Rapid specialization and stiffening of the primitive matrix in developing articular cartilage and meniscus.

Understanding early patterning events in the extracellular matrix (ECM) formation can provide a blueprint for regenerative strategies to better recapitulate the function of native tissues. Currently, there is little knowledge on the initial, incipient ECM of articular cartilage and meniscus, two load-bearing counterparts of the knee joint. This study elucidated distinctive traits of their developing ECMs by studying the composition and biomechanics of these two tissues in mice from mid-gestation (embryonic day 15.5) to neo-natal (post-natal day 7) stages. We show that articular cartilage initiates with the formation of a pericellular matrix (PCM)-like primitive matrix, followed by the separation into distinct PCM and territorial/interterritorial (T/IT)-ECM domains, and then, further expansion of the T/IT-ECM through maturity. In this process, the primitive matrix undergoes a rapid, exponential stiffening, with a daily modulus increase rate of 35.7% [31.9 39.6]% (mean [95% CI]). Meanwhile, the matrix becomes more heterogeneous in the spatial distribution of properties, with concurrent exponential increases in the standard deviation of micromodulus and the slope correlating local micromodulus with the distance from cell surface. In comparison to articular cartilage, the primitive matrix of meniscus also exhibits exponential stiffening and an increase in heterogeneity, albeit with a much slower daily stiffening rate of 19.8% [14.9 24.9]% and a delayed separation of PCM and T/IT-ECM. These contrasts underscore distinct development paths of hyaline versus fibrocartilage. Collectively, these findings provide new insights into how knee joint tissues form to better guide cell- and biomaterial-based repair of articular cartilage, meniscus and potentially other load-bearing cartilaginous tissues. STATEMENT OF SIGNIFICANCE: Successful regeneration of articular cartilage and meniscus is challenged by incomplete knowledge of early events that drive the initial formation of the tissues' extracellular matrix in vivo. This study shows that articular cartilage initiates with a pericellular matrix (PCM)-like primitive matrix during embryonic development. This primitive matrix then separates into distinct PCM and territorial/interterritorial domains, undergoes an exponential daily stiffening of ≈36% and an increase in micromechanical heterogeneity. At this early stage, the meniscus primitive matrix shows differential molecular traits and exhibits a slower daily stiffening of ≈20%, underscoring distinct matrix development between these two tissues. Our findings thus establish a new blueprint to guide the design of regenerative strategies to recapitulate the key developmental steps in vivo.

CXCL12 defines lung endothelial heterogeneity and promotes distal vascular growth.

There is a growing amount of data uncovering the cellular diversity of the pulmonary circulation and mechanisms governing vascular repair after injury. However, the molecular and cellular mechanisms contributing to the morphogenesis and growth of the pulmonary vasculature during embryonic development are less clear. Importantly, deficits in vascular development lead to significant pediatric lung diseases, indicating a need to uncover fetal programs promoting vascular growth. To address this, we used a transgenic mouse reporter for expression of Cxcl12, an arterial endothelial hallmark gene, and performed single-cell RNA sequencing on isolated Cxcl12-DsRed+ endothelium to assess cellular heterogeneity within pulmonary endothelium. Combining cell annotation with gene ontology and histological analysis allowed us to segregate the developing artery endothelium into functionally and spatially distinct subpopulations. Expression of Cxcl12 is highest in the distal arterial endothelial subpopulation, a compartment enriched in genes for vascular development. Accordingly, disruption of CXCL12 signaling led to, not only abnormal branching, but also distal vascular hypoplasia. These data provide evidence for arterial endothelial functional heterogeneity and reveal conserved signaling mechanisms essential for pulmonary vascular development.

Defining the spatial landscape of KRAS mutated congenital pulmonary airway malformations: a distinct entity with a spectrum of histopathologic features.

