clear association between structural changes at the molecular level and the measured magnetic relaxation times. In anisotropic, collagen-containing tissues, such as articular cartilage, the orientation dependency of nuclear magnetic relaxation can obscure the content of the images. Conversely, if the molecular origin of the phenomenon would be better understood, it would provide opportunities for diagnostics as well as treatment planning of degenerative changes in these tissues. We study the magnitude and orientation dependence of the nuclear magnetic relaxation due to dipole-dipole coupling of water protons in anisotropic, collagenous structures. The water-collagen interactions are modeled with molecular dynamics simulations of a small collagen-like peptide dissolved in water. We find that in the vicinity of the collagen-like peptide, the dipolar relaxation of water hydrogen nuclei is anisotropic, which can result in orientation-dependent relaxation times if the water remains close to the peptide. However, the orientation-dependency of the relaxation is different from the commonly observed magic-angle phenomenon in articular cartilage MRI.

Raman needle arthroscopy for in vivo molecular assessment of cartilage.

The development of treatments for osteoarthritis (OA) is burdened by the lack of standardized biomarkers of cartilage health that can be applied in clinical trials. We present a novel arthroscopic Raman probe that can "optically biopsy" cartilage and quantify key extracellular matrix (ECM) biomarkers for determining cartilage composition, structure, and material properties in health and disease. Technological and analytical innovations to optimize Raman analysis include (1) multivariate decomposition of cartilage Raman spectra into ECM-constituent-specific biomarkers (glycosaminoglycan [GAG], collagen [COL], water [H2 O] scores), and (2) multiplexed polarized Raman spectroscopy to quantify superficial zone (SZ) COL anisotropy via a partial least squares-discriminant analysis-derived Raman collagen alignment factor (RCAF). Raman measurements were performed on a series of ex vivo cartilage models: (1) chemically GAG-depleted bovine cartilage explants (n = 40), (2) mechanically abraded bovine cartilage explants (n = 30), (3) aging human cartilage explants (n = 14), and (4) anatomical-site-varied ovine osteochondral explants (n = 6). Derived Raman GAG score biomarkers predicted 95%, 66%, and 96% of the variation in GAG content of GAG-depleted bovine explants, human explants, and ovine explants, respectively (p < 0.001). RCAF values were significantly different for explants with abrasion-induced SZ COL loss (p < 0.001). The multivariate linear regression of Raman-derived ECM biomarkers (GAG and H2 O scores) predicted 94% of the variation in elastic modulus of ovine explants (p < 0.001). Finally, we demonstrated the first in vivo Raman arthroscopy assessment of an ovine femoral condyle through intraarticular entry into the synovial capsule. This study advances Raman arthroscopy toward a transformative low-cost, minimally invasive diagnostic platform for objective monitoring of treatment outcomes from emerging OA therapies.

Childhood Cartilage ECM Enhances the Chondrogenesis of Endogenous Cells and Subchondral Bone Repair of the Unidirectional Collagen-dECM Scaffolds in Combination with Microfracture.

Despite the formation of mechanically inferior fibrocartilage, microfracture (MF) still remains the gold standard to repair the articular cartilage defects in clinical settings. To date, although many tissue-engineering scaffolds have been developed to enhance the MF outcome, the clinical outcomes remain inconsistent. Decellularized extracellular matrix (dECM) is among the most promising scaffold for cartilage repair due to its inheritance of the natural cartilage components. However, the impact of dECM from different developmental stages on cellular chondrogenesis and therapeutic effect remains elusive, as the development of native cartilage involves the distinct temporal dependency of the ECM components and various growth factors. Herein, we hypothesized that the immature cartilage dECM at various developmental stages was inherently different, and would consequently impact the chondrogenic potential BMSCs. In this study, we fabricated three different unidirectional collagen-dECM scaffolds sourced from neonatal, childhood, and adolescent rabbit cartilage tissues, and identified the age-dependent biological variations, including DNA, cartilage-specific proteins, and growth factors; along with the mechanical and degradation differences. Consequently, the different local cellular microenvironments provided by these scaffolds led to the distinctive cell morphology, circularity, proliferation, chondrogenic genes expression, and chondrogenesis of BMSCs in vitro, and the different gross morphology, cartilage-specific protein production, and subchondral bone repair when in combination with microfracture in vivo. Together, this work highlights the immature cartilage dECM at different developmental stages that would result in the diversified effects to BMSCs, and childhood cartilage would be considered the optimal dECM source for the further development of dECM-based tissue engineering scaffolds in articular cartilage repair.

