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.

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.

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.

Purification and Isolation of Proteins from Hyaline Cartilage.

Proteins from hyaline or articular cartilage can be isolated and purified using a series of chemical extraction steps and various identification techniques including mass spectrometry and immunoblotting. The isolation and purification of proteins from cartilage will facilitate the study of specific proteins and multimeric complexes of cartilage proteins to better understand their functions in normal healthy cartilage as well as pathological conditions of cartilage. Cartilage tissue engineering efforts rely on the comprehensive understanding of the composition of cartilage and the function of each of the protein constituents.

Hyaline Cartilage Microtissues Engineered from Adult Dedifferentiated Chondrocytes: Safety and Role of WNT Signaling.

The repair of damaged articular cartilage is an unmet medical need. Chondrocyte-based cell therapy has been used to repair cartilage for over 20 years despite current limitations. Chondrocyte dedifferentiation upon expansion in monolayer is well known and is the main obstacle to their use as cell source for cartilage repair. Consequently, current approaches often lead to fibrocartilage, which is biomechanically different from hyaline cartilage and not effective as a long-lasting treatment. Here, we describe an innovative 3-step method to engineer hyaline-like cartilage microtissues, named Cartibeads, from high passage dedifferentiated chondrocytes. We show that WNT5A/5B/7B genes were highly expressed in dedifferentiated chondrocytes and that a decrease of the WNT signaling pathway was instrumental for full re-differentiation of chondrocytes, enabling production of hyaline matrix instead of fibrocartilage matrix. Cartibeads showed hyaline-like characteristics based on GAG quantity and type II collagen expression independently of donor age and cartilage quality. In vivo, Cartibeads were not tumorigenic when transplanted into SCID mice. This simple 3-step method allowed a standardized production of hyaline-like cartilage microtissues from a small cartilage sample, making Cartibeads a promising candidate for the treatment of cartilage lesions.

Cartilage-penetrating hyaluronic acid hydrogel preserves tissue content and reduces chondrocyte catabolism.

Articular cartilage injuries have a limited healing capacity and, due to inflammatory and catabolic activities, often experience progressive degeneration towards osteoarthritis. Current repair techniques generally provide short-term symptomatic relief; however, the regeneration of hyaline cartilage remains elusive, leaving both the repair tissue and surrounding healthy tissue susceptible to long-term wear. Therefore, methods to preserve cartilage following injury, especially from matrix loss and catabolism, are needed to delay, or even prevent, the deteriorative process. The goal of this study was to develop and evaluate a cartilage-penetrating hyaluronic-acid (HA) hydrogel to improve damaged cartilage biomechanics and prevent tissue degeneration. At time zero, the HA-based hydrogel provided a 46.5% increase in compressive modulus and a decrease in permeability after simulated degeneration of explants (collagenase application). Next, in a degenerative culture model (interleukin-1ß [IL-1ß] for 2 weeks), hydrogel application prior to or midway through the culture mitigated detrimental changes to compressive modulus and permeability observed in non-treated explants. Furthermore, localized loss of proteoglycan was observed in degenerative culture conditions alone (non-treated), but hydrogel administration significantly improved the retention of matrix elements. Finally, NITEGE staining and gene expression analysis showed the ability of the HA gel to decrease chondrocyte catabolic activity. These results highlight the importance of reinforcing damaged cartilage with a biomaterial system to both preserve tissue content and reduce catabolism associated with injury and inflammation.

Hyaline cartilage differentiation of fibroblasts in regeneration and regenerative medicine.

Amputation injuries in mammals are typically non-regenerative; however, joint regeneration is stimulated by BMP9 treatment, indicating the presence of latent articular chondrocyte progenitor cells. BMP9 induces a battery of chondrogenic genes in vivo, and a similar response is observed in cultures of amputation wound cells. Extended cultures of BMP9-treated cells results in differentiation of hyaline cartilage, and single cell RNAseq analysis identified wound fibroblasts as BMP9 responsive. This culture model was used to identify a BMP9-responsive adult fibroblast cell line and a culture strategy was developed to engineer hyaline cartilage for engraftment into an acutely damaged joint. Transplanted hyaline cartilage survived engraftment and maintained a hyaline cartilage phenotype, but did not form mature articular cartilage. In addition, individual hypertrophic chondrocytes were identified in some samples, indicating that the acute joint injury site can promote osteogenic progression of engrafted hyaline cartilage. The findings identify fibroblasts as a cell source for engineering articular cartilage and establish a novel experimental strategy that bridges the gap between regeneration biology and regenerative medicine.

