HSP70-A key regulator in chondrocyte homeostasis under naturally coupled hydrostatic pressure-thermal stimuli.

OBJECTIVE: During physical activities, chondrocytes experience coupled stimulation of hydrostatic pressure (HP) and a transient increase in temperature (T), with the latter varying within a physiological range from 32.5 °C to 38.7 °C. Previous short-term in vitro studies have demonstrated that the combined hydrostatic pressure-thermal (HP-T) stimuli more significantly enhance chondroinduction and chondroprotection of chondrocytes than isolated applications. Interestingly, this combined benefit is associated with a corresponding increase in HSP70 levels when HP and T are combined. The current study therefore explored the indispensable role of HSP70 in mediating the combined effects of HP-T stimuli on chondrocytes. DESIGN: In this mid-long-term study of in vitro engineered cartilage constructs, we assessed chondrocyte responses to HP-T stimuli using customized bioreactor in standard and HSP70-inhibited cultures. RESULTS: Surprisingly, under HSP70-inhibited conditions, the usually beneficial HP-T stimuli, especially its thermal component, exerted detrimental effects on chondrocyte homeostasis, showing a distinct and unfavorable shift in gene and protein expression patterns compared to non-HSP70-inhibited settings. Such effects were corroborated through mechanical testing and confirmed using a secondary cell source. A proteomic-based mechanistic analysis revealed a disruption in the balance between biosynthesis and fundamental cellular structural components in HSP70-inhibited conditions under HP-T stimuli. CONCLUSIONS: Our results highlight the critical role of sufficient HSP70 induction in mediating the beneficial effects of coupled HP-T stimulation on chondrocytes. These findings help pave the way for new therapeutic approaches to enhance physiotherapy outcomes and potentially shed light on the elusive mechanisms underlying the onset of cartilage degeneration, a long-standing enigma in orthopedics.

Impact of Molecular Dynamics of Polyrotaxanes on Chondrocytes in Double-Network Supramolecular Hydrogels under Physiological Thermomechanical Stimulation.

Hyaline cartilage, a soft tissue enriched with a dynamic extracellular matrix, manifests as a supramolecular system within load-bearing joints. At the same time, the challenge of cartilage repair through tissue engineering lies in replicating intricate cellular-matrix interactions. This study attempts to investigate chondrocyte responses within double-network supramolecular hybrid hydrogels tailored to mimic the dynamic molecular nature of hyaline cartilage. To this end, we infused noncovalent host-guest polyrotaxanes, by blending α-cyclodextrins as host molecules and polyethylene glycol as guests, into a gelatin-based covalent matrix, thereby enhancing its dynamic characteristics. Subsequently, chondrocytes were seeded into these hydrogels to systematically probe the effects of two concentrations of the introduced polyrotaxanes (instilling different levels of supramolecular dynamism in the hydrogel systems) on the cellular responsiveness. Our findings unveiled an augmented level of cellular mechanosensitivity for supramolecular hydrogels compared to pure covalent-based systems. This is demonstrated by an increased mRNA expression of ion channels (TREK1, TRPV4, and PIEZO1), signaling molecules (SOX9) and matrix-remodeling enzymes (LOXL2). Such outcomes were further elevated upon external application of biomimetic thermomechanical loading, which brought a stark increase in the accumulation of sulfated glycosaminoglycans and collagen. Overall, we found that matrix adaptability plays a pivotal role in modulating chondrocyte responses within double-network supramolecular hydrogels. These findings hold the potential for advancing cartilage engineering within load-bearing joints.

Unraveling cartilage degeneration through synergistic effects of hydrostatic pressure and biomimetic temperature increase.

Cartilage degeneration, typically viewed as an irreversible, vicious cycle, sees a significant reduction in two essential biophysical cues: the well-established hydrostatic pressure (HP) and the recently discovered transient temperature increase. Our study aimed to evaluate the combined influence of these cues on maintaining cartilage homeostasis. To achieve this, we developed a customized bioreactor, designed to mimic the specific hydrostatic pressure and transient thermal increase experienced during human knee physiological activities. This system enabled us to investigate the response of human 3D-cultured chondrocytes and human cartilage explants to either isolated or combined hydrostatic pressure and thermal stimuli. Our study found that chondroinduction (SOX9, aggrecan, and sulfated glycosaminoglycan) and chondroprotection (HSP70) reached maximum expression levels when hydrostatic pressure and transient thermal increase acted in tandem, underscoring the critical role of these combined cues in preserving cartilage homeostasis. These findings led us to propose a refined model of the vicious cycle of cartilage degeneration.