Resolving the Near-Infrared Spectrum of Articular Cartilage.

OBJECTIVE: Spectroscopic techniques, such as near-infrared (NIR) spectroscopy, are gaining significant research interest for characterizing connective tissues, particularly articular cartilage, because there is still a largely unmet need for rapid, accurate and objective methods for assessing tissue integrity in real-time during arthroscopic surgery. This study aims to identify the NIR spectral range that is optimal for characterizing cartilage integrity by (a) identifying the contribution of its major constituents (collagen and proteoglycans) to its overall spectrum using proxy constituent models and (b) determining constituent-specific spectral contributions that can be used for assessment of cartilage in its physiological state. DESIGN: The NIR spectra of cartilage matrix constituent models were measured and compared with specific molecular components of organic compounds in the NIR spectral range in order to identify their bands and molecular assignments. To verify the identified bands, spectra of the model compounds were compared with those of native cartilage. Since water obscures some bands in the NIR range, spectral measurements of the native cartilage were conducted under conditions of decreasing water content to amplify features of the solid matrix components. The identified spectral bands were then compared and examined in the resulting spectra of the intact cartilage samples. RESULTS: As water was progressively eliminated from cartilage, the specific contribution of the different matrix components was observed to correspond with those identified from the proxy cartilage component models. CONCLUSION: Spectral peaks in the regions 5500 to 6250 cm-1 and 8100 to 8600 cm-1 were identified to be effective for characterizing cartilage proteoglycan and collagen contents, respectively.

Cell-free scaffolds functionalized with bionic cartilage acellular matrix microspheres to enhance the microfracture treatment of articular cartilage defects.

Microfracture surgery remains the most popular treatment for articular cartilage lesions in the clinic, but often leads to the formation of inferior fibrocartilage tissue and damage to subchondral bone. To overcome these problems, extracellular matrix (ECM) scaffolds derived from decellularized natural cartilaginous tissues were introduced and showed excellent biological properties to direct the differentiation of bone marrow stem cells. However, besides the limited allogenic/allogenic supply and the risk of disease transfer from xenogeneic tissues, the effectiveness of ECM scaffolds always varied with a high variability of natural tissue quality. In this study, we developed composite scaffolds functionalized with a cell-derived ECM source, namely, bionic cartilage acellular matrix microspheres (BCAMMs), that support the chondrogenic differentiation of bone marrow cells released from microfracture. The scaffolds with BCAMMs at different developmental stages were investigated in articular cartilage regeneration and subchondral bone repair. Compared to microfracture, the addition of cell-free BCAMM scaffolds has demonstrated a great improvement of regenerated cartilage tissue quality in a rabbit model as characterized by a semi-quantitative analysis of cells, histology and biochemical assays as well as micro-CT images. Moreover, the variation in ECM properties was found to significantly affect the cartilage regeneration, highlighting the challenges of homogenous scaffolds in working with microfracture. Together, our results demonstrate that the biofunctionalized BCAMM scaffold with cell-derived ECM shows great potential to combine with microfracture for clinical translation to repair cartilage defects.

Early Detection of Cartilage Degeneration: A Comparison of Histology, Fiber Bragg Grating-Based Micro-Indentation, and Atomic Force Microscopy-Based Nano-Indentation.

