Repair Of Cartilage Injury Research Articles

Chitosan oligosaccharides packaged into rat adipose mesenchymal stem cells-derived extracellular vesicles facilitating cartilage injury repair and alleviating osteoarthritis

ObjectivesThis study aimed to investigate the roles of adipose mesenchymal stem cell (AMSC)-derived extracellular vesicles (EVs) binding with chitosan oligosaccharides (COS) in cartilage injury, as well as the related mechanisms.ResultsIL-1β treatment significantly inhibited the viability and migration of chondrocytes and enhanced cell apoptosis (P < 0.05), while chitosan oligosaccharides and extracellular vesicles-chitosan oligosaccharide conjugates (EVs-COS/EVs-COS conjugates) reversed the changes induced by IL-1β (P < 0.05), and the effects of extracellular vesicles-chitosan oligosaccharide conjugates were better than those of chitosan oligosaccharides (P < 0.05). After cartilage damage, IL-1β, OPN, and p53 were significantly upregulated, COL1A1, COL2A1, OCN, RUNX2, p-Akt/Akt, PI3K, c-Myc, and Bcl2 were markedly downregulated, and extracellular vesicles-chitosan oligosaccharide conjugates reversed the expression induced by cartilage injury. Through sequencing, 760 differentially expressed genes (DEGs) clustered into four expression patterns were associated with negative regulation of the canonical Wnt, PI3K-Akt, AMPK, and MAPK signaling pathways.ConclusionExtracellular vesicles-chitosan oligosaccharide conjugates may serve as a new cell-free biomaterial to facilitate cartilage injury repair and improve osteoarthritis.Graphical

Naringin in the repair of knee cartilage injury via the TGF-β/ALK5/Smad2/3 signal transduction pathway combined with an acellular dermal matrix.

ObjectiveBased on the expression changes in the TGF-β/ALK5/Smad2/3 signal transduction pathway, the repair of cartilage injury in the rabbit knee joint was investigated and evaluated by oral administration of naringin in combination with acellular dermal matrix implantation.MethodsFirst, twenty New Zealand white rabbits were randomly divided into five groups: a sham operation group (Sham group), a model group (Mod group), a naringin group (Nar group), an acellular dermal matrix group (ADM group), a naringin ​+ ​acellular dermal matrix group (Nar/ADM group). After the 12th week, the repaired tissues were assessed for histomorphology and repair content of the repaired site by observing the morphological characteristics of articular cartilage. The International Cartilage Repair Society (ICRS)'s macroscopic evaluation of the cartilage repair scale and the quantitative scoring repair effect of the modified O'Driscoll grading system were used as evaluation criteria. In addition, the structure of the rabbit knee joint was evaluated by micro-CT scan, histological staining (H & E staining, Alcian blue staining, Safranin-O staining) and immunohistochemical staining (TGF-β2 immunostaining, TGF-β3 immunostaining, Sox-9 immunostaining).Results① The observation of the repair morphology of joint defect tissues showed that the repair effects of the Nar and ADM groups were better than that of the Mod group, and the repair effect of Nar/ADM group was the best (P < 0.05). ② Quantitative scoring of joint defect tissue showed that the Nar/ADM group had the best repair efficacy in the quantitative scores of the above two scales compared with the other groups (P < 0.05). ③ Micro-CT scan showed that the ADM group had obvious repair of the defect structure, while the ADM/Nar group had blurred repair boundaries, and the layers of cartilage and subchondral bone were clear. ④ Histological staining (H & E staining, Alcian blue stain, Safranin-O staining) showed that the ADM group had a better effect on the repair of joint structure at the joint defect, the Nar group had a better effect on the repair of cartilage quality at the joint defect, and the ADM/Nar group had satisfactory results in both of the above aspects. ⑤ Immunohistochemical staining (TGF-β2 immunostaining, TGF-β3 immunostaining, Sox-9 immunostaining) revealed that the Nar group showed more abundant expression of the above proteins in articular cartilage defects than the Mod and ADM groups and that the Nar/ADM groups showed extensive TGF-β2, TGF-β3 and Sox-9 protein expression, with uniform expression and smooth distribution.ConclusionsOral administration of naringin, the active ingredient of Rhizoma Drynariae, combined with acellular dermal matrix can achieve better repair effects in both joint structure repair and cartilage quality repair at the defect site when repairing cartilage defects in rabbit knees, and the generation of this effect may be caused by the activation of the TGF-β/ALK5/Smad2/3 signal transduction pathway by naringin, resulting in the increased expression of TGF-β2, TGF-β3, and Sox-9 in cartilage defects.The Translational Potential of this ArticleNaringin combined with acellular dermal matrix can facilitate the repair of osteochondral defects and has potential for application in osteochondral tissue engineering.

