Implantation of hUCB-MSCs generates greater hyaline-type cartilage than microdrilling combined with high tibial osteotomy.

PURPOSE: To compare the outcomes of treating large cartilage defects in knee osteoarthritis using human allogeneic umbilical cord blood-derived mesenchymal stem cell (hUCB-MSC) implantation or arthroscopic microdrilling as a supplementary cartilage regenerative procedure combined with high tibial osteotomy (HTO). METHODS: This 1-year prospective comparative study included 25 patients with large, near full-thickness cartilage defects (International Cartilage Repair Society grade ≥ IIIB) in the medial femoral condyles and varus malalignment. Defects were treated with hUCB-MSC implantation or arthroscopic microdrilling combined with HTO. The primary outcomes were pain visual analogue scale and International Knee Documentation Committee subjective scores at 12, 24 and 48 weeks. Secondary outcomes included arthroscopic, histological and magnetic resonance imaging assessments at 1 year. RESULTS: Fifteen and 10 patients were treated via hUCB-MSC implantation and microdrilling, respectively. Baseline demographics, limb alignment and clinical outcomes did not significantly differ between the groups. Cartilage defects and total restored areas were significantly larger in the hUCB-MSC group (7.2 ± 1.9 vs. 5.2 ± 2.1 cm2, p = 0.023; 4.5 ± 1.4 vs. 3.0 ± 1.6 cm2, p = 0.035). The proportion of moderate-to-strong positive type II collagen staining was significantly higher in the hUCB-MSC group compared to that in the microdrilled group (93.3% vs. 60%, respectively). Rigidity upon probing resembled that of normal cartilage tissue more in the hUCB-MSC group (86.7% vs. 50.0%, p = 0.075). Histological findings revealed a higher proportion of hyaline cartilage in the group with implanted hUCB-MSC (p = 0.041). CONCLUSION: hUCB-MSC implantation showed comparable clinical outcomes to those of microdrilling as supplementary cartilage procedures combined with HTO in the short term, despite the significantly larger cartilage defect in the hUCB-MSC group. The repaired cartilage after hUCB-MSC implantation showed greater hyaline-type cartilage with rigidity than that after microdrilling. LEVEL OF EVIDENCE: Level II, Prospective Comparative Cohort Study.

Comparative Analysis of Hyaline Cartilage Characteristics and Chondrocyte Potential for Articular Cartilage Repair.

This study aimed to compare the histological, biochemical, and mechanical characteristics of hyaline cartilage in different regions and evaluate the potential of chondrocytes extracted from each region as donor sources for articular cartilage repair. The cartilage tissues of the femoral head and knee joint, ribs, nasal septum, thyroid, and xiphoid process of adult Bama pigs were isolated for histological, biochemical, and mechanical evaluation and analysis. The corresponding chondrocytes were isolated and evaluated for proliferation and redifferentiation capacity, using biochemical and histological analysis and RT-PCR experiments. Compared with articular cartilage, non-articular hyaline cartilage matrix stained more intensely in Safranin-O staining. Glycosaminoglycan and total collagen content were similar among all groups, while the highest content was measured in nasal septal cartilage. Regarding biomechanics, non-articular cartilage is similar to articular cartilage, but the elastic modulus and hardness are significantly higher in the middle region of costal cartilage. The chondrocytes extracted from different regions had no significant difference in morphology. Hyaline cartilage-like pellets were formed in each group after redifferentiation. The RT-PCR results revealed similar expressions of cartilage-related genes across the groups, albeit with lower expression of Col2 in the xiphoid chondrocytes. Conversely, higher expression of Col10 was observed in the chondrocytes from the rib, thyroid, and xiphoid cartilage. This study provides valuable preclinical data for evaluating heterotopic hyaline cartilage and chondrocytes for articular cartilage regeneration. The findings contribute to the selection of chondrocyte origins and advance the clinical translation of technology for cartilage regeneration.

Human Chondrocytes, Metabolism of Articular Cartilage, and Strategies for Application to Tissue Engineering.