The potential pathogenetic mechanisms underlying the varied morphology of congenital pulmonary airway malformations (CPAMs) have not been molecularly determined, but a subset have been shown to contain clusters of mucinous cells (MCC). These clusters are believed to serve as precursors for potential invasive mucinous adenocarcinoma, and they are associated with KRAS codon 12 mutations. To assess the universality of KRAS mutations in MCCs, we sequenced exon 2 of KRAS in 61 MCCs from 18 patients, and we found a KRAS codon 12 mutation in all 61 MCCs. Furthermore, all MCCs from a single patient always had the same KRAS mutation, and the same KRAS mutation was also found in non-mucinous lesional tissue. Next generation sequencing of seven MCCs showed no other mutations or copy number variations. Sequencing of 46 additional CPAMs with MCCs revealed KRAS mutations in non-mucinous lesional tissue in all cases. RNA in situ hybridization confirmed widespread distribution of cells with mutant KRAS RNA, even extending outside of the bronchiolar type epithelium. We identified 25 additional CPAMs with overall histologic architecture similar to CPAMs with KRAS mutations but without identifiable MCCs, and we found KRAS mutations in 17 (68%). The histologic features of these KRAS mutated CPAMs included type 1 and type 3 morphology, as well as lesions with an intermediate histologic appearance, and analysis revealed a strong correlation between the specific amino acid substitution and histomorphology. These findings, together with previously published model organism data, suggests that the formation of type 1 and 3 CPAMs is driven by mosaic KRAS mutations arising in the lung epithelium early in development and places them within the growing field of mosaic RASopathies. The presence of widespread epithelial mutation explains late metastatic disease in incompletely resected patients and reinforces the recommendation for complete resection of these lesions.

Molecular Engineering of Pericellular Microniche via Biomimetic Proteoglycans Modulates Cell Mechanobiology.

Molecular engineering of biological tissues using synthetic mimics of native matrix molecules can modulate the mechanical properties of the cellular microenvironment through physical interactions with existing matrix molecules, and in turn, mediate the corresponding cell mechanobiology. In articular cartilage, the pericellular matrix (PCM) is the immediate microniche that regulates cell fate, signaling, and metabolism. The negatively charged osmo-environment, as endowed by PCM proteoglycans, is a key biophysical cue for cell mechanosensing. This study demonstrated that biomimetic proteoglycans (BPGs), which mimic the ultrastructure and polyanionic nature of native proteoglycans, can be used to molecularly engineer PCM micromechanics and cell mechanotransduction in cartilage. Upon infiltration into bovine cartilage explant, we showed that localization of BPGs in the PCM leads to increased PCM micromodulus and enhanced chondrocyte intracellular calcium signaling. Applying molecular force spectroscopy, we revealed that BPGs integrate with native PCM through augmenting the molecular adhesion of aggrecan, the major PCM proteoglycan, at the nanoscale. These interactions are enabled by the biomimetic "bottle-brush" ultrastructure of BPGs and facilitate the integration of BPGs within the PCM. Thus, this class of biomimetic molecules can be used for modulating molecular interactions of pericellular proteoglycans and harnessing cell mechanosensing. Because the PCM is a prevalent feature of various cell types, BPGs hold promising potential for improving regeneration and disease modification for not only cartilage-related healthcare but many other tissues and diseases.

In utero adenine base editing corrects multi-organ pathology in a lethal lysosomal storage disease.

In utero base editing has the potential to correct disease-causing mutations before the onset of pathology. Mucopolysaccharidosis type I (MPS-IH, Hurler syndrome) is a lysosomal storage disease (LSD) affecting multiple organs, often leading to early postnatal cardiopulmonary demise. We assessed in utero adeno-associated virus serotype 9 (AAV9) delivery of an adenine base editor (ABE) targeting the Idua G→A (W392X) mutation in the MPS-IH mouse, corresponding to the common IDUA G→A (W402X) mutation in MPS-IH patients. Here we show efficient long-term W392X correction in hepatocytes and cardiomyocytes and low-level editing in the brain. In utero editing was associated with improved survival and amelioration of metabolic, musculoskeletal, and cardiac disease. This proof-of-concept study demonstrates the possibility of efficiently performing therapeutic base editing in multiple organs before birth via a clinically relevant delivery mechanism, highlighting the potential of this approach for MPS-IH and other genetic diseases.

Type V collagen regulates the structure and biomechanics of TMJ condylar cartilage: A fibrous-hyaline hybrid.