Thermochemotherapy Meets Tissue Engineering for Rheumatoid Arthritis Treatment

AbstractRheumatoid arthritis (RA) is an autoimmune disease that progresses from inflammation to cartilage destruction. Inspired by the similar characteristics of inflammatory granulation tissue to those of tumors, the newly emerged tumor therapy called thermochemotherapy is proposed to treat RA. Meanwhile, the repair of cartilage injury via tissue engineering is paid attention simultaneously. A first‐line antirheumatic drug (MTX; methotrexate) and transforming growth factor β1 (TGF‐β1) are loaded in nano‐Fe3O4 composite chitosan‐polyolefin to construct a multifunctional hydrogel (DN‐Fe‐MTX‐TGFβ1). The mechanical properties of the hydrogel are equivalent to that of articular cartilage to guarantee its role as a scaffold. A long‐term release ability and the magnetocaloric properties of the hydrogel assure its effect to provide sustained local thermochemotherapy. The effective ability of the hydrogel for both anti‐inflammation and cartilage repair is demonstrated. This work indicates a promising way to combine thermochemotherapy and tissue engineering for the effective treatment of RA for the first time.

Molecular Mechanisms of Chondrocyte Proliferation and Differentiation.

Cartilage is a kind of connective tissue that buffers pressure and is essential to protect joint movement. It is difficult to self-recover once cartilage is damaged due to the lack of blood vessels, lymph, and nerve tissues. Repair of cartilage injury is mainly achieved by stimulating chondrocyte proliferation and extracellular matrix (ECM) synthesis. Cartilage homeostasis involves the regulation of multiple growth factors and the transduction of cellular signals. It is a very complicated process that has not been elucidated in detail. In this review, we summarized a variety of signaling molecules related to chondrocytes function. Especially, we described the correlation between chondrocyte-specific regulatory factors and cell signaling molecules. It has potential significance for guiding the treatment of cartilage injury.

Preparation of high precision multilayer scaffolds based on Melt Electro-Writing to repair cartilage injury.

Rationale: Articular cartilage injury is quite common. However, post-injury cartilage repair is challenging and often requires medical intervention, which can be aided by 3D printed tissue engineering scaffolds. Specifically, the high accuracy of Melt Electro-Writing (MEW) technology facilitates the printing of scaffolds that imitate the structure and composition of natural cartilage to promote repair.Methods: MEW and Inkjet printing technology was employed to manufacture a composite scaffold that was then implanted into a cartilage injury site through microfracture surgery. While printing polycaprolactone (PCL) or PCL/hydroxyapatite (HA) scaffolds, cytokine-containing microspheres were sprayed alternately to form multiple layers containing transforming growth factor-β1 and bone morphogenetic protein-7 (surface layer), insulin-like growth factor-1 (middle layer), and HA (deep layer).Results: The composite biological scaffold was conducive to adhesion, proliferation, and differentiation of mesenchymal stem cells recruited from the bone marrow and blood. Meanwhile, the environmental differences between the scaffold's layers contributed to the regional heterogeneity of chondrocytes and secreted proteins to promote functional cartilage regeneration. The biological effect of the composite scaffold was validated both in vitro and in vivo.Conclusion: A cartilage repair scaffold was established with high precision as well as promising mechanical and biological properties. This scaffold can promote the repair of cartilage injury by using, and inducing the differentiation and expression of, autologous bone marrow mesenchymal stem cells.

