Functional joint regeneration is achieved using reintegration mechanism in Xenopus laevis.
ABSTRACT: A functional joint requires integration of multiple tissues: the apposing skeletal elements should form an interlocking structure, and muscles should insert into skeletal tissues via tendons across the joint. Whereas newts can regenerate functional joints after amputation, Xenopus laevis regenerates a cartilaginous rod without joints, a "spike." Previously we reported that the reintegration mechanism between the remaining and regenerated tissues has a significant effect on regenerating joint morphogenesis during elbow joint regeneration in newt. Based on this insight into the importance of reintegration, we amputated frogs' limbs at the elbow joint and found that frogs could regenerate a functional elbow joint between the remaining tissues and regenerated spike. During regeneration, the regenerating cartilage was partially connected to the remaining articular cartilage to reform the interlocking structure of the elbow joint at the proximal end of the spike. Furthermore, the muscles of the remaining part inserted into the regenerated spike cartilage via tendons. This study might open up an avenue for analyzing molecular and cellular mechanisms of joint regeneration using Xenopus.
Project description:Urodele amphibians, such as newts, can regenerate a functional limb, including joints, after amputation at any level along the proximal-distal axis of the limb. The blastema can regenerate the limb morphology largely independently of the stump after proximal-distal identity has been established, but the remaining and regenerated tissues must be structurally reintegrated (matched in size and shape). Here we used newt joint regeneration as a model to investigate reintegration, because a functionally interlocking joint requires structural integration between its opposing skeletal elements. After forelimbs were amputated at the elbow joint, the joint was regenerated between the remaining and regenerated skeletal elements. The regenerated cartilage was thick around the amputated joint to make a reciprocally interlocking joint structure with the remaining bone. Furthermore, during regeneration, the extracellular matrix of the remaining tissues was lost, suggesting that the remaining tissues might contribute to the morphogenesis of regenerating cartilage. Our results showed that the area of the regenerated cartilage matched the area of the apposed remaining cartilage, thus contributing to formation of a functional structure.
Project description:Purpose: One of the essential requirements in maintaining the normal joint motor function is the perfect tribological property of the articular cartilage. Many cartilage regeneration strategies have been developed for treatment in early stages of osteoarthritis, but there is little information on how repaired articular cartilage regains durability. The identification of biomarkers that can predict wear resistant property is critical to advancing the success of cartilage regeneration therapies. Proteoglycan 4 (PRG4) is a macromolecule distributing on the chondrocyte surface that contributes to lubrication. In this study, we investigate if PRG4 expression is associated with tribological properties of regenerated cartilage, and is able to predict its wear resistant status. Methods: Two different strategies including bone marrow enrichment plus microfracture (B/BME-MFX) and microfracture alone (B-MFX) of cartilage repair in sheep were used. PRG4 expression and a series of tribological parameters on regenerated cartilage were rigorously examined and compared. Results: Highly and continuously expression of PRG4 in regenerated cartilage surface was negatively correlated with each tribological parameter (P<0.0001, respectively). Multivariate analysis showed that PRG4 expression was the key predictor that contributed to the promotion of cartilage wear resistance. Conclusion: Higher PRG4 expression in regenerated cartilage is significantly associated with wear resistance improvement. PRG4 may be useful for predicting the wear resistant status of regenerated cartilage and determining the optimal cartilage repair strategy.
Project description:Synovial joints are among the most important structures that give us complex motor abilities as humans. Degenerative joint diseases, such as arthritis, cause loss of normal joint functioning and affect over 40 million people in the USA and approximately 350 million people worldwide. Therapies based on regenerative medicine hold the promise of effectively repairing or replacing damaged joints permanently. Here, for the first time, we introduce a model for synovial joint regeneration utilizing the chick embryo. In this model, a block of tissue that contains the prospective elbow is excised, leaving a window with strips of anterior and posterior tissue intact (window excision, WE). In contrast, we also slice out the same area containing the elbow and the distal piece of the limb is pinned back onto the stump (slice excision, SE). Interestingly, when the elbow is removed via WE, regeneration of the joint takes place, whereas the elbow joint does not regenerate following SE. In order to investigate whether the regeneration response recapitulates the developmental program of forming joints, we used GDF-5 and Autotaxin (Atx) as joint tissue specific markers, and Sox-9 and Col-9 as cartilage markers for in situ hybridization on sections at different time points after WE and SE surgeries. Re-expression of GDF-5 and Atx is observed in the WE samples by 60h after surgery. In contrast, the majority of the samples that underwent SE surgery did not express GDF-5 and Atx. Also, in SE fusion of cartilage elements takes place and the joint interzone does not form. This is indicated by continuous Col-9 expression in SE limbs, whereas Col-9 is downregulated at the joint interzone in the regenerating WE samples. This order and pattern of gene expression observed in regenerates is similar to the development of a joint suggesting that regeneration recapitulates development at the molecular level. This model defines some of the conditions required for inducing joint regeneration in an otherwise nonregenerating environment. This knowledge can be useful for designing new therapeutic approaches for joint loss or for conditions affecting joint integrity in humans.