We have determined the sensitivity and detection limit of a new fiber Bragg grating (FBG)-based optoelectronic micro-indenter for biomechanical testing of cartilage and compared the results to indentation-type atomic force microscopy (IT-AFM) and histological staining. As test samples, we used bovine articular cartilage, which was enzymatically degraded ex vivo for five minutes using different concentrations of collagenase (5, 50, 100 and 500 µg/mL) to mimic moderate extracellular matrix deterioration seen in early-stage osteoarthritis (OA). Picrosirius Red staining and polarization microscopy demonstrated gradual, concentration-dependent disorganization of the collagen fibrillar network in the superficial zone of the explants. Osteoarthritis Research Society International (OARSI) grading of histopathological changes did not discriminate between undigested and enzymatically degraded explants. IT-AFM was the most sensitive method for detecting minute changes in cartilage biomechanics induced by the lowest collagenase concentration, however, it did not distinguish different levels of cartilage degeneration for collagenase concentrations higher than 5 µg/mL. The FBG micro-indenter provided a better and more precise assessment of the level of cartilage degeneration than the OARSI histological grading system but it was less sensitive at detecting mechanical changes than IT-AFM. The FBG-sensor allowed us to observe differences in cartilage biomechanics for collagenase concentrations of 100 and 500 µg/mL. Our results confirm that the FBG sensor is capable of detecting small changes in articular cartilage stiffness, which may be associated with initial cartilage degeneration caused by early OA.

Wet-electrospun PHBV nanofiber reinforced carboxymethyl chitosan-silk hydrogel composite scaffolds for articular cartilage repair.

The objective of the study was to produce three-dimensional and porous nanofiber reinforced hydrogel scaffolds that can mimic the hydrated composite structure of the cartilage extracellular matrix. In this regard, wet-electrospun poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) nanofiber reinforced carboxymethyl chitosan-silk fibroin (PNFs/CMCht-SF) hydrogel composite scaffolds that were chemically cross-linked by poly(ethylene glycol) diglycidyl ether (PEGDE) were produced. To the best of our knowledge, this is the first study in cartilage regeneration where a three dimensional porous spongy composite scaffold was obtained by the dispersion of wet-electrospun nanofibers within a polymer matrix. All of the produced hydrogel composite scaffolds had an interconnected microporous structure with well-integrated PHBV nanofibers on the pore walls. The scaffold comprising an equal amount of PEGDE and polymer (PNFs/CMCht-SF1:PEGDE1) demonstrated comparable water content (91.4 ± 0.7%), tan δ (0.183 at 1 Hz) and compressive strength (457 ± 85 kPa) values to that of articular cartilage. Besides, based on the histological analysis, this hydrogel composite scaffold supported the chondrogenic differentiation of bone marrow mesenchymal stem cells. Consequently, this hydrogel composite scaffold presented a great promise for cartilage tissue regeneration.

The study of targeted blocking SDF-1/CXCR4 signaling pathway with three antagonists on MMPs, type II collagen, and aggrecan levels in articular cartilage of guinea pigs.

OBJECTIVE: To explore the possibility and mechanism of targeted blocking SDF-1/CXCR4 signaling pathway using three antagonists TN14003, T140, and AMD3100 in vivo, and to investigate the function of three antagonists in delay degeneration process of articular cartilage. METHODS: Ninety-six male Duncan-Hartley guinea pigs (6 months old) were divided into groups A, B, C, and D randomly. Alzet trace pump was implanted in the back subcutaneous tissue of pigs in group A, and TN14003 with concentration of 180 µg/ml was pumped every day. Alzet trace pump was implanted in the back subcutaneous tissue of pigs in group B, and T140 with concentration of 180 µg/ml was pumped every day. Alzet trace pump was implanted in the back subcutaneous tissue of pigs in group C, and AMD3100 with concentration of 180 µg/ml was pumped every day. Hartley guinea pigs in group D remained untreated as the blank control group. At 2, 4, 6, 8, 10, and 12 weeks of treatment, 5 to 8 animals in each group were randomly chosen for blood collection via cardiac puncture. SDF-1 content using enzyme-linked immunosorbent assay (ELISA). At 12 weeks, all guinea pigs were sacrificed by injecting pentobarbital sodium (30 mg/kg) into the peritoneal cavity. Cartilages from the tibial plateau in each group were harvested for PCR testing and western blot analysis. SPSS19.0 was used for data analysis. RESULTS: Result of ELISA: the serum levels of SDF-1 of groups A, B, and C decreased gradually with time. Significant drop of SDF-1 level was seen in group A while increased SDF-1 was shown in group D. At the same time, the serum levels of SDF-1 of the group A were significantly lower than that of group B; those of group B were significantly lower than that of group C, which was significantly lower than that of group D, and their difference is statistically significant (P < 0.05). Real time quantitative PCR result: The mRNA levels of MMPs in group A were significantly lower than group B, and those of group B were significantly lower than group C, which was significantly lower than group D, and there was statistically significant (P < 0.05). The mRNA levels of type II collagen, aggrecan in group A were significantly more than group B; those of group B were significantly more than group C, which was significantly more than group D, and the difference was statistically significant (P < 0.05). H&E staining result: cartilage of group C was more significantly degenerative than other groups. CONCLUSIONS: The three antagonists can target SDF-1/CXCR4 signaling pathway in vivo, reduce the expression and secretion of MMP-3, MMP-9, and MMP-13 in cartilage tissue, and reduce the degradation of collagen II and aggregating proteoglycan, thus delaying the degeneration of articular cartilage, of which TN14003 has the strongest regulatory effect. Targeted blockade of SDF-1/CXCR4 signaling pathway by TN14003 in vivo delays articular cartilage degeneration more effectively than T140 and AMD3100.