Hyaline cartilage, which is characterized by the absence of vascularization and innervation, has minimal self-repair potential in case of damage and defect formation in the chondral layer. Chondrocytes are specialized cells that ensure the synthesis of extracellular matrix components, namely type II collagen and aggregen. On their surface, they express integrins CD44, α1ß1, α3ß1, α5ß1, α10ß1, αVß1, αVß3, and αVß5, which are also collagen-binding components of the extracellular matrix. This article aims to contribute to solving the problem of the possible repair of chondral defects through unique methods of tissue engineering, as well as the process of pathological events in articular cartilage. In vitro cell culture models used for hyaline cartilage repair could bring about advanced possibilities. Currently, there are several variants of the combination of natural and synthetic polymers and chondrocytes. In a three-dimensional environment, chondrocytes retain their production capacity. In the case of mesenchymal stromal cells, their favorable ability is to differentiate into a chondrogenic lineage in a three-dimensional culture.

Development of cartilage tissue using a stirred bioreactor and human iPSC-derived limb bud mesenchymal cells.

Production of cartilaginous particles for regenerative medicine requires a large supply of chondrocytes and development of suitable production techniques. Previously, we successfully produced human induced pluripotent stem cell (hiPSC)-derived limb bud mesenchymal cells (ExpLBM cells) with a high chondrogenic differentiation potential that stably proliferate. It may be possible to use these cells in combination with a stirred bioreactor to develop a tissue-engineered cell culture technology with potential for scale-up to facilitate production of large amounts of cartilaginous particles. ExpLBM cells derived from 414C2 and Ff-I 14s04 (human leukocyte antigen homozygous) hiPSCs were seeded into a stirred bioreactor containing cartilage induction medium. To characterize the cartilaginous particles produced, we performed real-time quantitative reverse transcription-polymerase chain reaction and histological analyses. Additionally, we transplanted the cartilage tissue into osteochondral defects of immunocompromised rats to assess its functionality, and evaluated engraftment of the grafted tissue. We successfully produced large amounts of cartilaginous particles via cartilage induction culture in a stirred bioreactor. This tissue exhibited significantly increased expression levels of type II collagen (COL2), aggrecan (ACAN), and SRY-box transcription factor 9 (SOX9), as well as positive Safranin O and Toluidine blue staining, indicating that it possesses characteristics of hyaline cartilage. Furthermore, engrafted tissues in osteochondral knee defects of immunodeficient rats were positively stained for human vimentin, COL2, and ACAN as well as with Safranin O. In this study, we successfully generated large amounts of hiPSC-derived cartilaginous particles using a combination of tissue engineering techniques. This method is promising as a cartilage regeneration technology with potential for scale-up.

Converse modulation of Wnt/ß-catenin signaling during expansion and differentiation phases of Infrapatellar fat pad-derived MSCs for improved engineering of hyaline cartilage.

Mesenchymal stem cells (MSCs) are potential candidates in cell-based therapy for cartilage repair and regeneration. However, during chondrogenic differentiation, MSCs undergo undesirable hypertrophic maturation. This poses a risk of ossification in the neo-tissue formed that eventually impedes the clinical use of MSCs for cartilage repair. TGF-ß is a potent growth factor used for chondrogenic differentiation of MSCs, however, its role in hypertrophy remains ambiguous. In the present work, we decipher that TGF-ß activates Wnt/ß-catenin signaling through SMAD3 and increases the propensity of Infrapatellar fat pad derived MSCs (IFP-MSCs) towards hypertrophy. Notably, inhibiting TGF-ß induced Wnt/ß-catenin signaling suppresses hypertrophic progression and enhances chondrogenic ability of IFP-MSCs in plasma hydrogels. Additionally, we demonstrate that activating Wnt signaling during expansion phase, promotes proliferation and reduces senescence, while improving stemness of IFP-MSCs. Thus, conversely modulating Wnt signaling in vitro during expansion and differentiation phases generates hyaline-like cartilage with minimal hypertrophy. Importantly, pre-treatment of IFP-MSCs encapsulated in plasma hydrogel with Wnt modulators followed by subcutaneous implantation in nude mice resulted in formation of a cartilage tissue with negligible calcification. Overall, this study provides technological advancement on targeting Wnt/ß-catenin pathway in a 3D scaffold, while maintaining the standard chondro-induction protocol to overcome the challenges associated with the clinical use of MSCs to engineer hyaline cartilage.