Project description:A major goal of regenerative medicine is to stimulate tissue regeneration after traumatic injury. We previously discovered that treating digit amputation wounds with BMP2 in neonatal mice stimulates endochondral ossification to regenerate the stump bone. Here we show that treating the amputation wound with BMP9 stimulates regeneration of a synovial joint that forms an articulation with the stump bone. Regenerated structures include a skeletal element lined with articular cartilage and a synovial cavity, and we demonstrate that this response requires the Prg4 gene. Combining BMP2 and BMP9 treatments in sequence stimulates the regeneration of bone and joint. These studies provide evidence that treatment of growth factors can be used to engineer a regeneration response from a non-regenerating amputation wound.
Project description:Rotator cuff tears are common conditions that can alter shoulder mechanics and may lead to damage of intact joint tissues. These injuries are of particular concern in populations who perform tasks requiring repetitive overhead activity (e.g., athletes and laborers) and who are likely to return to aggressive pre-injury activity levels despite limited understanding of the potentially damaging effects on the remaining tissues. Therefore, we investigated the effect of returning to overuse activity following a supraspinatus tear on shoulder function and the mechanical properties of the remaining intact tendons and glenoid cartilage. Forty rats underwent 4 weeks of overuse activity to create a tendinopathic condition followed by detachment of the supraspinatus tendon and were then randomized into two groups: continued overuse or cage activity. Ambulatory measurements were performed throughout the 8 weeks prior to euthaniasia, and properties of the adjacent tendons and cartilage were evaluated. Results demonstrated that shoulder function was not compromised in the return to overuse group. However, alterations of the glenoid cartilage and biceps tendon properties occurred. Our results help define the contributory roles of common mechanical injury mechanisms and provide a framework by which physicians could better prescribe long-term treatment strategies for patients.
Project description:In spite of numerous investigations of regenerating salamander limbs, little attention has been paid to the details of how joints are reformed. An understanding of the process and mechanisms of joint regeneration in this model system for tetrapod limb regeneration would provide insights into developing novel therapies for inducing joint regeneration in humans. To this end, we have used the axolotl (Mexican Salamander) model of limb regeneration to describe the morphology and the expression patterns of marker genes during joint regeneration in response to limb amputation. These data are consistent with the hypothesis that the mechanisms of joint formation whether it be development or regeneration are conserved. We also have determined that defects in the epiphyseal region of both forelimbs and hind limbs in the axolotl are regenerated only when the defect is small. As is the case with defects in the diaphysis, there is a critical size above which the endogenous regenerative response is not sufficient to regenerate the joint. This non-regenerative response in an animal that has the ability to regenerate perfectly provides the opportunity to screen for the signaling pathways to induce regeneration of articular cartilage and joints.
Project description:This study was undertaken to examine whether bone morphogenetic protein 7 (BMP-7) induces ectopic cartilage formation in the rat tendon, and whether transplantation of tendon treated with BMP-7 promotes meniscal regeneration. Additionally, we analyzed the relative contributions of host and donor cells on the healing process after tendon transplantation in a rat model.BMP-7 was injected in situ into the Achilles tendon of rats, and the histologic findings and gene profile were evaluated. Achilles tendon injected with 1 ?g of BMP-7 was transplanted into a meniscal defect in rats. The regenerated meniscus and articular cartilage were evaluated at 4, 8, and 12 weeks. Achilles tendon from LacZ-transgenic rats was transplanted into the meniscal defect in wild-type rats, and vice versa.Injection of BMP-7 into the rat Achilles tendon induced the fibrochondrocyte differentiation of tendon cells and changed the collagen gene profile of tendon tissue to more closely approximate meniscal tissue. Transplantation of the rat Achilles tendon into a meniscal defect increased meniscal size. The rats that received the tendon treated with BMP-7 had a meniscus matrix that exhibited increased Safranin O and type II collagen staining, and showed a delay in articular cartilage degradation. Using LacZ-transgenic rats, we determined that the regeneration of the meniscus resulted from contribution from both donor and host cells.Our findings indicate that BMP-7 induces ectopic cartilage formation in rat tendons. Transplantation of Achilles tendon treated with BMP-7 promotes meniscus regeneration and prevents cartilage degeneration in a rat model of massive meniscal defect. Native cells in the rat Achilles tendon contribute to meniscal regeneration.