Main and Minor Types of Collagens in the Articular Cartilage: The Role of Collagens in Repair Tissue Evaluation in Chondral Defects.

Several collagen subtypes have been identified in hyaline articular cartilage. The main and most abundant collagens are type II, IX and XI collagens. The minor and less abundant collagens are type III, IV, V, VI, X, XII, XIV, XVI, XXII, and XXVII collagens. All these collagens have been found to play a key role in healthy cartilage, regardless of whether they are more or less abundant. Additionally, an exhaustive evaluation of collagen fibrils in a repaired cartilage tissue after a chondral lesion is necessary to determine the quality of the repaired tissue and even whether or not this repaired tissue is considered hyaline cartilage. Therefore, this review aims to describe in depth all the collagen types found in the normal articular cartilage structure, and based on this, establish the parameters that allow one to consider a repaired cartilage tissue as a hyaline cartilage.

Trends in Articular Cartilage Tissue Engineering: 3D Mesenchymal Stem Cell Sheets as Candidates for Engineered Hyaline-Like Cartilage.

Articular cartilage defects represent an inciting factor for future osteoarthritis (OA) and degenerative joint disease progression. Despite multiple clinically available therapies that succeed in providing short term pain reduction and restoration of limited mobility, current treatments do not reliably regenerate native hyaline cartilage or halt cartilage degeneration at these defect sites. Novel therapeutics aimed at addressing limitations of current clinical cartilage regeneration therapies increasingly focus on allogeneic cells, specifically mesenchymal stem cells (MSCs), as potent, banked, and available cell sources that express chondrogenic lineage commitment capabilities. Innovative tissue engineering approaches employing allogeneic MSCs aim to develop three-dimensional (3D), chondrogenically differentiated constructs for direct and immediate replacement of hyaline cartilage, improve local site tissue integration, and optimize treatment outcomes. Among emerging tissue engineering technologies, advancements in cell sheet tissue engineering offer promising capabilities for achieving both in vitro hyaline-like differentiation and effective transplantation, based on controlled 3D cellular interactions and retained cellular adhesion molecules. This review focuses on 3D MSC-based tissue engineering approaches for fabricating "ready-to-use" hyaline-like cartilage constructs for future rapid in vivo regenerative cartilage therapies. We highlight current approaches and future directions regarding development of MSC-derived cartilage therapies, emphasizing cell sheet tissue engineering, with specific focus on regulating 3D cellular interactions for controlled chondrogenic differentiation and post-differentiation transplantation capabilities.

Damage to the human lumbar cartilage endplate and its clinical implications.

The cartilaginous endplate (CEP) is a thin layer of hyaline cartilage, and plays an important role in the diffusion of nutrients into the intervertebral discs. Its damage may seriously affect the disc degeneration, and result in low back pain (LBP). However, the structural features of damaged CEPs have not been well characterized, and this hinders our understanding of the etiology of disc degeneration and pain. To present the structural features of micro-damaged CEPs in patients with disc degeneration and LBP that might even be regarded as an initial factor for disc degeneration, we performed a histological study of micro-damaged CEPs harvested from human lumbar intervertebral discs and analyzed its clinical implications. Human lumbar CEPs were excised from 35 patients (mean age 60.91 years) who had disc degeneration and LBP. Control tissue was obtained from 15 patients (mean age 54.67 years) with lumbar vertebral burst fractures. LBP and disability were assessed clinically, and all patients underwent anterior vertebral body fusion surgery. CEPs together with some adjacent nucleus pulposus (NP) were sectioned at 4 µm, and stained using H&E, Safranin O/Fast Green, and Alcian Blue. Immunostaining and PCR were used to identify various markers of degeneration, innervation, and inflammation. Histology demonstrated physical micro-damage in 14/35 CEPs from the disc degeneration group. Six major types of damage could be distinguished: fissure, traumatic nodes, vascular mimicry, incorporation of NP tissue within the CEP, incorporation of bone within the CEP, and incorporation of NP and bone within the CEP. Pain and disability scores (ODI: p = 0.0190; JOA: p = 0.0205; JOABPEQ: p = 0.0034) were significantly higher in those with micro-damaged CEPs (N = 14) than in those with non-damaged CEPs (N = 21). CEP damage was significantly associated with elevated MMP3 (p = 0.043), MMP13 (p = 0.0191), ADAMTS5 (p = 0.0253), TNF-α (p = 0.0011), and Substance P (p = 0.0028), and with reduced Sox9 (p = 0.0212), aggrecan (p = 0.0127), and type II collagen (p = 0.0139). In conclusion, we presented a new classification of human lumbar micro-damaged CEPs. Furthermore, we verify disc degeneration, innervation, and discogenic pain in micro-damaged CEPs.