Regulatory Effect of Kdm6b on Chondrocyte Metabolism in Mouse Cartilage Injury Repair Model

In order to study the expression of lysine-specific demethylase 6B, Kdm6b in differentiation of chondrocyte and chondrogenesis and the regulation effect of Kdm6b on chondrocyte anabolism, a mouse model of osteochondral defects was established in this study. The mice were subjected to antibiotic treatment and intestinal microbiota reconstruction by the method of specific knock out Kdm6b of embryonic cartilage. The mice were divided into Col2al-CreER Kdm6b experimental group and Kdm6b control group and then the destabilization of the medial meniscus operation was performed to model osteoarthritis in mice, which accelerated the abnormal metabolism of cartilage. In addition, transgenic mice with induced chondrocyte specific knockout of Kdm6b were constructed and embryonic samples were collected to study the effects of Kdm6b knockout on cartilage development. At the same time, the proliferation and apoptosis of chondrocytes after kdm6b gene knockout were also studied based on the obvious abnormalities in embryonic chondrogenesis. The results showed that 3 weeks after the mouse osteochondral defect modelling surgery, the mice in the Col2al-CreER; Kdm6b group still had an area without complete healing in the femoral trochle defect, while the defect areas in the Kdm6b group of mice were completely filled. After 12 weeks of mouse osteoarthritis modelling, mice in the Kdm6b group showed cartilage staining loss and cartilage defect. The mice in the Col2al-CreER Kdm6b group were more severe, and even a large area of wear and destruction of the cartilage layer appeared. However, after the growth plate thickness of mice in the Col2al-CreER Kdm6b group was observed, it was found that there was no statistical significance compared with the Kdm6b control group. qRT-PCR analysis showed that compared with the Kdm6b control group, the Col10al gene expression in the primary chondrocytes of the Col2a1-CreER Kdm6b group was significantly decreased. In addition, qRT-PCR and Western Blot sequencing verified that the expressions of Sox9, Col2al, Acan and other proteins related to anabolism were significantly reduced after Kdm6b knockout. Therefore, it can be concluded that Kdm6b directly regulated the expression of genes related to chondrocyte anabolism, and Kdm6b knockout could inhibit the anabolism of chondrocytes, impair the synthesis of cartilage matrix, and accelerate the progress of osteoarthritis.

Effects of cartilage progenitor cells and microRNA-140 on repair of osteoarthritic cartilage injury

To summarize the effect of cartilage progenitor cells (CPCs) and microRNA-140 (miR-140) on the repair of osteoarthritic cartilage injury, and analyze their clinical prospects. The recent researches regarding the CPCs, miR-140, and repair of cartilage in osteoarthritis (OA) disease were extensively reviewed and summarized. CPCs possess the characteristics of self-proliferation, expression of stem cell markers, and multi-lineage differentiation potential, and their chondrogenic ability is superior to other tissues-derived mesenchymal stem cells. CPCs are closely related to the development of OA, but the autonomic activation and chondrogenic ability of CPCs around the osteoarthritic cartilage lesion cannot meet the requirements of complete cartilage repair. miR-140 specifically express in cartilage, and has the potential to activate CPCs by inhibiting key molecules of Notch signaling pathway and enhance its chondrogenic ability, thus promoting the repair of osteoarthritic cartilage injury. Intra-articular delivery of drugs is one of the main methods of OA treatment, although intra-articular injection of miR-140 has a significant inhibitory effect on cartilage degeneration in rats, it also exhibit some limitations such as non-targeted aggregation, low bioavailability, and rapid clearance. So it is a good application prospect to construct a carrier with good safety, cartilage targeting, and high-efficiency for miR-140 based on articular cartilage characteristics. In addition, CPCs are mainly dispersed in the cartilage surface, while OA cartilage injury also begins from this layer, it is therefore essential to emphasize early intervention of OA. miR-140 has the potential to activate CPCs and promote the repair of cartilage injury in early OA, and it is of great clinical significance to further explore the role of miR-140 in OA etiology and to develop new OA treatment strategies based on miR-140.

Sustained Release SDF-1α/TGF-β1-Loaded Silk Fibroin-Porous Gelatin Scaffold Promotes Cartilage Repair.