Enhanced osteochondral repair with hyaline cartilage formation using an extracellular matrix-inspired natural scaffold.

Osteochondral defects pose a great challenge and a satisfactory strategy for their repair has yet to be identified. In particular, poor repair could result in the generation of fibrous cartilage and subchondral bone, causing the degeneration of osteochondral tissue and eventually leading to repair failure. Herein, taking inspiration from the chemical elements inherent in the natural extracellular matrix (ECM), we proposed a novel ECM-mimicking scaffold composed of natural polysaccharides and polypeptides for osteochondral repair. By meticulously modifying natural biopolymers to form reversible guest-host and rigid covalent networks, the scaffold not only exhibited outstanding biocompatibility, cell adaptability, and biodegradability, but also had excellent mechanical properties that can cater to the environment of osteochondral tissue. Additionally, benefiting from the drug-loading group, chondrogenic and osteogenic drugs could be precisely integrated into the specific zone of the scaffold, providing a tissue-specific microenvironment to facilitate bone and cartilage differentiation. In rabbit osteochondral defects, the ECM-inspired scaffold not only showed a strong capacity to promote hyaline cartilage formation with typical lacuna structure, sufficient mechanical strength, good elasticity, and cartilage-specific ECM deposition, but also accelerated the regeneration of quality subchondral bone with high bone mineralization density. Furthermore, the new cartilage and subchondral bone were heterogeneous, a trait that is typical of the natural landscape, reflecting the gradual progression from cartilage to subchondral bone. These results suggest the potential value of this bioinspired osteochondral scaffold for clinical applications.

The Dual-Responsive Interaction of Particulated Hyaline Cartilage and Plasma Rich in Growth Factors (PRGF) in the Repair of Cartilage Defects: An In Vitro Study.

The treatment of chondral and osteochondral defects is challenging. These types of lesions are painful and progress to osteoarthritis over time. Tissue engineering offers tools to address this unmet medical need. The use of an autologous cartilage construct consisting of hyaline cartilage chips embedded in plasma rich in growth factors (PRGF) has been proposed as a therapeutic alternative. The purpose of this study was to dig into the potential mechanisms behind the in vitro remodelling process that might explain the clinical success of this technique and facilitate its optimisation. Chondrocyte viability and cellular behaviour over eight weeks of in vitro culture, type II collagen synthesis, the dual delivery of growth factors by hyaline cartilage and PRGF matrix, and the ultrastructure of the construct and its remodelling were characterised. The main finding of this research is that the cartilage fragments embedded in the three-dimensional PRGF scaffold contain viable chondrocytes that are able to migrate into the fibrin network, proliferate and synthesise extracellular matrix after the second week of in vitro culture. The characterization of this three-dimensional matrix is key to unravelling the molecular kinetics responsible for its efficacy.

Dual-tissue transplantation versus osteochondral autograft transplantation in the treatment of osteochondral defects: a porcine model study.

BACKGROUND: Osteochondral injury is a common sports injury, and hyaline cartilage does not regenerate spontaneously when injured. However, there is currently no gold standard for treating osteochondral defects. Osteochondral autograft transplantation (OAT) is widely used in clinical practice and is best used to treat small osteochondral lesions in the knee that are < 2 cm2 in size. Autologous dual-tissue transplantation (ADTT) is a promising method with wider indications for osteochondral injuries; however, ADTT has not been evaluated in many studies. This study aimed to compare the radiographic and histological results of ADTT and OAT for treating osteochondral defects in a porcine model. METHODS: Osteochondral defects were made in the bilateral medial condyles of the knees of 12 Dian-nan small-ear pigs. The 24 knees were divided into the ADTT group (n = 8), OAT group (n = 8), and empty control group (n = 8). At 2 and 4 months postoperatively, the knees underwent gross evaluation based on the International Cartilage Repair Society (ICRS) score, radiographic assessment based on CT findings and the magnetic resonance observation of cartilage repair tissue (MOCART) score, and histological evaluation based on the O'Driscoll histological score of the repair tissue. RESULTS: At 2 months postoperatively, the ICRS score, CT evaluation, MOCART score, and O'Driscoll histological score were significantly better in the OAT group than the ADTT group (all P < 0.05). At 4 months postoperatively, the ICRS score, CT evaluation, MOCART score, and O'Driscoll histological score tended to be better in the OAT group than the ADTT group, but these differences did not reach statistical significance (all P > 0.05). CONCLUSIONS: In a porcine model, ADTT and OAT are both effective treatments for osteochondral defects in weight bearing areas. ADTT may be useful as an alternative procedure to OAT for treating osteochondral defects.