The Janus Role of Adhesion in Chondrogenesis.

Tackling the first stages of the chondrogenic commitment is essential to drive chondrogenic differentiation to healthy hyaline cartilage and minimize hypertrophy. During chondrogenesis, the extracellular matrix continuously evolves, adapting to the tissue adhesive requirements at each stage. Here, we take advantage of previously developed nanopatterns, in which local surface adhesiveness can be precisely tuned, to investigate its effects on prechondrogenic condensation. Fluorescence live cell imaging, immunostaining, confocal microscopy and PCR analysis are used to follow the condensation process on the nanopatterns. Cell tracking parameters, condensate morphology, cell-cell interactions, mechanotransduction and chondrogenic commitment are evaluated in response to local surface adhesiveness. Results show that only condensates on the nanopatterns of high local surface adhesiveness are stable in culture and able to enter the chondrogenic pathway, thus highlighting the importance of controlling cell-substrate adhesion in the tissue engineering strategies for cartilage repair.

Propionibacterium acnes induces cartilaginous endplate degeneration by promoting MIF expression via the NF-κB pathway.

BACKGROUND: Propionibacterium acnes (P. acnes) is a novel pathogenic factor that contributes to cartilaginous endplate (CEP) degeneration. However, the underlying mechanism of P. acnes-induced CEP degeneration remains unclear. The objective of this study is to investigate the underlying mechanism of P. acnes-induced CEP degeneration. METHODS: We first examined MIF expression in degenerated human CEP samples by immunohistochemistry. We developed a P. acnes-induced rat model and detected MIF expression using immunohistochemistry. Additionally, we investigated the mechanism of P. acnes-induced CEP degeneration in CEP cells using western blotting and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). RESULTS: We found that compared with the normal human CEP, the expression of MIF was increased in the degenerated human CEP. In a rat model, P. acnes induced CEP degeneration and upregulated MIF expression significantly. More importantly, we revealed the underlying mechanism of P. acnes-induced CEP degeneration in the rat CEP cells. Firstly, P. acnes induced the expression of MIF in a concentration-dependent manner. Then, MIF upregulated the expression of MMP-13 and promoted the secretion of IL-6 and IL-1ß. Finally, P. acnes may promote MIF expression via NF-κB pathway rather than ERK1/2 pathway. CONCLUSION: P. acnes-induced MIF expression via NF-κB pathway may be the underlying mechanism of CEP degeneration.

Mechanisms of TGFß3 Action as a Therapeutic Agent for Promoting the Synthesis of Extracellular Matrix Proteins in Hyaline Cartilage.

Hyaline cartilage is a nonvascular connective tissue covering the joint surface. It consists mostly of the extracellular matrix proteins and a small number of highly differentiated chondrocytes. At present, various techniques for repairing joint surfaces damage, for example, the use of modified cell cultures and biodegradable scaffolds, are under investigation. Molecular mechanisms of cartilage tissue proliferation have been also actively studied in recent years. TGFß3, which plays a critical role in the proliferation of normal cartilage tissue, is one of the most important protein among cytokines and growth factors affecting chondrogenesis. By interacting directly with receptors on the cell membrane surface, TGFß3 triggers a cascade of molecular interactions involving transcription factor Sox9. In this review, we describe the effects of TGFß3 on the receptor complex activation and subsequent intracellular trafficking of Smad proteins and analyze the relation between these processes and upregulation of expression of major extracellular matrix genes, such as col2a1 and acan.