Enhancing Robustness of Adhesive Hydrogels through PEG-NHS Incorporation.

Tissue wounds are a significant challenge for the healthcare system, affecting millions globally. Current methods like suturing and stapling have limitations as they inadequately cover the wound, fail to prevent fluid leakage, and increase the risk of infection. Effective solutions for diverse wound conditions are still lacking. Adhesive hydrogels, on the other hand, can be a potential alternative for wound care. They offer benefits such as firm sealing without leakage, easy and rapid application, and the provision of mechanical support and flexibility. However, the in vivo durability of hydrogels is often compromised by excessive swelling and unforeseen degradation, which limits their widespread use. In this study, we addressed the durability issues of the adhesive hydrogels by incorporating acrylamide polyethylene glycol N-hydroxysuccinimide (PEG-NHS) moieties (max. 2 wt %) into hydrogels based on hydroxy ethyl acrylamide (HEAam). The results showed that the addition of PEG-NHS significantly enhanced the adhesion performance, achieving up to 2-fold improvement on various soft tissues including skin, trachea, heart, lung, liver, and kidney. We further observed that the addition of PEG-NHS into the adhesive hydrogel network improved their intrinsic mechanical properties. The tensile modulus of these hydrogels increased up to 5-fold, while the swelling ratio decreased up to 2-fold in various media. These hydrogels also exhibited improved durability under the enzymatic and oxidative biodegradation induced conditions without causing any toxicity to the cells. To evaluate its potential for clinical applications, we used PEG-NHS based hydrogels to address tracheomalacia, a condition characterized by inadequate mechanical support of the airway due to weak/malacic cartilage rings. Ex vivo study confirmed that the addition of PEG-NHS to the hydrogel network prevented approximately 90% of airway collapse compared to the case without PEG-NHS. Overall, this study offers a promising approach to enhance the durability of adhesive hydrogels by the addition of PEG-NHS, thereby improving their overall performances for various biomedical applications.

Low-oxygen tension augments chondrocyte sensitivity to biomimetic thermomechanical cues in cartilage-engineered constructs.

Chondrocytes respond to various biophysical cues, including oxygen tension, transient thermal signals, and mechanical stimuli. However, understanding how these factors interact to establish a unique regulatory microenvironment for chondrocyte function remains unclear. Herein, we explore these interactions using a joint-simulating bioreactor that independently controls the culture's oxygen concentration, evolution of temperature, and mechanical loading. Our analysis revealed significant coupling between these signals, resulting in a remarkable ∼14-fold increase in collagen type II (COL2a) and aggrecan (ACAN) mRNA expression. Furthermore, dynamic thermomechanical stimulation enhanced glycosaminoglycan and COL2a protein synthesis, with the magnitude of the biosynthetic changes being oxygen dependent. Additionally, our mechanistic study highlighted the crucial role of SRY-box transcription factor 9 (SOX9) as a major regulator of chondrogenic response, specifically expressed in response to combined biophysical signals. These findings illuminate the integration of various mechanobiological cues by chondrocytes and provide valuable insights for improving the extracellular matrix content in cartilage-engineered constructs.

Mimicking Loading-Induced Cartilage Self-Heating in Vitro Promotes Matrix Formation in Chondrocyte-Laden Constructs with Different Mechanical Properties.