Particulated Costal Hyaline Cartilage Allograft With Subchondral Drilling Improves Joint Space Width and Second-Look Macroscopic Articular Cartilage Scores Compared With Subchondral Drilling Alone in Medial Open-Wedge High Tibial Osteotomy.

PURPOSE: To compare the articular cartilage regeneration based on second-look arthroscopy in patients who underwent medial open-wedge high tibial osteotomy (MOWHTO) combined with particulated costal hyaline cartilage allograft (PCHCA) implantation with those who underwent MOWHTO and subchondral drilling (SD). Moreover, we compared the clinical and radiographic outcomes between the groups. METHODS: From January 2014 to November 2020, patients with full-thickness cartilage defect on the medial femoral condyle who underwent MOWHTO combined with PCHCA (group A) or SD (group B) were reviewed. Fifty-one knees were matched after propensity score matching. The status of regenerated cartilage was classified according to the International Cartilage Repair Society-Cartilage Repair Assessment (ICRS-CRA) grading system and Koshino staging system, based on second-look arthroscopic findings. Clinically, the Knee Injury and Osteoarthritis Outcome Score, the Western Ontario and McMaster Universities Osteoarthritis Index, and range of motion were compared. Radiographically, we compared the differences in the minimum joint space width (JSW) and change in JSW. RESULTS: The average age was 55.5 years (range, 42-64 years), and the average follow-up period was 27.1 months (range, 24-48 months). Group A showed a significantly better cartilage status than group B based on the ICRS-CRA grading system and Koshino staging system (P < .001 and <.001, respectively). There were no significant differences in clinical and radiographic outcomes between groups. In group A, the minimum JSW at the last follow-up was significantly increased than that before surgery (P = .013), and a significantly greater increase in JSW was observed in group A (P = .025). CONCLUSIONS: When performed with MOWHTO, the combination of SD and PCHCA was associated with superior articular cartilage regeneration on the ICRS-CRA grading and Koshino staging on second-look arthroscopy performed at a minimum of 2 years follow-up than SD alone. However, there was no difference in clinical outcomes. LEVEL OF EVIDENCE: Level III, retrospective comparative study.

Anatomy, molecular structures, and hyaluronic acid - Gelatin injectable hydrogels as a therapeutic alternative for hyaline cartilage recovery: A review.

Cartilage damage caused by trauma or osteoarthritis is a common joint disease that can increase the social and economic burden in society. Due to its avascular characteristics, the poor migration ability of chondrocytes, and a low number of progenitor cells, the self-healing ability of cartilage defects has been significantly limited. Hydrogels have been developed into one of the most suitable biomaterials for the regeneration of cartilage because of its characteristics such as high-water absorption, biodegradation, porosity, and biocompatibility similar to natural extracellular matrix. Therefore, the present review article presents a conceptual framework that summarizes the anatomical, molecular structure and biochemical properties of hyaline cartilage located in long bones: articular cartilage and growth plate. Moreover, the importance of preparation and application of hyaluronic acid - gelatin hydrogels for cartilage tissue engineering are included. Hydrogels possess benefits of stimulating the production of Agc1, Col2α1-IIa, and SOX9, molecules important for the synthesis and composition of the extracellular matrix of cartilage. Accordingly, they are believed to be promising biomaterials of therapeutic alternatives to treat cartilage damage.

Engineering Large Airways.