Glucocorticoids induce osteoporosis mediated by glucocorticoid receptor-dependent and -independent pathways.

Clinically, glucocorticoids (GCs) are widely used to treat inflammation-related diseases; however, their long-term use causes side effects, such as osteoporosis and predisposition to bone fractures, known as glucocorticoid-induced osteoporosis (GIOP). Nr3c1 is the major glucocorticoid receptor, and its downstream signaling pathway is involved in regulating various intracellular physiological processes, including those related to bone cells; however, its mechanism in glucocorticoid-induced osteoporosis (GIOP) remains unclear. In this study, a zebrafish nr3c1-mutant was successfully generated using CRISPR/Cas9 technology to investigate the role of nr3c1 in GIOP. Mutations in nr3c1 altered cartilage development and significantly decreased bone mineralization area. Additionally, qRT-PCR results showed that the expression of extracellular matrix-, osteoblast-, and osteoclast-related genes was altered in the nr3c1-mutant. The GC-Nr3c1 pathway regulates the expression of extracellular matrix-, osteoblast-, and osteoclast-related genes via Nr3c1-dependent and Nr3c1-independent pathways. A dual-luciferase reporter assay further revealed that GCs and Nr3c1 transcriptionally regulate matrix metalloproteinase 9 (mmp9), alkaline phosphatase (alp), and acid phosphatase 5a (acp5a). This study reveals that GCs/Nr3c1 affect the expression of genes involved in bone metabolism and provides a basis to determine the role of GIOP and Nr3c1 in bone metabolism and development. We also identified a new effector target for the clinical treatment of GIOP.

Cytokine Directed Chondroblast Trans-Differentiation: JAK Inhibition Facilitates Direct Reprogramming of Fibroblasts to Chondroblasts.

Osteoarthritis (OA) is a degenerative disease of the hyaline articular cartilage. This disease is progressive and may lead to disability. Researchers proposed many regenerative approaches to treat osteoarthritis, including stem cells. Trans-differentiation of a fully differentiated cell state directly into another different differentiated cell state avoids the disadvantages of fully reprogramming cells to induced pluripotent stem cells (iPSCs) in terms of faster reprogramming of the needed cells. Trans-differentiation also reduces the risk of tumor formation by avoiding the iPSC state. OSKM factors (Oct4, Sox2, Klf4, and cMyc) accompanied by the JAK-STAT pathway inhibition, followed by the introduction of specific differentiation factors, directly reprogrammed mouse embryonic fibroblasts to chondroblasts. Our results showed the absence of intermediate induced pluripotent stem cell formation. The resulting aggregates showed clear hyaline and hypertrophic cartilage. Tumor formation was absent in sub-cutaneous capsules transplanted in SCID mice.

A cell-free approach with a supporting biomaterial in the form of dispersed microspheres induces hyaline cartilage formation in a rabbit knee model.

The objective of this study was to test a regenerative medicine strategy for the regeneration of articular cartilage. This approach combines microfracture of the subchondral bone with the implant at the site of the cartilage defect of a supporting biomaterial in the form of microspheres aimed at creating an adequate biomechanical environment for the differentiation of the mesenchymal stem cells that migrate from the bone marrow. The possible inflammatory response to these biomaterials was previously studied by means of the culture of RAW264.7 macrophages. The microspheres were implanted in a 3 mm-diameter defect in the trochlea of the femoral condyle of New Zealand rabbits, covering them with a poly(l-lactic acid) (PLLA) membrane manufactured by electrospinning. Experimental groups included a group where exclusively PLLA microspheres were implanted, another group where a mixture of 50/50 microspheres of PLLA (hydrophobic and rigid) and others of chitosan (a hydrogel) were used, and a third group used as a control where no material was used and only the membrane was covering the defect. The histological characteristics of the regenerated tissue have been evaluated 3 months after the operation. We found that during the regeneration process the microspheres, and the membrane covering them, are displaced by the neoformed tissue in the regeneration space toward the subchondral bone region, leaving room for the formation of a tissue with the characteristics of hyaline cartilage.