The Optimal Regimen for the Treatment of Temporomandibular Joint Injury Using Low-Intensity Pulsed Ultrasound in Rats with Chronic Sleep Deprivation.

Low-intensity pulsed ultrasound (LIPUS) is an emerging physical therapy for the treatment of early temporomandibular joint injury and has a good effect on promoting cartilage and subchondral bone tissue repair. However, the best LIPUS intensity and treatment duration remain unclear. This study is aimed at observing the preventive and therapeutic effects of different modes of LIPUS and at identifying the optimal LIPUS treatment regimen for temporomandibular joint injury. In the present study, rat models of temporomandibular joint injury were established using a chronic sleep deprivation (CSD) method, and the effect of LIPUS as intensities of 30, 45, and 60 mW/cm2 was observed at 7, 14, and 21 days. After CSD, the condylar cartilage of the rats demonstrated variable degrees of surface roughening, collagen fiber disarrangement or even partial exfoliation, decreased proteoglycan synthesis and cartilage thickness, decreased chondrocyte proliferation, decreased type 2 collagen (COL-2) expression, and increased matrix metalloproteinase- (MMP-) 3 expression at all three time points. When the rats with CSD received different intensities of LIPUS treatment, the pathological changes were alleviated to various extents. The groups receiving 45 mW/cm2 LIPUS showed the most significant relief of cartilage damage, and this significant effect was observed on days 14 and 21. These results demonstrated that LIPUS can effectively inhibit CSD-induced condylar cartilage damage in rats, and LIPUS treatment at an intensity of 45 mW/cm2 for at least 2 weeks is the optimal regimen for temporomandibular joint injury.

MiR-9 facilitates cartilage regeneration of osteoarthritis in rabbits through regulating Notch signaling pathway.