A key issue facing trachea replacement attempts has been the discrepancy of the mechanical properties between the native tracheal tissue and that of the replacement construct; this difference is often one of the major causes for implant failure in vivo and within clinical efforts. The trachea is composed of distinct structural regions, with each component fulfilling a different role in maintaining overall tracheal stability. The trachea's horseshoe-shaped hyaline cartilage rings, smooth muscle and annular ligament collectively produce an anisotropic tissue that allows for longitudinal extensibility and lateral rigidity. Therefore, any tracheal substitute must be mechanically robust in order to withstand intra-thoracic pressure changes that occur during respiration. Conversely, they must also be able to deform radially to allow for changes in the cross-sectional area during coughing and swallowing. These complicated native tissue characteristics, coupled with a lack of standardised protocols to accurately quantify tracheal biomechanics as guidance for implant design, constitute a significant hurdle for tracheal biomaterial scaffold fabrication. This chapter aims to highlight the pressure forces exerted on the trachea and how they can influence tracheal construct design and also the biomechanical properties of the three main components of the trachea and how to mechanically assess them.

Applied Compressive Strain Governs Hyaline-like Cartilage versus Fibrocartilage-like ECM Produced within Hydrogel Constructs.

The goal of cartilage tissue engineering (CTE) is to regenerate new hyaline cartilage in joints and treat osteoarthritis (OA) using cell-impregnated hydrogel constructs. However, the production of an extracellular matrix (ECM) made of fibrocartilage is a potential outcome within hydrogel constructs when in vivo. Unfortunately, this fibrocartilage ECM has inferior biological and mechanical properties when compared to native hyaline cartilage. It was hypothesized that compressive forces stimulate fibrocartilage development by increasing production of collagen type 1 (Col1), an ECM protein found in fibrocartilage. To test the hypothesis, 3-dimensional (3D)-bioprinted hydrogel constructs were fabricated from alginate hydrogel impregnated with ATDC5 cells (a chondrogenic cell line). A bioreactor was used to simulate different in vivo joint movements by varying the magnitude of compressive strains and compare them with a control group that was not loaded. Chondrogenic differentiation of the cells in loaded and unloaded conditions was confirmed by deposition of cartilage specific molecules including glycosaminoglycans (GAGs) and collagen type 2 (Col2). By performing biochemical assays, the production of GAGs and total collagen was also confirmed, and their contents were quantitated in unloaded and loaded conditions. Furthermore, Col1 vs. Col2 depositions were assessed at different compressive strains, and hyaline-like cartilage vs. fibrocartilage-like ECM production was analyzed to investigate how applied compressive strain affects the type of cartilage formed. These assessments showed that fibrocartilage-like ECM production tended to reduce with increasing compressive strain, though its production peaked at a higher compressive strain. According to these results, the magnitude of applied compressive strain governs the production of hyaline-like cartilage vs. fibrocartilage-like ECM and a high compressive strain stimulates fibrocartilage-like ECM formation rather than hyaline cartilage, which needs to be addressed by CTE approaches.

Hypoxia Promotes Cartilage Regeneration in Cell-Seeded 3D-Printed Bioscaffolds Cultured with a Bespoke 3D Culture Device.

In this study, we investigated the effect of oxygen tension on the expansion of ADMSCs and on their differentiation toward their chondrocytic phenotype, regenerating a lab-based cartilaginous tissue with superior characteristics. Controversial results with reference to MSCs that were cultured under different hypoxic levels, mainly in 2D culturing settings combined with or without other biochemical stimulus factors, prompted our team to study the role of hypoxia on MSCs chondrogenic differentiation within an absolute 3D environment. Specifically, we used 3D-printed honeycomb-like PCL matrices seeded with ADMSCs in the presence or absence of TGF and cultured with a prototype 3D cell culture device, which was previously shown to favor nutrient/oxygen supply, cell adhesion, and infiltration within scaffolds. These conditions resulted in high-quality hyaline cartilage that was distributed uniformly within scaffolds. The presence of the TGF medium was necessary to successfully produce cartilaginous tissues with superior molecular and increased biomechanical properties. Despite hypoxia's beneficial effect, it was overall not enough to fully differentiate ADMSCs or even promote cell expansion within 3D scaffolds alone.