Safranin O without fast green is the best staining method for testing the degradation of macromolecules in a cartilage extracellular matrix for the determination of the postmortem interval.

Methods for the determination of the postmortem interval (PMI) include methods that monitor the postmortem changes of cells and molecules in different tissues. The rate of pathological degradation of macromolecules in the extracellular matrix (ECM) of hyaline cartilage could be verified by assessing the intensity of collagen and proteoglycan (PG) staining. In the presented in vitro pilot study, this methodology was used for the first time to determine PMI. The osteochondral samples of three donors were stored at 11 °C and 35 °C and analyzed on day 1, day 12, and day 36 postmortem. The intensity of staining using Masson's trichrome and Sirius red for collagen, and Alcian blue and Safranin O dyes for PG was estimated ten times according to the modified Bern grading scale. Statistical analysis showed that the Safranin O without Fast green method is the most appropriate (raters agreement 0.5541) for up to 36 days postmortem, and that the influence of time is more important (p = 0.023) than the influence of temperature (p = 0.061) on the degradation of the ECM macromolecules. The described method, which is simple and can be performed in any histological laboratory, should be verified in corpore conditions, on a large number of donors, and using an objective method for assessing the intensity of cartilage macromolecule staining for PMI determination.

Membrane-Free Stem Cell Components Inhibit Interleukin-1α-Stimulated Inflammation and Cartilage Degradation in vitro and in vivo: A Rat Model of Osteoarthritis.

Membrane-free stem cell components (MFSCC) from basal adipose tissue-derived stem cells (ADSCs) are unknown for the treatment strategies in osteoarthritis (OA). OA has been considered to be associated with inflammatory damage and cartilage degradation. In this study, we intended to investigate the molecular mechanism of the anti-inflammation and cartilage protection effect of MFSCC in vitro (rat primary chondrocytes) and in vivo (rat OA model). The MFSCC treatment significantly inhibited interleukin-1α (IL-1α) stimulated inflammation and cartilage degradation. The MFSCC considerably reduced the levels of inflammatory factors such as iNOS, COX-2, NO, and PGE2 and was suppressed NF-κB and MAPKs signaling pathways in IL-1α-stimulated rat chondrocytes. Additionally, biomarkers of OA such as MMP-9, COMP, and CTX-II decreased in the monosodium iodoacetate (MIA)-induced rat OA model by MFSCC treatment. In conclusion, the MFSCC was established to suppress IL-1α induced inflammation and cartilage degradation in vitro and in vivo. These findings provide new insight for understanding OA therapy using membrane-free stem cell approaches.

Redifferentiated Chondrocytes in Fibrin Gel for the Repair of Articular Cartilage Lesions.

BACKGROUND: Autologous chondrocyte implantation, which uses passaged chondrocytes, commonly leads to the formation of fibrocartilage. When chondrocytes are passaged to increase cell numbers, they lose their phenotype and ability to form hyaline cartilage. The use of transforming growth factor ß (TGFß) to redifferentiate passaged chondrocytes has been validated in vitro; however, it is unknown if redifferentiated chondrocytes will enhance defect repair when implanted in vivo. Furthermore, fibrin gel is used in orthopaedic surgery as a fixative and scaffold and could be an appropriate carrier to enhance retention of cells in the repair site. PURPOSE: To investigate if passaged redifferentiated chondrocytes in fibrin gel have the ability to form cartilage tissue and if these redifferentiated cells will enhance the formation of hyaline cartilage in vivo when implanted into critical-size osteochondral defects. STUDY DESIGN: Controlled laboratory study. METHODS: Rabbit and human chondrocytes were serially passaged twice in monolayer culture. Twice-passaged cells were used directly (dedifferentiated) or redifferentiated in high-density culture with TGFß3. Dedifferentiated or redifferentiated cells were mixed with fibrin gel to form fibrin clots, which were cultured in vitro to assess the use of fibrin gel as a scaffold or implanted in vivo in a critical-size osteochondral defect in New Zealand White rabbit knee joints. Rabbits were sacrificed 6 weeks after implantation, and tissues were assessed histologically and by immunohistochemistry. RESULTS: Redifferentiation of passaged chondrocytes by means of 3-dimensional culture in the presence of TGFß3 improved the formation of cartilaginous tissues in vitro, and culture in fibrin gel did not affect the cell phenotype. Implantation of dedifferentiated cells in vivo resulted in fibrocartilaginous repair tissues. Redifferentiated chondrocyte implants resulted in granulation tissues containing the hyaline cartilage marker collagen type 2. CONCLUSION: Redifferentiated chondrocytes will maintain their chondrogenic differentiation in fibrin clots. Implanted redifferentiated chondrocytes show a different reparative response than dedifferentiated chondrocytes and do not appear to enhance repair at an early time point. Another study of longer duration is required to assess tissue maturation over time. CLINICAL RELEVANCE: Redifferentiation of passaged chondrocytes with TGFß3 before implantation does not improve defect repair in the first 6 weeks.