OBJECTIVE: To explore the effect of micro ribonucleic acid (miR-9) on cartilage regeneration of osteoarthritis in rabbits through the regulation of the Notch signaling pathway. MATERIALS AND METHODS: A total of 30 specific pathogen-free Sprague-Dawley rabbits were randomly divided into control group (healthy rabbits, n=10), model group (osteoarthritis model, n=10) and miR-9 group (osteoarthritis model + miR-9 interference, n=10). The degeneration degree of rabbit knee articular cartilage in three groups was assessed through the Mankin's score. The morphology of cartilage tissues was observed under an optical microscope. Expressions of the Notch1, B-cell lymphoma-2 (Bcl-2), Bcl-2 associated X protein (Bax) proteins, and collagen II (CII) in chondrocytes were detected via the immunohistochemical assay. RESULTS: In rabbits of control group, the articular cartilage had a smooth surface and complete structure, and the cells were arranged orderly with a clear tidal line. A large number of articular chondrocytes died in model group, while it was improved in miR-9 group. The Mankin's score was 3.52±0.79 points in control group, 6.73±0.95 points in model group, and 5.37±0.61 points in miR-9 group, showing significant differences among the three groups (p<0.05). Results of immunohistochemistry showed that the protein expressions of Notch1 and Bax were higher in model group, but lower in control group and miR-9 group (p<0.05). The protein expression of Bcl-2 was lower in model group but was upregulated in control group and miR-9 group (p<0.05). The results of Reverse Transcription-Polymerase Chain Reaction (RT-PCR) revealed that the expressions of Notch1 and Bax in control group were lower than those in model group and miR-9 group (p<0.05), while the expression of Bcl-2 in model group was lower than that in control group and miR-9 group (p<0.05). According to the results of immunohistochemistry, the CII optical density (OD) value was 0.18±0.013, 0.25±0.05 and 0.22±0.009 in control group, model group, and miR-9 group, respectively. It could be seen that the CII OD value was the highest in model group, indicating that the CII expression in articular cartilage in osteoarthritis was negatively correlated with the severity of osteoarthritis. CONCLUSIONS: MiR-9, through the down-regulation of the expressions of Notch and Bax, can activate the Bcl-2 to promote the differentiation and regeneration of chondrocytes. It can facilitate the cartilage regeneration of osteoarthritis in rabbits through the mediation of the CII expression.

Arthroscopic Determination of Cartilage Proteoglycan Content and Collagen Network Structure with Near-Infrared Spectroscopy.

Conventional arthroscopic evaluation of articular cartilage is subjective and insufficient for assessing early compositional and structural changes during the progression of post-traumatic osteoarthritis. Therefore, in this study, arthroscopic near-infrared (NIR) spectroscopy is introduced, for the first time, for in vivo evaluation of articular cartilage thickness, proteoglycan (PG) content, and collagen orientation angle. NIR spectra were acquired in vivo and in vitro from equine cartilage adjacent to experimental cartilage repair sites. As reference, digital densitometry and polarized light microscopy were used to evaluate superficial and full-thickness PG content and collagen orientation angle. To relate NIR spectra and cartilage properties, ensemble neural networks, each with two different architectures, were trained and evaluated by using Spearman's correlation analysis (ρ). The ensemble networks enabled accurate predictions for full-thickness reference properties (PG content: ρin vitro, Val= 0.691, ρin vivo= 0.676; collagen orientation angle: ρin vitro, Val= 0.626, ρin vivo= 0.574) from NIR spectral data. In addition, the networks enabled reliable prediction of PG content in superficial (25%) cartilage (ρin vitro, Val= 0.650, ρin vivo= 0.613) and cartilage thickness (ρin vitro, Val= 0.797, ρin vivo= 0.596). To conclude, NIR spectroscopy could enhance the detection of initial cartilage degeneration and thus enable demarcation of the boundary between healthy and compromised cartilage tissue during arthroscopic surgery.

Light Absorptive Properties of Articular Cartilage, ECM Molecules, Synovial Fluid, and Photoinitiators as Potential Barriers to Light-Initiated Polymer Scaffolding Procedures.

OBJECTIVE: Many in vivo procedures to repair chondral defects use ultraviolet (UV)-photoinitiated in situ polymerization within the cartilage matrix. Chemical species that absorb UV light might reduce the effectiveness of these procedures by acting as light absorption barriers. This study evaluated whether any of the individual native biochemical components in cartilage and synovial fluid interfered with the absorption of light by common scaffolding photosensitizers. MATERIALS: UV-visible spectroscopy was performed on each major component of cartilage in solution, on bovine synovial fluid, and on four photosensitizers, riboflavin, Irgacure 2959, quinine, and riboflavin-5'-phosphate. Molar extinction and absorption coefficients were calculated at wavelengths of maximum absorbance and 365 nm. Intact articular cartilage was also examined. RESULTS: The individual major biochemical components of cartilage, Irgacure 2959, and quinine did not exhibit a significant absorption at 365 nm. Riboflavin and riboflavin-5'-phosphate were more effectual light absorbers at 365 nm, compared with the individual native species. Intact cartilage absorbed a significantly greater amount of UV light in comparison with the native species. CONCLUSION: Our results indicate that none of the individual native species in cartilage will interfere with the absorption of UV light at 365 nm by these commonly used photoinitiators. Intact cartilage slices exhibited significant light absorption at 365 nm, while also having distinct absorbance peaks at wavelengths less than 300 nm. Determining the UV absorptive properties of the biomolecules native to articular cartilage and synovial fluid will aid in optimizing scaffolding procedures to ensure sufficient scaffold polymerization at a minimum UV intensity.