Continuous delivery of growth factors to the injury site is crucial to creating a favorable microenvironment for cartilage injury repair. In the present study, we fabricated a novel sustained-release scaffold, stromal-derived factor-1α (SDF-1α)/transforming growth factor-β1 (TGF-β1)-loaded silk fibroin-porous gelatin scaffold (GSTS). GSTS persistently releases SDF-1α and TGF-β1, which enhance cartilage repair by facilitating cell homing and chondrogenic differentiation. Scanning electron microscopy showed that GSTS is a porous microstructure and the protein release assay demonstrated the sustainable release of SDF-1α and TGF-β1 from GSTS. Bone marrow-derived mesenchymal stem cells (MSCs) maintain high in vitro cell activity and excellent cell distribution and phenotype after seeding into GSTS. Furthermore, MSCs acquired enhanced chondrogenic differentiation capability in the TGF-β1-loaded scaffolds (GSTS or GST: loading TGF-β1 only) and the conditioned medium from SDF-1α-loaded scaffolds (GSTS or GSS: loading SDF-1α only) effectively promoted MSCs migration. GSTS was transplanted into the osteochondral defects in the knee joint of rats, and it could promote cartilage regeneration and repair the cartilagedefects at 12 weeks after transplantation. Our study shows that GSTS can facilitate in vitro MSCs homing, migration, chondrogenic differentiation and SDF-1α and TGF-β1 have a synergistic effect on the promotion of in vivo cartilage forming. This SDF-1α and TGF-β1 releasing GSTS have promising therapeutic potential in cartilage repair.

Mechanism research on a bioactive resveratrol- PLA-gelatin porous nano-scaffold in promoting the repair of cartilage defect.

BackgroundArticular cartilage defects are difficult to treat, but drug-loaded tissue engineering scaffolds provide a possible treatment option for these types of injuries.PurposeIn this study, we designed a bioactive resveratrol–PLA–gelatin porous nano-scaffold using electrospinning, freeze drying, and uniform dispersion techniques to repair articular cartilage defects, and then investigated the possible mechanism behind the successful repair.MethodsWe established an articular cartilage defect rat model with a 2 mm diameter wound in the middle of the knee joint femoral condyle non-weight-bearing area, with a depth reaching the full thickness of the subchondral bone. Postmodel specimens and micro computed tomography (CT) were used to observe any macroscopic morphological changes in the articular cartilage and subchondral bone, whereas multiple staining methods were used to observe all microcosmic morphological changes. Gross scores and Mankin scores were used to evaluate the repair condition. Immunohistochemical staining was employed to detect protein expression.ResultsWhen the repair included the resveratrol–PLA–gelatin porous nano-scaffold, the repaired cartilage and subchondral bone were in better condition. The expression levels of SIRT1, type II collagen, and PI3K/AKT signaling pathway-related proteins (AKT, VEGF, PTEN, Caspase 9, and MMP13) changed significantly. The expression levels of SIRT1,AKT and type II collagen proteins increased significantly, while the expression levels of VEGF, PTEN, Caspase9 and MMP13 proteins decreased significantly compared with the repair included blank porous PLA–gelatin nano-scaffold and without scaffold.ConclusionWe designed a bioactive resveratrol–PLA–gelatin porous nano-scaffold with better performance, which promoted the repair of cartilage injury as a whole, and explained its possible mechanism in accelerating cartilage repair via the PI3K/AKT signaling pathway.

Chondrogenesis of Adipose-derived Stem Cells with Chondrocytes in Low Serum towards Clinical Application

Currently, fetal bovine serum (FBS) have been widely use in culture media to promote human cell proliferation. However, the usage of FBS for cell therapy in clinical application was associated with the risk of viral and prion transmission as well as immune rejection. To provide an option for this risk, this study was conducted to determine the effect of adipose derived stem cells (ADSCs) co-culture with chondrocyte in promoting cell proliferation and chondrogenesis toward FBS free condition. ADSCs co-cultured with chondrocyte at the ratio of 1:1, 2:1 and 1:2 were tested. Cell morphology changes, cell proliferation and gene expression level of stemness (Oct4, FGF-4, Nanog) and chondrogenic (Collagen Type II, ACP) were assessed. The results showed ADSCs in all co-culture groups changed morphology from fibroblastic spindle to polygonal shape which resembled chondrocytes. The morphological changes were accompanied with increased expression of chondrogenic genes; denoted chondrogenesis process. While maintaining expression of stemness genes indicated continuation of cell proliferation. From the three co-culture groups tested; ADSCs and chondrocytes (1:1 ratio) have been shown to exert better effects in promoting cell proliferation and chondrogenesis. In conclusion, ADSCs could replace FBS to grow sufficient number of chondrogenic cells to repair cartilage injury in the near future. Further in vivo study should be performed to test the effectiveness of this co-culture technique in cartilage injury repair.