Adeno-Associated Virus-Delivered Fibroblast Growth Factor 18 Gene Therapy Promotes Cartilage Anabolism.

OBJECTIVE: To determine the characterization of chondrogenic properties of adeno-associated virus type 2 (AAV2)-delivered hFGF18, via analysis of effects on primary human chondrocyte proliferation, gene expression, and in vivo cartilage thickness changes in the tibia and meniscus. DESIGN: Chondrogenic properties of AAV2-FGF18 were compared with recombinant human FGF18 (rhFGF18) in vitro relative to phosphate-buffered saline (PBS) and AAV2-GFP negative controls. Transcriptome analysis was performed using RNA-seq on primary human chondrocytes treated with rhFGF18 and AAV2-FGF18, relative to PBS. Durability of gene expression was assessed using AAV2-nLuc and in vivo imaging. Chondrogenesis was evaluated by measuring weight-normalized thickness in the tibial plateau and the white zone of the anterior horn of the medial meniscus in Sprague-Dawley rats. RESULTS: AAV2-FGF18 elicits chondrogenesis by promoting proliferation and upregulation of hyaline cartilage-associated genes, including COL2A1 and HAS2, while downregulating fibrocartilage-associated COL1A1. This activity translates to statistically significant, dose-dependent increases in cartilage thickness in vivo within the area of the tibial plateau, following a single intra-articular injection of the AAV2-FGF18 or a regimen of 6 twice-weekly injections of rhFGF18 protein relative to AAV2-GFP. In addition, we observed AAV2-FGF18-induced and rhFGF18-induced increases in cartilage thickness of the anterior horn of the medial meniscus. Finally, the single-injection AAV2-delivered hFGF18 offers a potential safety advantage over the multi-injection protein treatment as evidenced by reduced joint swelling over the study period. CONCLUSION: AAV2-delivered hFGF18 represents a promising strategy for the restoration of hyaline cartilage by promoting extracellular matrix production, chondrocyte proliferation, and increasing articular and meniscal cartilage thickness in vivo after a single intra-articular injection.

Incidence of Compression-Induced Microinjuries in the Cartilage Endplate of the Spine.

STUDY DESIGN: In vitro biomechanical study. OBJECTIVE: This study investigated the incidence of microstructural endplate injuries caused by cyclic compression loading. The covarying effects of joint posture, loading duration, and peak compression variation were assessed. SUMMARY OF BACKGROUND DATA: The endplate is physiologically and functionally important for the maintenance of spine health. Despite the ability to radiographically diagnose and classify macroscopic endplate injuries, the mechanical mechanisms of injury initiation and progression remain largely unknown. METHODS: One hundred and fourteen porcine cervical spinal units were examined. All spinal units were exposed to preconditioning tests, followed by cyclic compression testing that differed by posture (flexed, neutral), loading duration (1000, 3000, 5000 cycles), and peak compression variation (10%, 20%, 40%). Microstructural injuries were examined via immunofluorescence staining for collagen I ( i.e. , subchondral bone) and collagen II ( i.e. , hyaline cartilage endplate). From the 678 acquired images, the incidence of node, avulsion, cartilage, and circumferential pore microinjuries were determined. The distribution of microinjuries between postures, spinal levels, and vertebrae were evaluated along with the associations of incidence and size of injuries with loading duration and variation. RESULTS: The incidence of avulsion injuries was significantly greater in caudal endplates (92%, P =0.006). No other injuries differed between vertebrae ( P ≥0.804) and no significant differences were observed between spinal units ( P ≥0.158). With respect to posture, 100% ( P <0.001) and 90% ( P <0.001) of avulsion and node injuries, respectively, occurred in flexed postures, whereas 82% ( P <0.001) of cartilage microinjuries occurred with neutral postures. Loading duration was significantly associated with microinjury incidence ( P <0.001) and lesion size ( P ≤0.003). CONCLUSION: Mechanical factors such as posture did not appreciably affect the incidence of endplate injury, but microinjury types were differently distributed between flexed and neutral postures. The duration of compression was shown to have an important role in the incidence of microinjury and lesion size.

