Facilitate Cartilage Regeneration Research Articles

3D-bioprinted anti-senescence organoid scaffolds repair cartilage defect and prevent joint degeneration via miR-23b/ELOVL5-mediated metabolic rewiring

IntroductionOsteoarthritis (OA) is the most prevalent degenerative disease worldwide and commonly occurs among the elderly. At present, there is no effective treatment for OA. When OA develops to the end stage, arthroplasty is generally operated to improve the life quality of patients. Targeting senescence has been considered as a therapeutic approach for OA in recent years. Methods & resultsIn this study, we have identified P16 gene as a novel target for anti-senescence strategy and harnessed small interfering RNA (siRNA) technology to fabricate P16-siRNA encapsulated PLGA microspheres. We combined this with our previously successful three-dimensional (3D) culture of functionally bioengineered chondrogenic synovial mesenchymal stromal cell (SMSC) organoids. This served as the primary component of the bio-ink for 3D bioprinting, culminating in the creation of P16-siRNA PLGA µS and SMSC organoid hydrogel-polymer composite scaffold (PPSOH). Its superior capabilities of cartilage repair were confirmed through in vivo and in vitro experimentations. In the rabbit model of knee cartilage defects, PPSOH not only restored the superior characteristics of native healthy hyaline cartilage but also maintained post-implanted joint function. It prevented development of joint degeneration resulting from cartilage defects by mitigating intra-articular inflammatory responses and cellular senescence. Our usage of miRNA and RNA sequencing reveals that PPSOH targets the miR-23b/ELOVL5 axis and rewired cellular metabolism, therefore regulating glycolysis and fatty acid oxidation. This effectively alleviates cellular senescence, promotes relief from OA, and facilitates cartilage regeneration. ConclusionPPSOH scaffold could effectively alleviate cellular senescence, foster relief from OA, and facilitate cartilage regeneration. Further experiments disclosed that PPSOH targets the miR-23b/ELOVL5 axis and rewired cellular metabolism by regulating glycolysis and fatty acid oxidation. Therefore, PPSOH could be used as potential therapy for cartilage repair in osteoarthritic patients.

Lithium-Containing Biomaterials Stimulate Cartilage Repair through Bone Marrow Stromal Cells-Derived Exosomal miR-455-3p and Histone H3 Acetylation.

The repair of damaged cartilage still remains a great challenge in clinic. It is demonstrated that bone marrow stromal cells (BMSCs)-chondrocytes communication is of great significance for cartilage repair. Moreover, BMSCs have been confirmed to enhance biological function of chondrocytes via exosome-mediated paracrine pathway. Lithium-containing scaffolds have been reported to effectively promote cartilage regeneration; however, whether lithium-containing biomaterial could facilitate cartilage regeneration through regulating BMSCs-derived exosomes has not been illustrated. In the study, the model lithium-substituted bioglass ceramic (Li-BGC) is selected and regulatory effects of BMSCs-derived exosomes after Li-BGC treatment (Li-BGC-Exo) are systemically evaluated. The data reveal that Li-BGC-Exo notably promotes chondrogenesis, which attributes to the upregulated exosomal miR-455-3p transfer, consequently leads to suppression of histone deacetylase 2 (HDAC2) and enhanced histone H3 acetylation in chondrocytes. Notably, BMSCs-derived exosomes after LiCltreatment(LiCl-Exo) exhibits the similar regulatory effect with Li-BGC-Exo, indicating that the pro-chondrogenesis capability of them is mainly owing to the lithium ions. Furthermore, the in vivo study proves that LiCl-Exo remarkably facilitates cartilage regeneration. The research may provide novel possibility for the intrinsic mechanism of chondrogenesis trigged by lithium-containing biomaterials, and suggests that application of lithium-containing scaffolds may be a promising strategy for cartilage regeneration.

Clinical phase I/II trial of SVF therapy for cartilage regeneration: A cellular therapy with novel 3D MRI imaging for evaluating chondral defect of knee osteoarthritis.