Clinical curative effect of repair surgery for treatment of articular cartilage injury

Objective: This study aims to determine the clinical efficacy of articular cartilage injury repair surgery. Methods: A total of 112 patients with knee articular cartilage injury who received treatment at our hospital from August 2015 to May 2017 were selected as research subjects. The patients were divided into control and experimental groups (56 patients in each group) through random even-odd number method and treated with arthroscopic joint debridement and arthroscopic microfracture repair surgery, respectively. Clinical treatment effect, HSS score, complication, and surgical result satisfaction were observed and compared between the two groups. Results: Patients in the experimental group exhibited significantly higher total effective rate than those in the control group (P<0.05). The HSS score in the experimental group was higher than that in the control group, and more patients in the former group showed HSS score higher than 85 or between 60-85 and less patients had HSS score less than 60 (P<0.05). Patients in the control group experienced less satisfaction on the result of surgery and higher complication incidence rate than those in the experimental group; the difference was statistically significant between the two groups (P<0.05). Conclusion: Arthroscopic microfracture surgery exerts satisfactory treatment effect for knee articular cartilage injury and can significantly improve patient satisfaction regarding surgery result, improve the articular function and physical action ability of patients, and reduce the incidence rate of complications. Hence, this technique is a feasible option and must be promoted for clinical application.

Application of growth factor in repair of articular cartilage injury

The joint is an important athletic organ, and has special structure, i.e. no blood supply in the mature articular cartilage. Hence, once articular cartilage is damaged and is difficult to self-repair. Nowadays, there are several clinical methods for articular cartilage injury, such as micro-fracture, transplant of articular cartilage, replacement of articular cartilage and cartilage tissue engineering. But the repairing effects of these methods are unsatisfactory. Growth factors are biologically active substances that are synthesized by cells in vivo and can accelerate cell growth, promote cell proliferation and induce cell migration etc. There are many growth factors participate in the development of cartilage, such as fibroblast growth factor(FGF), bone morphogenetic protein(BMP), insulin-like growth factor(IGF) and so on. Some research showed that the addition of exogenous growth factor in articular cartilage repair can effectively promote the repair of articular cartilage injury. In this paper, the main growth factors used for articular cartilage injury repair were reviewed, and the functions of these factors in the development and the repair of articular cartilage were discussed. The problems of growth factors in the application of articular cartilage repair were analyzed. Key words: Articular cartilage injury; Repair; Chondrocyte; Growth factor

Adipose-derived stem cells in cartilage regeneration: current perspectives.

Repair of cartilage injuries represents a musculoskeletal medicine criticism because of the poor ability to self-renewal of adult cartilage. Therefore, research focuses on developing new regenerative strategies combining chondrocytes or stem cells, scaffolds and growth factors. Because of the low proliferation capability of explanted chondrocytes, new chondrogenesis models, employing human adipose-derived stem cells (ASCs), have been investigated. ASCs are readily accessible with no morbidity and display the capability to differentiate into several cell lineages, including the spontaneous chondrogenic differentiation when entrapped in collagen gel scaffolds. Recent studies also defined some biomolecular mechanisms involved in ASC chondrogenesis in vitro, and their regenerative properties in bioengineered scaffolds and in the presence of growth factors. However, further investigations are required to validate these exciting preclinical results for the application of bioenginereed ASCs in the clinical practice.

Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) Based Electrospun 3D Scaffolds for Delivery of Autogeneic Chondrocytes and Adipose-Derived Stem Cells: Evaluation of Cartilage Defects in Rabbit.