Sequential Zonal Chondrogenic Differentiation of Mesenchymal Stem Cells in Cartilage Matrices.

IMPACT STATEMENT: The higher regenerative capacity of fetal articular cartilage compared with the adult is rooted in differences in cell density and matrix composition. We hypothesized that the zonal organization of articular cartilage can be engineered by encapsulation of mesenchymal stem cells in a single superficial zone-like matrix followed by sequential addition of zone-specific growth factors within the matrix, similar to the process of fetal cartilage development. The results demonstrate that the zonal organization of articular cartilage can potentially be regenerated using an injectable, monolayer cell-laden hydrogel with sequential release of growth factors.

Tissue-specific extracellular matrix scaffolds for the regeneration of spatially complex musculoskeletal tissues.

Biological scaffolds generated from tissue-derived extracellular matrix (ECM) are commonly used clinically for soft tissue regeneration. Such biomaterials can enhance tissue-specific differentiation of adult stem cells, suggesting that structuring different ECMs into multi-layered scaffolds can form the basis of new strategies for regenerating damaged interfacial tissues such as the osteochondral unit. In this study, mass spectrometry is used to demonstrate that growth plate (GP) and articular cartilage (AC) ECMs contain a unique array of regulatory proteins that may be particularly suited to bone and cartilage repair respectively. Applying a novel iterative freeze-drying method, porous bi-phasic scaffolds composed of GP ECM overlaid by AC ECM are fabricated, which are capable of spatially directing stem cell differentiation in vitro, promoting the development of graded tissues transitioning from calcified cartilage to hyaline-like cartilage. Evaluating repair 12-months post-implantation into critically-sized caprine osteochondral defects reveals that these scaffolds promote regeneration in a manner distinct to commercial control-scaffolds. The GP layer supports endochondral bone formation, while the AC layer stimulates the formation of an overlying layer of hyaline cartilage with a collagen fiber architecture better recapitulating the native tissue. These findings support the use of a bi-layered, tissue-specific ECM derived scaffolds for regenerating spatially complex musculoskeletal tissues.

Gait Mechanics and T1ρ MRI of Tibiofemoral Cartilage 6 Months after ACL Reconstruction.

PURPOSE: Aberrant walking biomechanics after anterior cruciate ligament reconstruction (ACLR) are hypothesized to be associated with deleterious changes in knee cartilage. T1ρ magnetic resonance imaging (MRI) is sensitive to decreased proteoglycan density of cartilage. Our purpose was to determine associations between T1ρ MRI interlimb ratios (ILR) and walking biomechanics 6 months after ACLR. METHODS: Walking biomechanics (peak vertical ground reaction force (vGRF), vGRF loading rate, knee extension moment, knee abduction moment) were extracted from the first 50% of stance phase in 29 individuals with unilateral ACLR. T1ρ MRI ILR (ACLR limb/uninjured limb) was calculated for regions of interest in both medial and lateral femoral (LFC) and medial and lateral tibial condyles. Separate, stepwise linear regressions were used to determine associations between biomechanical outcomes and T1ρ MRI ILR after accounting for walking speed and meniscal/chondral injury (P ≤ 0.05). RESULTS: Lesser peak vGRF in the ACLR limb was associated with greater T1ρ MRI ILR for the LFC (posterior ΔR = 0.14, P = 0.05; central ΔR = 0.15, P = 0.05) and medial femoral condyle (central ΔR = 0.24, P = 0.01). Lesser peak vGRF loading rate in the ACLR limb (ΔR = 0.21, P = 0.02) and the uninjured limb (ΔR = 0.27, P = 0.01) was associated with greater T1ρ MRI ILR for the anterior LFC. Lesser knee abduction moment for the injured limb was associated with greater T1ρ MRI ILR for the anterior LFC (ΔR = 0.16, P = 0.04) as well as the posterior medial tibial condyle (ΔR = 0.13, P = 0.04). CONCLUSION: Associations between outcomes related to lesser mechanical loading during walking and greater T1ρ MRI ILR were found 6 months after ACLR. Although preliminary, our results suggest that underloading of the ACLR limb at 6 months after ACLR may be associated with lesser proteoglycan density in the ACLR limb compared with the uninjured limb.