Articular cartilage presents a mechanically sensitive tissue. Chondrocytes, the sole cell type residing in the tissue, perceive and react to physical cues as signals that significantly modulate their behavior. Hyaline cartilage is a connective tissue with high dissipative capabilities, able to increase its temperature during daily activities, thus providing a dynamic thermal milieu for the residing chondrocytes. This condition, self-heating, which is still chiefly ignored among the scientific community, adds a new thermal dimension in cartilage mechanobiology. Motivated by the lack of studies exploring this dynamic temperature increase as a potential stimulus in cartilage-engineered constructs, we aimed to elucidate whether loading-induced evolved temperature serves as an independent or complementary regulatory cue for chondrocyte function. In particular, we evaluated the chondrocytes' response to thermal and/or mechanical stimulation in two types of scaffolds exhibiting dissipation levels close to healthy and degenerated articular cartilage. It was found, in both scaffold groups, that the combination of dynamic thermal and mechanical stimuli induced superior effects in the expression of major chondrogenic genes, such as SOX9 and LOXL2, compared to either signal alone. Similar effects were also observed in proteoglycan accumulation over time, along with increased mRNA transcription and synthesis of TRPV4, and for the first time demonstrated in chondrocytes, TREK1 ion channels. Conversely, the chondrogenic response of cells to isolated thermal or mechanical cues was generally scaffold-type dependent. Nonetheless, the significance of thermal stimulus as a chondro-inductive signal was better supported in both studied groups. Our data indicates that the temperature evolution is necessary for chondrocytes to more effectively perceive and translate applied mechanical loading.

A guide to preclinical evaluation of hydrogel-based devices for treatment of cartilage lesions.

The drive to develop cartilage implants for the treatment of major defects in the musculoskeletal system has resulted in a major research thrust towards developing biomaterial devices for cartilage repair. Investigational devices for the restoration of articular cartilage are considered as significant risk materials by regulatory bodies and therefore proof of efficacy and safety prior to clinical testing represents a critical phase of the multidisciplinary effort to bridge the gap between bench and bedside. To date, review articles have thoroughly covered different scientific facets of cartilage engineering paradigm, but surprisingly, little attention has been given to the preclinical considerations revolving around the validation of a biomaterial implant. Considering hydrogel-based cartilage products as an example, the present review endeavors to provide a summary of the critical prerequisites that such devices should meet for cartilage repair, for successful implantation and subsequent preclinical validation prior to clinical trials. Considerations pertaining to the choice of appropriate animal model, characterization techniques for the quantitative and qualitative outcome measures, as well as concerns with respect to GLP practices are also extensively discussed. This article is not meant to provide a systematic review, but rather to introduce a device validation-based roadmap to the academic investigator, in anticipation of future healthcare commercialization. STATEMENT OF SIGNIFICANCE: There are significant challenges around translation of in vitro cartilage repair strategies to approved therapies. New biomaterial-based devices must undergo exhaustive investigations to ensure their safety and efficacy prior to clinical trials. These considerations are required to be applied from early developmental stages. Although there are numerous research works on cartilage devices and their in vivo evaluations, little attention has been given into the preclinical pathway and the corresponding approval processes. With a focus on hydrogel devices to concretely illustrate the preclinical path, this review paper intends to highlight the various considerations regarding the preclinical validation of hydrogel devices for cartilage repair, from regulatory considerations, to implantation strategies, device performance aspects and characterizations.

Thoughts on cartilage tissue engineering: A 21st century perspective.

In mature individuals, hyaline cartilage demonstrates a poor intrinsic capacity for repair, thus even minor defects could result in progressive degeneration, impeding quality of life. Although numerous attempts have been made over the past years for the advancement of effective treatments, significant challenges still remain regarding the translation of in vitro cartilage engineering strategies from bench to bedside. This paper reviews the latest concepts on engineering cartilage tissue in view of biomaterial scaffolds, tissue biofabrication, mechanobiology, as well as preclinical studies in different animal models. The current work is not meant to provide a methodical review, rather a perspective of where the field is currently focusing and what are the requirements for bridging the gap between laboratory-based research and clinical applications, in light of the current state-of-the-art literature. While remarkable progress has been accomplished over the last 20 years, the current sophisticated strategies have reached their limit to further enhance healthcare outcomes. Considering a clinical aspect together with expertise in mechanobiology, biomaterial science and biofabrication methods, will aid to deal with the current challenges and will present a milestone for the furtherance of functional cartilage engineering.