The management of chondral defects has long been a challenge because of the poor self-healing capacity of articular cartilage. Many approaches ranging from symptomatic treatment to structural cartilage regeneration have obtained very limited satisfactory results. Cartilage tissue engineering, which involves an optimized combination of novel scaffolds, cell sources and growth factors, has emerged as a promising strategy for cartilage regeneration and repair. In this study, the cellular morphologies and the adhesion, migration and proliferation capabilities of adipose-derived stem cells (ASCs) and chondrocytes seeded on 3D scaffolds composed of electrospun poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P3HB4HB) were evaluated. Next, TGF-β1/scaffolds with 4:1 co-culture of ASCs and chondrocytes were implanted into the full thickness cartilage defects in rabbit knee for 16 weeks. ASCs and chondrocytes seeded on the scaffolds showed better adhesion, migration and proliferation than that on petri dishes in vitro. Importantly, implantation with TGF-β1/scaffolds with delivery of ASCs and chondrocytes revealed desirable in vivo healing outcomes. These results demonstrate that ASCs have great potential in the field of tissue engineering. It is possible that the improvement in ASC-seeded electrospun 3D P3HB4HB scaffolds may ultimately lead to improved repair of cartilage injuries.

Implant size and mechanical properties influence the failure of the adhesive bond between cartilage implants and native tissue in a finite element analysis

Implanted cartilage replacements (ICRs) are a promising approach to restore the functionality of joints with partial- and full-thickness articular cartilage lesions. Two major hurdles hindering successful repair of cartilage injuries with ICRs are their inadequate mechanical properties and fixation into the defect area. While the ICR geometry and mechanical properties are expected to affect the loads and deformations at the adhesive interface between the implant and native cartilage, little is known about the relationship between these implant characteristics and the quality of the fixation. The objective of this study was to evaluate the effect of implant size, thickness, modulus, surface coefficient of friction and Poisson's ratio on the failure of a fibrin adhesive interface. These factors were evaluated in an idealized finite element model of the medial compartment of the human knee with the damage and failure of fibrin at the implant/cartilage interface represented by a cohesive zone model. Both axial compression and sliding were included in the loading conditions. The results demonstrated that ICR size and material properties have a significant effect on the failure of the fibrin that adheres the implant to the native tissue. Lack of anchorage to underlying bone, larger implant sizes, higher surface coefficient of friction and higher compliance of the implant can increase the chance of implant loosening and delamination. In the future, these results may guide implant design and cartilage repair techniques.

Differentiation of adipose-derived stem cells into ear auricle cartilage in rabbits

Adipose-derived stem cells have been reported as a novel candidate for the repair of cartilage injuries in vivo. In order to assess their differentiation ability, adipose-derived stem cells isolated from rabbit fat tissue were injected into the midportion of a surgically created rabbit ear auricle cartilage defect. After several months, the auricles were resected, histopathologically assessed and compared with a control group. Histopathological examination of auricles removed three, four and five months after injection showed islands of new cartilage formation at the site of the surgically induced defect. Six months after injection, we observed well-formed, mature cartilaginous plates that completely filled the defect in the native cartilage. In the control group, there was no significant growth of new cartilage. The results of this study suggest the great potential of adipose-derived stem cells to repair damaged cartilage tissue in vivo.