Quadriceps weakness associates with greater T1ρ relaxation time in the medial femoral articular cartilage 6 months following anterior cruciate ligament reconstruction.

PURPOSE: Quadriceps weakness following anterior cruciate ligament reconstruction (ACLR) is linked to decreased patient-reported function, altered lower extremity biomechanics and tibiofemoral joint space narrowing. It remains unknown if quadriceps weakness is associated with early deleterious changes to femoral cartilage composition that are suggestive of posttraumatic osteoarthritis development. The purpose of the cross-sectional study was to determine if quadriceps strength was associated with T1ρ relaxation times, a marker of proteoglycan density, of the articular cartilage in the medial and lateral femoral condyles 6 months following ACLR. It is hypothesized that individuals with weaker quadriceps would demonstrate lesser proteoglycan density. METHODS: Twenty-seven individuals (15 females, 12 males) with a patellar tendon autograft ACLR underwent isometric quadriceps strength assessments in 90°of knee flexion during a 6-month follow-up exam. Magnetic resonance images (MRI) were collected bilaterally and voxel by voxel T1ρ relaxation times were calculated using a five-image sequence and a monoexponential equation. Following image registration, the articular cartilage for the weight-bearing surfaces of the medial and lateral femoral condyles (MFC and LFC) were manually segmented and further sub-sectioned into posterior, central and anterior regions of interest (ROI) based on the corresponding meniscal anatomy viewed in the sagittal plane. Univariate linear regression models were used to determine the association between quadriceps strength and T1ρ relaxation times in the entire weight-bearing MFC and LFC, as well as the ROI in each respective limb. RESULTS: Lesser quadriceps strength was significantly associated with greater T1ρ relaxation times in the entire weight-bearing MFC (R2 = 0.14, P = 0.05) and the anterior-MFC ROI (R2 = 0.22, P = 0.02) of the ACLR limb. A post hoc analysis found lesser strength and greater T1ρ relaxation times were significantly associated in a subsection of participants (n = 18) without a concomitant medial tibiofemoral compartment meniscal or chondral injury in the entire weight-bearing MFC, as well as anterior-MFC and central-MFC ROI of the ACLR and uninjured limb. CONCLUSIONS: The association between weaker quadriceps and greater T1ρ relaxation times in the MFC suggests deficits in lower extremity muscle strength may be related to cartilage composition as early as 6 months following ACLR. Maximizing quadriceps strength in the first 6 months following ACLR may be critical for promoting cartilage health early following ACLR. LEVEL OF EVIDENCE: Prognostic level 1.

Bionic cartilage acellular matrix microspheres as a scaffold for engineering cartilage.

Extracellular matrix (ECM) scaffolds made from decellularized natural cartilage have been successfully used in cartilage lesion repair, but allogeneic cartilage donors are always in shortage and xenogeneic cartilage tissues may have the risk of unknown disease transfer. In this study, we constructed artificial bionic cartilage microspheres by encapsulating MSCs in collagen microspheres and cultured in a chondrogenic-inducing medium. Then, acellular matrix microsphere (BCAMM) scaffolds were fabricated from the cultured microspheres at three different developmental stages. A novel technique was introduced to fabricate BCAMM scaffolds, which enabled the production and utilization of the scaffolds in a short time. Due to the differences in surface morphologies and biological compositions, the three BCAMM scaffolds showed different chondrogenic effects. The 10-day BCAMM (10-BCAMM) scaffold showed the best overall results, successfully inducing MSC chondrogenesis without any additional fetal bovine serum or induction components (TGF-ß or dexamethasone). In comparison, the 5-day BCAMM (5-BCAMM) scaffold showed potential osteogenic effects. The advantages of micron-sized BCAMMs are outlined, specifically in the easier decellularization process without grinding, homogeneous cell seeding and infiltration, chondrogenic induction and better fitting to the irregular lesion shape.