Background: The clinical applications of stromal vascular fraction (SVF) therapy for osteoarthritis (OA) have attracted academic and clinical attention. However, data of the effects of stromal vascular fraction therapy on regeneration of degenerated cartilage are limited in the literature. Meanwhile, there is a great need for a simple and non-invasive evaluation method to analyze the changes of joint cartilage qualitatively and quantitatively in clinical trials. This study entitled "stromal vascular fraction Therapy for Human Knee Osteoarthritis" was registered in ClinicalTrial.gov # NCT05019378. Materials and Methods: We designed and conducted a single center, open labeled clinical phase I/II study, and 6 osteoarthritis patients with both knee cartilage defect I-II were enrolled in this study. The two knees of each patient were randomly assigned to autologous stromal vascular fraction treatment group or non-treatment control group to evaluate the safety and therapeutic effect of stromal vascular fraction therapy for human knee osteoarthritis. We have also established a novel protocol to provide 3D MRI imaging for human knee cartilage enabling us to qualitatively and quantitatively evaluate cartilage degeneration and regeneration in this study. Results: The qualitative and quantitative evaluation of 3D Magnetic Resonance Imaging (MRI) imaging of knee cartilage demonstrated that the stromal vascular fraction therapy reduced the cartilage defects; and significant increase of cartilage value both in defect cartilage area and whole cartilage area of treated group and significant increase of thickness and area of both femoral and tibia cartilage in vertical sections of the stromal vascular fraction treated Group at 12 and 24W post treatment in cartilage defect I-II osteoarthritis patients. Conclusion: This clinical phase I/II study indicated that stromal vascular fraction therapy is a safe clinical procedure and provided evidence that the stromal vascular fraction therapy significantly facilitated cartilage regeneration, opening the opportunity to a phase III trial investigating authentic efficacy of the procedure. This study is the first qualitative and quantitative evaluation of the efficacy of autologous stromal vascular fraction cellular therapy on cartilage regeneration. Through early and definite diagnosis of knee osteoarthritis patients, and providing safe and efficient therapy to facilitate cartilage regeneration, we will be able to control or reverse cartilage degeneration and completely change the epidemiology of osteoarthritis worldwide.

EE338 Cost-Effectiveness Analysis of Arthroscopic Injection of a Bioadhesive Hydrogel Implant in Conjunction with Microfracture for the Treatment of Focal Chondral Defects of the Knee: An Australian Perspective

JointRep is a bioadhesive hydrogel arthroscopically injected to facilitate cartilage regeneration. The cost-effectiveness of JointRep with microfracture surgery compared to microfracture alone was evaluated from the Australian healthcare system perspective, in patients with symptomatic focal osteochondral defects (Outerbridge Grade 3 or 4) of the knee who had failed conservative treatment and were indicated for surgery.

Cost-effectiveness analysis of arthroscopic injection of a bioadhesive hydrogel implant in conjunction with microfracture for the treatment of focal chondral defects of the knee – an Australian perspective

Aim JointRep is a bioadhesive hydrogel arthroscopically injected to facilitate cartilage regeneration. The cost-effectiveness of JointRep with microfracture surgery compared to microfracture alone was evaluated from the Australian healthcare system perspective, in patients with symptomatic focal chondral defects (Outerbridge Grade 3 or 4) of the knee who had failed conservative treatment and were indicated for surgery. Materials and methods A de novo Markov model comprising two health states- ‘Alive’ and ‘Dead’ was developed. Model transition probability was based on the general population mortality rates. Clinical outcomes were assessed using the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) scores, a validated patient-reported tool measuring pain, stiffness, and physical function. The utility was derived by mapping WOMAC scores to EQ-5D scores using a published algorithm. Cost inputs were based on published Australian costs from AR-DRGs, Medicare Benefits Schedule, and Prostheses List. Model outcomes included costs, Quality-adjusted life years (QALYs), and incremental cost-effectiveness ratio (ICER). Base-case analysis was conducted for a time horizon of 3 years and a cycle length of 1 year. Cost and health outcomes were discounted at 5% per annum. Sensitivity and scenario analyses were also conducted. Results Total QALYs were estimated to be higher for JointRep with microfracture surgery (2.61) compared to microfracture surgery alone (1.66), an incremental gain of 0.95 QALY. JointRep with microfracture surgery was associated with an incremental cost of $6,022 compared to microfracture surgery alone, thus leading to an ICER of $6,328. Results were substantially robust to varying parameters in the sensitivity analyses conducted, alternative model settings and assumptions in scenario analyses. Limitations The clinical inputs used in the model were based on data from short duration, non-randomized, post-market clinical trial. Conclusions JointRep with microfracture surgery is a cost-effective treatment option compared to microfracture alone from the Australian health care system perspective.