Dynamic Compressive Loading Improves Cartilage Repair in an In Vitro Model of Microfracture: Comparison of 2 Mechanical Loading Regimens on Simulated Microfracture Based on Fibrin Gel Scaffolds Encapsulating Connective Tissue Progenitor Cells.

BACKGROUND: Microfracture of focal chondral defects often produces fibrocartilage, which inconsistently integrates with the surrounding native tissue and possesses inferior mechanical properties compared with hyaline cartilage. Mechanical loading modulates cartilage during development, but it remains unclear how loads produced in the course of postoperative rehabilitation affect the formation of the new fibrocartilaginous tissue. PURPOSE: To assess the influence of different mechanical loading regimens, including dynamic compressive stress or rotational shear stress, on an in vitro model of microfracture repair based on fibrin gel scaffolds encapsulating connective tissue progenitor cells. STUDY DESIGN: Controlled laboratory study. METHODS: Cylindrical cores were made in bovine hyaline cartilage explants and filled with either (1) cartilage plug returned to original location (positive control), (2) fibrin gel (negative control), or (3) fibrin gel with encapsulated connective tissue progenitor cells (microfracture mimic). Constructs were then subjected to 1 of 3 loading regimens: (1) no loading (ie, unloaded), (2) dynamic compressive loading, or (3) rotational shear loading. On days 0, 7, 14, and 21, the integration strength between the outer chondral ring and the central insert was measured with an electroforce mechanical tester. The central core component, mimicking microfracture neotissue, was also analyzed for gene expression by real-time reverse-transcription polymerase chain reaction, glycosaminoglycan, and double-stranded DNA contents, and tissue morphology was analyzed histologically. RESULTS: Integration strengths between the outer chondral ring and central neotissue of the cartilage plug and fibrin + cells groups significantly increased upon exposure to compressive loading compared with day 0 controls (P = .007). Compressive loading upregulated expression of chondrogenesis-associated genes (SRY-related HGMG box-containing gene 9 [SOX9], collagen type II α1 [COL2A1], and increased ratio of COL2A1 to collagen type I α1 [COL1A1], an indicator of more hyaline phenotype) in the neotissue of the fibrin + cells group compared with the unloaded group at day 21 (SOX9, P = .0032; COL2A1, P < .0001; COL2A1:COL1A1, P = .0308). Fibrin + cells constructs exposed to shear loading expressed higher levels of chondrogenic genes compared with the unloaded condition, but the levels were not as high as those for the compressive loading condition. Furthermore, catabolic markers (MMP3 and ADAMTS 5) were significantly upregulated by shear loading (P = .0234 and P < .0001, respectively) at day 21 compared with day 0. CONCLUSION: Dynamic compressive loading enhanced neotissue chondrogenesis and maturation in a simulated in vitro model of microfracture, with generation of more hyaline-like cartilage and improved integration with the surrounding tissue. CLINICAL RELEVANCE: Controlled loading after microfracture may be beneficial in promoting the formation of more hyaline-like cartilage repair tissue; however, the loading regimens applied in this in vitro model do not yet fully reproduce the complex loading patterns created during clinical rehabilitation. Further optimization of in vitro models of cartilage repair may ultimately inform rehabilitation protocols.