Directed induction of chondrogenic cells from mouse dermal fibroblast culture

Repair of cartilage injury with hyaline cartilage has been a challenging clinical problem. Articular cartilage damage sometimes heals with fibrocartilage, which is different from hyaline cartilage. Fibrocartilage is a type of scar tissue that expresses types I and II collagen. In contrast, hyaline cartilage does not express type I collagen. When aiming to induce hyaline chondrogenic cells directly from dermal fibroblasts, in addition to activation of cartilage-specific matrix genes, elimination of expression of type I collagen is needed for generation of hyaline cartilage. Otherwise, the presence of type I collagen impairs cartilage extracellular matrix architecture, which leads to formation of fibrocartilage. The generation of induced pluripotent stem cells has provided a tool for reprogramming dermal fibroblasts to an undifferentiated state by ectopic expression of reprogramming factors. We found that retroviral expression of two reprogramming factors (c-Myc and Klf4) and one chondrogenic factor (SOX9) induces polygonal chondrogenic cells directly from adult dermal fibroblast cultures. Induced cells expressed marker genes for chondrocytes but not fibroblasts; the promoters of type I collagen genes were extensively methylated. Transduction of c-Myc, Klf4, and SOX9 produced two types of cells: chondrogenically reprogrammed cells and partially reprogrammed intermediate cells. Chondrogenically reprogrammed cells generated stable homogenous hyaline cartilage-like tissue without tumor formation when subcutaneously injected into nude mice. Hyaline cartilage-like tissue expressed type II collagen but not type I collagen. On the other hand, partially reprogrammed intermediate cells expressed type I collagen and produced tumor when injected into nude mice. Induced chondrogenic cells did not undergo pluripotent state during induction from dermal fibroblast culture, as time-lapse observation did not detect GFP reporter expression during induction from dermal fibroblasts prepared from transgenic mice in which GFP is inserted into the Nanog locus. These results suggest that chondrogenic cells induced by this approach are free from a risk of teratoma formation which associates with cells prepared through generation of iPS cells followed by redifferentiation into the target cell type. The dox-inducible induction system demonstrated that induced cells are able to respond to chondrogenic medium by expressing endogenous Sox9 and maintain chondrogenic potential after substantial reduction of transgene expression. This approach could lead to the preparation of hyaline cartilage directly from skin, without going through pluripotent stem cells, in future regenerative medicine.

Generation of hyaline cartilaginous tissue from mouse adult dermal fibroblast culture by defined factors

Repair of cartilage injury with hyaline cartilage continues to be a challenging clinical problem. Because of the limited number of chondrocytes in vivo, coupled with in vitro de-differentiation of chondrocytes into fibrochondrocytes, which secrete type I collagen and have an altered matrix architecture and mechanical function, there is a need for a novel cell source that produces hyaline cartilage. The generation of induced pluripotent stem (iPS) cells has provided a tool for reprogramming dermal fibroblasts to an undifferentiated state by ectopic expression of reprogramming factors. Here, we show that retroviral expression of two reprogramming factors (c-Myc and Klf4) and one chondrogenic factor (SOX9) induces polygonal chondrogenic cells directly from adult dermal fibroblast cultures. Induced cells expressed marker genes for chondrocytes but not fibroblasts, i.e., the promoters of type I collagen genes were extensively methylated. Although some induced cell lines formed tumors when subcutaneously injected into nude mice, other induced cell lines generated stable homogenous hyaline cartilage–like tissue. Further, the doxycycline-inducible induction system demonstrated that induced cells are able to respond to chondrogenic medium by expressing endogenous Sox9 and maintain chondrogenic potential after substantial reduction of transgene expression. Thus, this approach could lead to the preparation of hyaline cartilage directly from skin, without generating iPS cells.

Matrix‐induced autologous chondrocyte implantation in sheep: objective assessments including confocal arthroscopy

The assessment of cartilage repair has largely been limited to macroscopic observation, magnetic resonance imaging (MRI), or destructive biopsy. The aims of this study were to establish an ovine model of articular cartilage injury repair and to examine the efficacy of nondestructive techniques for assessing cartilage regeneration by matrix-induced autologous chondrocyte implantation (MACI). The development of nondestructive assessment techniques facilitates the monitoring of repair treatments in both experimental animal models and human clinical subjects. Defects (Ø 6 mm) were created on the trochlea and medial femoral condyle of 21 sheep randomized into untreated controls or one of two treatment arms: MACI or collagen-only membrane. Each group was divided into 8-, 10-, and 12-week time points. Repair outcomes were examined using laser scanning confocal arthroscopy (LSCA), MRI, histology, macroscopic ICRS grading, and biomechanical compression analysis. Interobserver analysis of the randomized blinded scoring of LSCA images validated our scoring protocol. Pearson correlation analysis demonstrated the correlation between LSCA, MRI, and ICRS grading. Testing of overall treatment effect independent of time point revealed significant differences between MACI and control groups for all sites and assessment modalities (Asym Sig < 0.05), except condyle histology. Biomechanical analysis suggests that while MACI tissue may resemble native tissue histologically in the early stages of remodeling, the biomechanical properties remain inferior at least in the short term. This study demonstrates the potential of a multisite sheep model of articular cartilage defect repair and its assessment via nondestructive methods.