This study queried the role of type V collagen in the post-natal growth of temporomandibular joint (TMJ) condylar cartilage, a hybrid tissue with a fibrocartilage layer covering a secondary hyaline cartilage layer. Integrating outcomes from histology, immunofluorescence imaging, electron microscopy and atomic force microscopy-based nanomechanical tests, we elucidated the impact of type V collagen reduction on TMJ condylar cartilage growth in the type V collagen haploinsufficiency and inducible knockout mice. Reduction of type V collagen led to significantly thickened collagen fibrils, decreased tissue modulus, reduced cell density and aberrant cell clustering in both the fibrous and hyaline layers. Post-natal growth of condylar cartilage involves the chondrogenesis of progenitor cells residing in the fibrous layer, which gives rise to the secondary hyaline layer. Loss of type V collagen resulted in reduced proliferation of these cells, suggesting a possible role of type V collagen in mediating the progenitor cell niche. When the knockout of type V collagen was induced in post-weaning mice after the start of physiologic TMJ loading, the hyaline layer exhibited pronounced thinning, supporting an interplay between type V collagen and occlusal loading in condylar cartilage growth. The phenotype in hyaline layer can thus be attributed to the impact of type V collagen on the mechanically regulated progenitor cell activities. In contrast, knee cartilage does not contain the progenitor cell population at post-natal stages, and develops normal structure and biomechanical properties with the loss of type V collagen. Therefore, in the TMJ, in addition to its established role in regulating the assembly of collagen I fibrils, type V collagen also impacts the mechanoregulation of progenitor cell activities in the fibrous layer. We expect such knowledge to establish a foundation for understanding condylar cartilage matrix development and regeneration, and to yield new insights into the TMJ symptoms in patients with classic Ehlers-Danlos syndrome, a genetic disease due to autosomal mutation of type V collagen.

Decorin regulates cartilage pericellular matrix micromechanobiology.

In cartilage tissue engineering, one key challenge is for regenerative tissue to recapitulate the biomechanical functions of native cartilage while maintaining normal mechanosensitive activities of chondrocytes. Thus, it is imperative to discern the micromechanobiological functions of the pericellular matrix, the ~ 2-4 µm-thick domain that is in immediate contact with chondrocytes. In this study, we discovered that decorin, a small leucine-rich proteoglycan, is a key determinant of cartilage pericellular matrix micromechanics and chondrocyte mechanotransduction in vivo. The pericellular matrix of decorin-null murine cartilage developed reduced content of aggrecan, the major chondroitin sulfate proteoglycan of cartilage and a mild increase in collagen II fibril diameter vis-à-vis wild-type controls. As a result, decorin-null pericellular matrix showed a significant reduction in micromodulus, which became progressively more pronounced with maturation. In alignment with the defects of pericellular matrix, decorin-null chondrocytes exhibited decreased intracellular calcium activities, [Ca2+]i, in both physiologic and osmotically evoked fluidic environments in situ, illustrating impaired chondrocyte mechanotransduction. Next, we compared [Ca2+]i activities of wild-type and decorin-null chondrocytes following enzymatic removal of chondroitin sulfate glycosaminoglycans. The results showed that decorin mediates chondrocyte mechanotransduction primarily through regulating the integrity of aggrecan network, and thus, aggrecan-endowed negative charge microenvironment in the pericellular matrix. Collectively, our results provide robust genetic and biomechanical evidence that decorin is an essential constituent of the native cartilage matrix, and suggest that modulating decorin activities could improve cartilage regeneration.

Early changes in cartilage pericellular matrix micromechanobiology portend the onset of post-traumatic osteoarthritis.