Project description:Although meniscus defects and degeneration are strongly correlated with the later development of osteoarthritis, the promise of regenerative medicine strategies is to prevent and/or delay the disease's progression. Meniscal reconstruction has been shown in animal models with tendon grafting and transplantation of mesenchymal stem cells (MSCs); however, these procedures have not shown the same efficacy in clinical studies. Here, our aim was to investigate the ability of tendon grafts pretreated with exogenous synovial-derived MSCs to prevent cartilage degeneration in a rat partial meniscus defect model. We removed the anterior half of the medial meniscus and grafted autologous Achilles tendons with or without a 10-minute pretreatment of the tendon with synovial MSCs. The meniscus and surrounding cartilage were evaluated at 2, 4, and 8 weeks (n = 5). Tendon grafts increased meniscus size irrespective of synovial MSCs. Histological scores for regenerated menisci were better in the tendon + MSC group than in the other two groups at 4 and 8 weeks. Both macroscopic and histological scores for articular cartilage were significantly better in the tendon + MSC group at 8 weeks. Implanted synovial MSCs survived around the grafted tendon and native meniscus integration site by cell tracking assays with luciferase+, LacZ+, DiI+, and/or GFP+ synovial MSCs and/or GFP+ tendons. Flow cytometric analysis showed that transplanted synovial MSCs retained their MSC properties at 7 days and host synovial tissue also contained cells with MSC characteristics. Synovial MSCs promoted meniscus regeneration augmented by autologous Achilles tendon grafts and prevented cartilage degeneration in rats.
Project description:Traumatic and degenerative lesions of articular cartilage usually progress to osteoarthritis (OA), a leading cause of disability in humans. MicroRNAs (miRNAs) can regulate the differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs) and play important roles in the expression of genes related to OA. However, their functional roles in OA remain poorly understood. Here, we have examined miR-449a, which targets sirtuin 1 (SIRT1) and lymphoid enhancer-binding factor-1 (LEF-1), and observed its effects on damaged cartilage. The levels of chondrogenic markers and miR-449a target genes increased during chondrogenesis in anti-miR-449a-transfected hBMSCs. A locked nucleic acid (LNA)-anti-miR-449a increased cartilage regeneration and expression of type II collagen and aggrecan on the regenerated cartilage surface in acute defect and OA models. Furthermore, intra-articular injection of LNA-anti-miR-449a prevented disease progression in the OA model. Our study indicates that miR-449a may be a novel potential therapeutic target for age-related joint diseases like OA.
Project description:Large rotator cuff tears (supraspinatus and infraspinatus) are common in patients who perform overhead activities (laborers, athletes). In addition, following large cuff tears, these patients commonly attempt to return to pre-injury activity levels. However, there is a limited understanding of the damaging effects on the uninjured joint tissues when doing so. Therefore, the objective of this study was to investigate the effect of returning to overuse activity following a supraspinatus and infraspinatus tear on shoulder function and the structural and biological properties of the intact tendons and glenoid cartilage. Forty rats underwent 4 weeks of overuse followed by detachment of the supraspinatus and infraspinatus tendons and were then randomized into two groups: return to overuse or cage activity. Ambulatory measurements were performed over time and structural and biological properties of the adjacent tendons and cartilage were evaluated. Results demonstrated that animals returning to overuse activity did not have altered shoulder function but despite this, did have altered cartilage and tendon properties. These mechanical changes corresponded to altered transcriptional regulation of chondrogenic genes within cartilage and tendon. This study helps define the mechanical and biological mechanisms leading to joint damage and provides a framework for treating active cuff tear patients.