Development of a novel hybrid bioactive hydrogel for future clinical applications.

Three-dimensional hydrogels are ideal for tissue engineering applications due to their structural integrity and similarity to native soft tissues; however, they can lack mechanical stability. Our objective was to develop a bioactive and mechanically stable hydrogel for clinical application. Auricular cartilage was decellularised using a combination of hypertonic and hypotonic solutions with and without enzymes to produce acellular tissue. Methacryloyl groups were crosslinked with alginate and PVA main chains via 2-aminoethylmathacrylate and the entire macromonomer further crosslinked with the acellular tissue. The resultant hydrogels were characterised for its physicochemical properties (using NMR), in vitro degradation (via GPC analysis), mechanical stability (compression tests) and in vitro biocompatibility (co-culture with bone marrow-derived mesenchymal stem cells). Following decellularisation, the cartilage tissue showed to be acellular at a significant level (DNA content 25.33 ng/mg vs. 351.46 ng/mg control tissue), with good structural and molecular integrity of the retained extra cellular matrix (s-GAG= 0.19 µg/mg vs. 0.65 µg/mg ±0.001 control tissue). Proteomic analysis showed that collagen subtypes and proteoglycans were retained, and SEM and TEM showed preserved matrix ultra-structure. The hybrid hydrogel was successfully cross-linked with biological and polymer components, and it was stable for 30 days in simulated body fluid (poly dispersal index for alginate with tissue was stable at 1.08 and for PVA with tissue was stable at 1.16). It was also mechanically stable (Young's modulus of 0.46 ± 0.31 KPa) and biocompatible, as it was able to support the development of a multi-cellular feature with active cellular proliferation in vitro. We have shown that it is possible to successfully combine biological tissue with both a synthetic and natural polymer and create a hybrid bioactive hydrogel for clinical application.

Regulation of decellularized tissue remodeling via scaffold-mediated lentiviral delivery in anatomically-shaped osteochondral constructs.

Cartilage-derived matrix (CDM) has emerged as a promising scaffold material for tissue engineering of cartilage and bone due to its native chondroinductive capacity and its ability to support endochondral ossification. Because it consists of native tissue, CDM can undergo cellular remodeling, which can promote integration with host tissue and enables it to be degraded and replaced by neotissue over time. However, enzymatic degradation of decellularized tissues can occur unpredictably and may not allow sufficient time for mechanically competent tissue to form, especially in the harsh inflammatory environment of a diseased joint. The goal of the current study was to engineer cartilage and bone constructs with the ability to inhibit aberrant inflammatory processes caused by the cytokine interleukin-1 (IL-1), through scaffold-mediated delivery of lentiviral particles containing a doxycycline-inducible IL-1 receptor antagonist (IL-1Ra) transgene on anatomically-shaped CDM constructs. Additionally, scaffold-mediated lentiviral gene delivery was used to facilitate spatial organization of simultaneous chondrogenic and osteogenic differentiation via site-specific transduction of a single mesenchymal stem cell (MSC) population to overexpress either chondrogenic, transforming growth factor-beta 3 (TGF-ß3), or osteogenic, bone morphogenetic protein-2 (BMP-2), transgenes. Controlled induction of IL-1Ra expression protected CDM hemispheres from inflammation-mediated degradation, and supported robust bone and cartilage tissue formation even in the presence of IL-1. In the absence of inflammatory stimuli, controlled cellular remodeling was exploited as a mechanism for fusing concentric CDM hemispheres overexpressing BMP-2 and TGF-ß3 into a single bi-layered osteochondral construct. Our findings demonstrate that site-specific delivery of inducible and tunable transgenes confers spatial and temporal control over both CDM scaffold remodeling and neotissue composition. Furthermore, these constructs provide a microphysiological in vitro joint organoid model with site-specific, tunable, and inducible protein delivery systems for examining the spatiotemporal response to pro-anabolic and/or inflammatory signaling across the osteochondral interface.