The pericellular matrix (PCM) of cartilage is a structurally distinctive microdomain surrounding each chondrocyte, and is pivotal to cell homeostasis and cell-matrix interactions in healthy tissue. This study queried if the PCM is the initiation point for disease or a casualty of more widespread matrix degeneration. To address this question, we queried the mechanical properties of the PCM and chondrocyte mechanoresponsivity with the development of post-traumatic osteoarthritis (PTOA). To do so, we integrated Kawamoto's film-assisted cryo-sectioning with immunofluorescence-guided AFM nanomechanical mapping, and quantified the microscale modulus of murine cartilage PCM and further-removed extracellular matrix. Using the destabilization of the medial meniscus (DMM) murine model of PTOA, we show that decreases in PCM micromechanics are apparent as early as 3 days after injury, and that this precedes changes in the bulk ECM properties and overt indications of cartilage damage. We also show that, as a consequence of altered PCM properties, calcium mobilization by chondrocytes in response to mechanical challenge (hypo-osmotic stress) is significantly disrupted. These aberrant changes in chondrocyte micromechanobiology as a consequence of DMM could be partially blocked by early inhibition of PCM remodeling. Collectively, these results suggest that changes in PCM micromechanobiology are leading indicators of the initiation of PTOA, and that disease originates in the cartilage PCM. This insight will direct the development of early detection methods, as well as small molecule-based therapies that can stop early aberrant remodeling in this critical cartilage microdomain to slow or reverse disease progression. STATEMENT OF SIGNIFICANCE: Post-traumatic osteoarthritis (PTOA) is one prevalent musculoskeletal disease that afflicts young adults, and there are no effective strategies for early detection or intervention. This study identifies that the reduction of cartilage pericellular matrix (PCM) micromodulus is one of the earliest events in the initiation of PTOA, which, in turn, impairs the mechanosensitive activities of chondrocytes, contributing to the vicious loop of cartilage degeneration. Rescuing the integrity of PCM has the potential to restore normal chondrocyte mechanosensitive homeostasis and to prevent further degradation of cartilage. Our findings enable the development of early OA detection methods targeting changes in the PCM, and treatment strategies that can stop early aberrant remodeling in this critical microdomain to slow or reverse disease progression.

Cyclophilin B control of lysine post-translational modifications of skin type I collagen.

Covalent intermolecular cross-linking of collagen is essential for tissue stability. Recent studies have demonstrated that cyclophilin B (CypB), an endoplasmic reticulum (ER)-resident peptidyl-prolyl cis-trans isomerase, modulates lysine (Lys) hydroxylation of type I collagen impacting cross-linking chemistry. However, the extent of modulation, the molecular mechanism and the functional outcome in tissues are not well understood. Here, we report that, in CypB null (KO) mouse skin, two unusual collagen cross-links lacking Lys hydroxylation are formed while neither was detected in wild type (WT) or heterozygous (Het) mice. Mass spectrometric analysis of type I collagen showed that none of the telopeptidyl Lys was hydroxylated in KO or WT/Het mice. Hydroxylation of the helical cross-linking Lys residues was almost complete in WT/Het but was markedly diminished in KO. Lys hydroxylation at other sites was also lower in KO but to a lesser extent. A key glycosylation site, α1(I) Lys-87, was underglycosylated while other sites were mostly overglycosylated in KO. Despite these findings, lysyl hydroxylases and glycosyltransferase 25 domain 1 levels were significantly higher in KO than WT/Het. However, the components of ER chaperone complex that positively or negatively regulates lysyl hydroxylase activities were severely reduced or slightly increased, respectively, in KO. The atomic force microscopy-based nanoindentation modulus were significantly lower in KO skin than WT. These data demonstrate that CypB deficiency profoundly affects Lys post-translational modifications of collagen likely by modulating LH chaperone complexes. Together, our study underscores the critical role of CypB in Lys modifications of collagen, cross-linking and mechanical properties of skin.

Variable piezoelectricity of electrospun chitin.

Investigations into the piezoelectricity of natural polymers is a continuing area of interest due to their potential role in the complex interplay of mechanical and electrical forces present in biological organisms. Their synthetic counterparts, when electrospun using the air gap electrospinning method, are known to have increased crystallinity and tensile strength as compared to randomly aligned nanofibers composed of the same constituent polymers. Using the air gap electrospinning method with the naturally-occurring, semi-crystalline polymer chitin, the nanofibers were determined to have a 300% increase in tensile strength over randomly collected ones. Additionally, a 400% increase in piezoelectric response in the aligned nanofiber chitin mats was measured. The increased tensile strength and piezoelectricity in aligned chitin nanofibers is a consequence of an increase in α-chitin crystallinity in the nanofibers induced by the air gap collection method.

Biomechanical properties of murine TMJ articular disc and condyle cartilage via AFM-nanoindentation.