SPATIAL TRANSCRIPTOMICS REVEAL UNIQUE MOLECULAR FINGERPRINTS OF CHONDROGENESIS DURING EMBRYONIC LIMB DEVELOPMENT

Purpose: Cartilage has a poor self-healing capacity. Thus, it is highly challenging to treat the cartilage-associated diseases such as osteoarthritis. In this regard, tissue engineering and stem cell therapy may offer valuable means to facilitate cartilage regeneration. Nevertheless, it is challenging to induce chondrogenesis of stem cells without off-target differentiation, largely due to complicated signaling pathways, gene regulatory networks, and cell-cell interactions involved in this process.

Chondroinductive Peptides for Cartilage Regeneration.

Inducing and maintaining a hyaline cartilage phenotype are the greatest challenge for cartilage regeneration. Synthetic chondroinductive biomaterials might be the answer to the unmet clinical need for a safe, stable, and cost-effective material capable of inducing true hyaline cartilage formation. The past decade witnessed an emergence of peptides to achieve chondrogenesis, as peptides have the advantages of versatility, high target specificity, minimized toxicity and immunogenicity, and ease of synthesis. In this study, we review peptides as the basis for creating promising synthetic chondroinductive biomaterials for in situ scaffold-based cartilage regeneration. We provide a thorough review of peptides evaluated for cartilage regeneration while distinguishing between peptides reported to induce chondrogenesis independently, and peptides reported to act in synergy with other growth factors to induce cartilage regeneration. In addition, we highlight that most peptide studies have been in vitro, and appropriate controls are not always present. A few rigorously performed in vitro studies have proceeded to in vivo studies, but the peptides in those in vivo studies were mainly introduced through systemic, subcutaneous, or intra-articular injections, with a paucity of studies employing in situ defects with appropriate controls. Clinical translation of peptides will require the evaluation of these peptides in well-controlled in vivo cartilage defect studies. In the decade ahead, we may be poised to leverage peptides to design devices that are safe, reproducible, cost-efficient, and scalable biomaterials, which are themselves chondroinductive to achieve true hyaline cartilage regeneration without the need for growth factors and other small molecules. Impact statement The regeneration of articular cartilage into its original structural, functional, and organizational hyaline phenotype remains a significant problem in the tissue engineering and orthopedic community. While cell-based solutions have shown promising outcomes, there are realistic translational challenges inherent to cell therapies. Alternatively, biomaterials have been widely studied and used as scaffolds to support and facilitate cartilage regeneration; however, the key technical challenge is to independently induce cartilage regeneration. The search for chondroinductive compounds and materials is an emerging area of research with peptides at its heart, which presents a timely opportunity to review and highlight peptides with cartilage regenerative activity and to fill gaps from previous reviews. The content of this review will serve as a valuable guide for researchers pursuing the discovery of new chondroinductive peptides or looking into incorporating the most promising existing peptides in their work.

Biomimetic cartilage-lubricating polymers regenerate cartilage in rats with early osteoarthritis.

The early stages of progressive degeneration of cartilage in articular joints are a hallmark of osteoarthritis. Healthy cartilage is lubricated by brush-like cartilage-binding nanofibres with a hyaluronan backbone and two key side chains (lubricin and lipid). Here, we show that hyaluronan backbones grafted with lubricin-like sulfonate-rich polymers or with lipid-like phosphocholine-rich polymers together enhance cartilage regeneration in a rat model of early osteoarthritis. These biomimetic brush-like nanofibres show a high affinity for cartilage proteins, form a lubrication layer on the cartilage surface and efficiently lubricate damaged human cartilage, lowering its friction coefficient to the low levels typical of native cartilage. Intra-articular injection of the two types of nanofibre into rats with surgically induced osteoarthritic joints led to cartilage regeneration and to the abrogation of osteoarthritis within 8 weeks. Biocompatible injectable lubricants that facilitate cartilage regeneration may offer a translational strategy for the treatment of early osteoarthritis.