Exploring the Mechanism of Microfracture in the Treatment of Porcine Full-Thickness Cartilage Defect.

BACKGROUND: Microfracture has the most extensive clinical application because of its advantages of a single operation, unified process, and low operation cost. Because research on the repair mechanism of microfractures in the treatment of cartilage defects is not in-depth, this study aimed to elucidate the mechanism. PURPOSE: To identify the characteristic cell subsets at different repair stages after microfracture, systematically analyze the repair process of the defect area after microfracture, and investigate the mechanism of fibrocartilage repair. STUDY DESIGN: Descriptive laboratory study. METHODS: Full-thickness articular cartilage defects and microfractures was established in the right knee of Bama miniature pigs. Single-cell transcriptional assays were used to identify the characteristics of cells isolated from healthy articular cartilage and regenerated tissues. RESULTS: Microfractures induced mature fibrous repair in the full-thickness cartilage defect six months after surgery, while early stages of repair occurred within six weeks. Based on single-cell sequencing results, eight subsets and specific marker genes were identified. Two processes may occur after microfracture: normal hyaline cartilage regeneration and abnormal fibrocartilage repair. Regulatory chondrocytes, proliferative chondrocytes and cartilage progenitor cells (CPCs) may play important roles in the normal regeneration process. During abnormal repair, CPCs and skeletal stem cells may have different functions, and macrophages and endothelial cells may play important regulatory roles in the formation of fibrochondrocytes. CONCLUSIONS: Using single-cell transcriptome sequencing, this study investigated the tissue regeneration process and identified key cell subsets after microfracture. CLINICAL RELEVANCE: These results provide future targets for optimizing the repair effect of microfracture.

Articular Cartilage Repair After Implantation of Hyaline Cartilage Beads Engineered From Adult Dedifferentiated Chondrocytes: Cartibeads Preclinical Efficacy Study in a Large Animal Model.

BACKGROUND: Chondrocyte-based cell therapy to repair cartilage has been used for >25 years despite current limitations. This work presents a new treatment option for cartilage lesions. HYPOTHESIS: High-quality hyaline cartilage microtissues called Cartibeads are capable of treating focal chondral lesions once implanted in the defect, by complete fusion of Cartibeads among themselves and their integration with the surrounding native cartilage and subchondral bone. STUDY DESIGN: Controlled laboratory study. METHODS: Cartibeads were first produced from human donors and characterized using histology (safranin O staining of glycosaminoglycan [GAG] and immunohistochemistry of collagen I and II) and GAG dosage. Cartibeads from 6 Göttingen minipigs were engineered and implanted in an autologous condition in the knee (4 or 5 lesions per knee). One group was followed up for 3 months and the other for 6 months. Feasibility and efficacy were measured using histological analysis and macroscopic and microscopic scores. RESULTS: Cartibeads revealed hyaline features with strong staining of GAG and collagen II. High GAG content was obtained: 24.6-µg/mg tissue (wet weight), 15.52-µg/mg tissue (dry weight), and 35 ± 3-µg GAG/bead (mean ± SD). Histological analysis of Göttingen minipigs showed good integration of Cartibeads grafts at 3 and 6 months after implantation. The Bern Score of the histological assay comparing grafted versus empty lesions was significant at 3 months (grafted, n = 10; nongrafted, n = 4; score, 3.3 and 5.3, respectively) and 6 months (grafted, n = 11; nongrafted, n = 3; score, 1.6 and 5.1). CONCLUSION: We developed an innovative 3-step method allowing, for the first time, the use of fully dedifferentiated adult chondrocytes with a high number of cell passage (owing to the extensive amplification in culture). Cartibeads engineered from chondrocytes hold potential as an advanced therapy medicinal product for treating cartilage lesions with established efficacy. CLINICAL RELEVANCE: This successful preclinical study, combined with standardized manufacturing of Cartibeads according to good manufacturing practice guidelines, led to the approval of first-in-human clinical trial by the ethics committee and local medical authority. The generated data highlighted a promising therapy to treat cartilage lesions from a small amount of starting biopsy specimen. With our innovative cell amplification technology, very large lesions can be treated, and older active patients can benefit from it.