This study aims to quantify the biomechanical properties of murine temporomandibular joint (TMJ) articular disc and condyle cartilage using AFM-nanoindentation. For skeletally mature, 3-month old mice, the surface of condyle cartilage was found to be significantly stiffer (306±84kPa, mean±95% CI) than those of the superior (85±23kPa) and inferior (45±12kPa) sides of the articular disc. On the disc surface, significant heterogeneity was also detected across multiple anatomical sites, with the posterior end being the stiffest and central region being the softest. Using SEM, this study also found that the surfaces of disc are composed of anteroposteriorly oriented collagen fibers, which are sporadically covered by thinner random fibrils. Such fibrous nature results in both an F-D3/2 indentation response, which is a typical Hertzian response for soft continuum tissue under a spherical tip, and a linear F-D response, which is typical for fibrous tissues, further signifying the high degree of tissue heterogeneity. In comparison, the surface of condyle cartilage is dominated by thinner, randomly oriented collagen fibrils, leading to Hertzian-dominated indentation responses. As the first biomechanical study of murine TMJ, this work will provide a basis for future investigations of TMJ tissue development and osteoarthritis in various murine TMJ models.

Self-similar Hierarchical Wrinkles as a Potential Multifunctional Smart Window with Simultaneously Tunable Transparency, Structural Color, and Droplet Transport.

Smart window has immense potential for energy savings in architectural and vehicular applications, while most studies focus on the tunability of a single property of optical transmittance. Here we explore harnessing dynamically tunable hierarchical wrinkles for design of a potential multifunctional smart window with combined structural color and water droplet transport control. The self-similar hierarchical wrinkles with both nanoscale and microscale features are generated on a prestrained poly(dimethylsiloxane) elastomer through sequential strain release and multistep oxygen plasma treatment. We show that the hierarchically wrinkled elastomer displays both opaqueness and iridescent structural color. We find that restretching/releasing the elastomer leads to the reversible and repeatable switch from opaqueness to transparency, arising from the flattening of large wrinkles (micrometer scale), while a nonvanishing structural color occurs due to the nondisappearing small wrinkles (nanoscale). The unique features of combined reversible large wrinkles and irreversible small wrinkles during hierarchical wrinkling are well reproduced by corresponding finite element simulation. The criteria for generating self-similar hierarchical wrinkles is revealed through a simplified theoretical model and validated by experiments. In addition to its tunable optical property, we further show its ability in control of water droplet transport on demand through mechanical stretching and release. We find that an initially pinned water droplet on the tilted hierarchically wrinkled surface starts to slide when the surface is stretched, and becomes pinned again upon strain release. Such a process is reversible and repeatable. The hierarchically wrinkled surface could find broad potential applications not only in multifunctional smart windows with additional features of aesthetics and water collection, but in microfluidics, design of slippery surfaces, and directional water transportation.

AFM-Nanomechanical Test: An Interdisciplinary Tool That Links the Understanding of Cartilage and Meniscus Biomechanics, Osteoarthritis Degeneration, and Tissue Engineering.

Our objective is to provide an in-depth review of the recent technical advances of atomic force microscopy (AFM)-based nanomechanical tests and their contribution to a better understanding and diagnosis of osteoarthritis (OA), as well as the repair of tissues undergoing degeneration during OA progression. We first summarize a range of technical approaches for AFM-based nanoindentation, including considerations in both experimental design and data analysis. We then provide a more detailed description of two recently developed modes of AFM-nanoindentation, a high-bandwidth nanorheometer system for studying poroviscoelasticity and an immunofluorescence-guided nanomechanical mapping technique for delineating the pericellular matrix (PCM) and territorial/interterritorial matrix (T/IT-ECM) of surrounding cells in connective tissues. Next, we summarize recent applications of these approaches to three aspects of joint-related healthcare and disease: cartilage aging and OA, developmental biology and OA pathogenesis in murine models, and nanomechanics of the meniscus. These studies were performed over a hierarchy of length scales, from the molecular, cellular to the whole tissue level. The advances described here have contributed greatly to advancing the fundamental knowledge base for improved understanding, detection, and treatment of OA.