A Collagen-Conducting Polymer Composite with Enhanced Chondrogenic Potential.

Conducting polymers (CPs) have demonstrated promise for promoting tissue repair, yet their ability to facilitate cartilage regeneration has yet to be thoroughly investigated. Integrating CPs into common scaffolds for tissue regeneration, such as collagen, would enable mechanistic studies on the potential for CPs to promote cartilage repair. Here, we combine absorbable collagen sponges (ACS) with the CP PEDOT-S and show that the PEDOT-S-collagen composite (PEDOT-ACS) has enhanced chondrogenic potential compared to the collagen sponge alone. PEDOT-S was incorporated through a simple incubation process. Changes to scaffold topography, elastic modulus, swelling ratio, and surface charge were measured to analyze how PEDOT-S affected the material properties of the scaffold. Changes in rat bone marrow mesenchymal stem cell (rBMSC) functionality were assessed with cell viability and glycosaminoglycan production assays. Macrostructure and microstructure of the scaffold remained largely unaffected by PEDOT-S modification, as observed through SEM images and quantification of scaffold porosity. Zeta potential, swelling ratio, and dry elastic modulus of the collagen scaffold were significantly changed by the incorporation of PEDOT-S. Seeding cells on PEDOT-ACS improved cell viability and enhanced glycosaminoglycan production. We demonstrate a practical approach to generate PEDOT-S composites with comparable physical properties to pristine collagen scaffolds. We show that PEDOT-ACS can influence cell functionality and serve as a promising model system for mechanistic investigations on the roles of bioelectronic signaling in the repair of cartilage and other tissue types.

Extracellular matrix imitation utilizing nanofibers-embedded biomimetic scaffolds for facilitating cartilage regeneration

Repair of cartilage legions remains a big challenge for clinicians due to the limit self-regeneration ability of cartilage. A SF-based scaffold that reinforced by the SF–chitin hybrid nanofibers and impregnated with TGF-β1 is developed to mimic extracellular matrix and facilitate cartilage regeneration. The composite scaffold with an excellent biocompatibility exhibited the suitable elasticity and hardness after incorporation of the SF–chitin hybrid nanofibers. The attachment and proliferation of chondrocytes was also improved by a rough morphology created by the SF–chitin hybrid nanofibers. As a chondrogenic growth factor, TGF-β1 was incorporated into the composite scaffold and modulated the performance of scaffolds by inducing cells homing. In the treatment of rat cartilaginous defects, the SF–chitin hybrid nanofiber-functionalized composite scaffold loaded with TGF-β1 remarkably accelerated the cartilage regeneration. Thus, the SF–chitin hybrid nanofiber/TGF-β1-functionalized scaffold fabricated in this study promotes the adhesion and proliferation of chondrocytes in vitro, and cartilage regeneration in vivo, thus offering a suitable strategy for the repair of cartilage defects.

Human Amniotic Mesenchymal Stem Cell Sheets Encapsulating Cartilage Particles Facilitate Repair of Rabbit Osteochondral Defects