Histological and Radiological Assessment of Endogenously Generated Repair Tissue In Vivo Following a Chondral Harvest.

OBJECTIVE: To examine repair tissue formed approximately 15 months after a chondral harvest in the human knee. DESIGN: Sixteen individuals (12 males, 4 females, mean age 36 ± 9 years) underwent a chondral harvest in the trochlea as a pre-requisite for autologous chondrocyte implantation (ACI) treatment. The harvest site was assessed via MRI at 14.3 ± 3.2 months and arthroscopy at 15 ± 3.5 months (using the Oswestry Arthroscopy Score [O-AS] and the International Cartilage Repair Society Arthroscopy Score [ICRS-AS]). Core biopsies (1.8 mm diameter, n = 16) of repair tissue obtained at arthroscopy were assessed histologically (using the ICRS II and OsScore histology scores) and examined via immunohistochemistry for the presence of collagen types I and II. RESULTS: The mean O-AS and ICRS-AS of the repaired harvest sites were 7.2 ± 3.2 and 10.1 ± 3.5, respectively, with 80.3% ± 26% repair fill depth on MRI. The histological quality of the repair tissue formed was variable, with some hyaline cartilage present in 50% of the biopsies; where this occurred, it was associated with a significantly higher ICRS-AS than those with no hyaline cartilage present (median 11 vs. 7.5, P = 0.049). Collagen types I and II were detected in 12/14 and 10/13 biopsies, respectively. CONCLUSIONS: We demonstrate good-quality structural repair tissue formed following cartilage harvest in ACI, suggesting this site can be useful to study endogenous cartilage repair in humans. The trochlea is less commonly affected by osteoarthritis; therefore, location may be critical for spontaneous repair. Understanding the mechanisms and factors influencing this could improve future treatments for cartilage defects.

Hyperelastic characterization reveals proteoglycans drive the nanoscale strain-stiffening response in hyaline cartilage.

Degenerative diseases such as osteoarthritis (OA) result in deterioration of cartilage extracellular matrix (ECM) components, significantly compromising tissue function. For measurement of mechanical properties at micron resolution, atomic force microscopy (AFM) is a leading technique in biomaterials research, including in the study of OA. It is common practice to determine material properties by applying classical Hertzian contact theory to AFM data. However, errors are consequential because the application of a linear elastic contact model to tissue ignores the fact that soft materials exhibit nonlinear properties even at small strains, influencing the biological conclusions of clinically-relevant studies. Additionally, nonlinear material properties are not well characterized, limiting physiological relevance of Young's modulus. Here, we probe the ECM of hyaline cartilage with AFM and explore the application of Hertzian theory in comparison to five hyperelastic models: NeoHookean, Mooney-Rivlin, Arruda-Boyce, Fung, and Ogden. The Fung and Ogden models achieved the best fits of the data, but the Fung model demonstrated robust sensitivity during model validation, demonstrating its ideal application to cartilage ECM and potentially other connective tissues. To develop a biological understanding of the Fung nonlinear parameter, we selectively degraded ECM components to target collagens (purified collagenase), hyaluronan (bacterial hyaluronidase), and glycosaminoglycans (chondroitinase ABC). We found significant differences in both Fung parameters in response to enzymatic treatment, indicating that proteoglycans drive the nonlinear response of cartilage ECM, and validating biological relevance of these phenomenological parameters. Our findings add value to the biomechanics community of using two-parameter material models for microindentation of soft biomaterials.

Isolation of Chondrons from Hyaline Cartilage.

In native healthy hyaline cartilage, the chondrocytes are surrounded by a pericellular matrix that has a distinct composition and function compared to the hyaline cartilage extracellular matrix. The chondrocyte together with its pericellular matrix is called a chondron. The type VI collagen, which is the main component of the pericellular matrix, is resistant to enzymatic digestion by pure collagenase and dispase that do digest the extracellular matrix. Therefore, this combination of enzymes can be used to enzymatically isolate chondrons from hyaline cartilage. Chondrons have a high potential for cartilage tissue engineering. This chapter describes in detail how chondrons can be isolated from hyaline cartilage for further use.