Background: Human amniotic mesenchymal stem cells (hAMSCs) are being widely applied in various fields. Therefore, hAMSCs represent a promising candidate to facilitate cartilage regeneration. Nonetheless, no studies have investigated the application of hAMSC sheets to repair cartilage defects in vivo. Purpose: To evaluate hAMSC sheets encapsulating cartilage particles to promote repair of rabbit osteochondral defects. Study Design: Controlled laboratory study. Methods: hAMSC sheets were constructed with passage 3 hAMSCs. The phenotypic and structural characteristics of hAMSC sheets were evaluated by flow cytometry and scanning electron microscopy, respectively. The potential for chondrogenic differentiation of hAMSC sheets was assessed by cartilage-specific marker staining, immunohistochemistry, and mRNA and protein expression (SOX9, COLII, and ACAN). Osteochondral defects (diameter, 3.5 mm; depth, 3 mm) were created in the left patellar grooves of 20 New Zealand White rabbits (female or male). The defects were treated with hAMSC sheet/cartilage particles (n = 5), cartilage particles (n = 5), hAMSC sheets (n = 5), or fibrin glue (n = 5). Macroscopic and histological evaluations of the regenerated tissue were conducted after 3 months. The survival time and differentiation of transplanted hAMSCs in the defect area were evaluated by immunofluorescence. Results: hAMSC sheets had a multilayered structure, with cells stacked layer by layer. Importantly, hAMSC sheets highly expressed phenotypic markers of mesenchymal stem cells. Cartilage-specific marker staining and immunohistochemistry were positive, and mRNA and protein expression was higher in the chondrogenically induced hAMSC sheet group than in the hAMSC sheet group (P < .05). hAMSC sheet/cartilage particles formed a large amount of hyaline-like cartilage in the defect area. In addition, macroscopic and histological scores were significantly higher than those in the other groups. Integration with surrounding normal cartilage and subchondral bone regeneration in the hAMSC sheet/cartilage particles group were better when compared with the other groups. A large number of human nuclear-specific antigen-positive cells were observed in the defect area of hAMSC sheet/cartilage particles and hAMSC sheet groups. Moreover, some positive cells expressed SOX9. Conclusion: hAMSC sheets encapsulating cartilage particles facilitate osteochondral defect repair. Clinical Relevance: Delivery of cells in the form of a cell sheet in conjunction with cartilage particles provides a novel approach for cell-based cartilage regeneration.

Identifying the Potential of Qur’anic Recitation on the Proliferation of Chondrocytes Derived from Rabbit Articular Cartilage: Work in Progress

Introduction: In Islamic practice, the use of Qur’anic recitation in treatment can be traced back to the times of Prophet Muhammad (PBUH). This preliminary study aims to identify the potential of Qur’anic recitation of Surah Al-Fatihah on the proliferation of chondrocytes derived from rabbit articular cartilage. Cartilage tissue engineering offers an alternative way to facilitate cartilage regeneration in-vitro. Materials and Methods: The cellular model was established using a serially cultured and expanded chondrocytes in-vitro. The model was assigned into three groups. The first group was exposed to the Surah Al-Fatihah, recited 17 times based on the five times daily prayer unit (Raka’ah) obligated upon Muslims. The second group was exposed to an Arabic poem recitation. The third group was not exposed to any sound and served as the control. All groups were subjected to the growth profile analysis. The analysis was conducted at different passages starting from passage 0 to passage 3. Results: The results showed that the cells proliferation based on the growth kinetic analysis is higher for the cells exposed with Qur’anic recitation as compared to the Arabic poem and control groups. Conclusions: The proliferation process of the rabbit articular cartilage might be influenced with the use of Qur’anic recitation and as well as Arabic poem recitation. Exposure to the Western poem recitation and mute sound will be added for future study. It is hoped that this study could shed some light on the potential use of the Qur’anic recitation to facilitate cartilage regeneration in tissue engineering studies.

Tracking mesenchymal stem cell contributions to regeneration in an immunocompetent cartilage regeneration model.

It is currently controversially discussed whether mesenchymal stem cells (MSC) facilitate cartilage regeneration in vivo by a progenitor- or a nonprogenitor-mediated mechanism. Here, we describe a potentially novel unbiased in vivo cell tracking system based on transgenic donor and corresponding immunocompetent marker-tolerant recipient mouse and rat lines in inbred genetic backgrounds. Tolerance of recipients was achieved by transgenic expression of an immunologically neutral but physicochemically distinguishable variant of the marker human placental alkaline phosphatase (ALPP). In this dual transgenic system, donor lines ubiquitously express WT, heat-resistant ALPP protein, whereas recipient lines express a heat-labile ALPP mutant (ALPPE451G) resulting from a single amino acid substitution. Tolerance of recipient lines to ALPP-expressing cells and tissues was verified by skin transplantation. Using this model, we show that intraarticularly injected MSC contribute to regeneration of articular cartilage in full-thickness cartilage defects mainly via a nonprogenitor-mediated mechanism.

Material characterization of microsphere-based scaffolds with encapsulated raw materials

“Raw materials,” or materials capable of serving both as building blocks and as signals, which are often but not always natural materials, are taking center stage in biomaterials for contemporary regenerative medicine. In osteochondral tissue engineering, a field leveraging the underlying bone to facilitate cartilage regeneration, common raw materials include chondroitin sulfate (CS) for cartilage and β-tricalcium phosphate (TCP) for bone. Building on our previous work with gradient scaffolds based on microspheres, here we delved deeper into the characterization of individual components. In the current study, the release of CS and TCP from poly(d,l-lactic-co-glycolic acid) (PLGA) microsphere-based scaffolds was evaluated over a time period of 4weeks. Raw material encapsulated groups were compared to ‘blank’ groups and evaluated for surface topology, molecular weight, and mechanical performance as a function of time. The CS group may have led to increased surface porosity, and the addition of CS improved the mechanical performance of the scaffold. The finding that CS was completely released into the surrounding media by 4weeks has a significant impact on future in vivo studies, given rapid bioavailability. The addition of TCP seemed to contribute to the rough external appearance of the scaffold. The current study provides an introduction to degradation patterns of homogenous raw material encapsulated scaffolds, providing characterization data to advance the field of microsphere-based scaffolds in tissue engineering.

Effect of Longan polysaccharides on proliferation and phenotype maintenance in rabbit articular chondrocytes in vitro.

For autologous chondrocyte implantation (ACI) to restore cartilage defect, limited cell numbers and dedifferentiation of chondrocytes are the major difficulties. An alternative is the use of growth factors, but the high cost and potential tumorigenesis are the major obstacles. To ensure successful ACI therapy, it is of significance to find effective substituted pro-chondrogenic agent. Polysaccharides from plant extract have low toxicity and few undesirable side effects, which were reported to facilitate cartilage regeneration. In this study, we investigated the effect of Longan polysaccharides (LP) on rabbit articular chondrocytes through examination of the cell proliferation, morphology, viability, glycosaminoglycan synthesis and cartilage-specific gene expression. Results showed that close to the positive group which used the growth factor of TGF-β, LP could effectively promote chondrocytes growth and enhance secretion and synthesis of cartilage extracellular matrix by up-regulating expression levels of aggrecan, collagen II and sox9 compared to the negative control. Expression of collagen I gene was effectively down-regulated, demonstrating the inhibition of chondrocytes dedifferentiation by LP. Hypertrophy that might lead to chondrocyte ossification was also undetectable in LP groups. Range of 4.69-18.76µg/ml was recommended dose of LP, among which the most profound response was observed with 9.38μg/ml. All the evidences revealed that LP may replace the growth factors to be applied in ACI therapy. This study might provide a basis for development of a novel agent in the treatment of articular cartilage defect.

Periodic Heat Shock Accelerated the Chondrogenic Differentiation of Human Mesenchymal Stem Cells in Pellet Culture

Osteoarthritis (OA) is one of diseases that seriously affect elderly people's quality of life. Human mesenchymal stem cells (hMSCs) offer a potential promise for the joint repair in OA patients. However, chondrogenic differentiation from hMSCs in vitro takes a long time (∼6 weeks) and differentiated cells are still not as functionally mature as primary isolated chondrocytes, though chemical stimulations and mechanical loading have been intensively studied to enhance the hMSC differentiation. On the other hand, thermal stimulations of hMSC chondrogenesis have not been well explored. In this study, the direct effects of mild heat shock (HS) on the differentiation of hMSCs into chondrocytes in 3D pellet culture were investigated. Periodic HS at 41°C for 1 hr significantly increased sulfated glycosaminoglycan in 3D pellet culture at Day 10 of chondrogenesis. Immunohistochemical and Western Blot analyses revealed an increased expression of collagen type II and aggrecan in heat-shocked pellets than non heat-shocked pellets on Day 17 of chondrogenesis. In addition, HS also upregulated the expression of collagen type I and X as well as heat shock protein 70 on Day 17 and 24 of differentiation. These results demonstrate that HS accelerated the chondrogenic differentiation of hMSCs and induced an early maturation of chondrocytes differentiated from hMSCs. The results of this study will guide the design of future protocols using thermal treatments to facilitate cartilage regeneration with human mesenchymal stem cells.