Biology of platelet-rich plasma and its clinical application in cartilage repair

Platelet-rich plasma (PRP) is an autologous concentrated cocktail of growth factors and inflammatory mediators, and has been considered to be potentially effective for cartilage repair. In addition, the fibrinogen in PRP may be activated to form a fibrin matrix to fill cartilage lesions, fulfilling the initial requirements of physiological wound healing. The anabolic, anti-inflammatory and scaffolding effects of PRP based on laboratory investigations, animal studies, and clinical trials are reviewed here. In vitro, PRP is found to stimulate cell proliferation and cartilaginous matrix production by chondrocytes and adult mesenchymal stem cells (MSCs), enhance matrix secretion by synoviocytes, mitigate IL-1β-induced inflammation, and provide a favorable substrate for MSCs. In preclinical studies, PRP has been used either as a gel to fill cartilage defects with variable results, or to slow the progression of arthritis in animal models with positive outcomes. Findings from current clinical trials suggest that PRP may have the potential to fill cartilage defects to enhance cartilage repair, attenuate symptoms of osteoarthritis and improve joint function, with an acceptable safety profile. Although current evidence appears to favor PRP over hyaluronan for the treatment of osteoarthritis, the efficacy of PRP therapy remains unpredictable owing to the highly heterogeneous nature of reported studies and the variable composition of the PRP preparations. Future studies are critical to elucidate the functional activity of individual PRP components in modulating specific pathogenic mechanisms.     

Mesenchymal stem cells for cartilage regeneration in dogs

Articular cartilage damage and osteoarthritis (OA) are common orthopedic diseases in both humans and dogs. Once damaged, the articular cartilage seldom undergoes spontaneous repair because of its avascular, aneural, and alymphatic state, and the damage progresses to a chronic and painful situation. Dogs have distinctive characteristics compared to other laboratory animal species in that they share an OA pathology with humans. Dogs can also require treatment for naturally developed OA;therefore, effective treatment methods for OA are desired in veterinary medicine as well as in human medicine. Recently, interest has grown in regenerative medicine that includes the use of mesenchymal stem cells (MSCs). In cartilage repair, MSCs are a promising therapeutic tool due to their self-renewal capacity, ability to differentiate into cartilage, potential for trophic factor production, and capacity for immunomodulation. The MSCs from dogs (canine MSCs;cMSCs) share various characteristics with MSCs from other animal species, but they show some deviations, particularly in their differentiation ability and surface epitope expression. In vivo studies of cMSCs have demonstrated that intraarticular cMSC injection into cartilage lesions results in excellent hyaline cartilage regeneration. In clinical situations, cMSCs have shown great therapeutic effects, including amelioration of pain and lameness in dogs suffering from OA. However, some issues remain, such as a lack of regulations or guidelines and a need for unified methods for the use of cMSCs. This review summarizes what is known about cMSCs, including their in vitro characteristics, their therapeutic effects in cartilage lesion treatment in preclinical in vivo studies, their clinical efficacy for treatment of naturally developed OA in dogs, and the current limitations of cMSC studies.     

Bioengineered chondrocyte sheets may be potentially useful for the treatment of partial thickness defects of articular cartilage

Some treatments for full thickness defects of articular cartilage, such as cultured chondrocyte transplantation, have already been done. However, to overcome osteoarthritis, we must further study the partial thickness defect of articular cartilage. It is much more difficult to repair a partial thickness defect because few repairing cells can address such injured sites. We herein show that bioengineered layered chondrocyte sheets using temperature-responsive culture dishes may be a potentially useful treatment for partial thickness defects. We evaluated the property of these sheets using real-time PCR and histological findings, and allografted these sheets to evaluate the effect of treatment using a rabbit partial model. In conclusion, layered chondrocyte sheets were able to maintain the cartilageous phenotype, and could be attached to the sites of cartilage damage which acted as a barrier to prevent a loss of proteoglycan from these sites and to protect them from catabolic factors in the joint.     

Maturation-dependent spontaneous healing of partial thickness cartilage defects in infantile rats

Partial-thickness articular cartilage defects (PTCDs) do not heal spontaneously and are thought to be a predisposing factor for the development of osteoarthritis. Younger and smaller animals have a better healing capacity for many types of injuries including those to articular cartilage. Our aim was to examine the longitudinal histological changes of immature murine articular cartilage after the creation of small PTCDs and to compare them to PTCDs in mature cartilage. Single linear PTCDs were created in 3-week-old and 16-week-old rats in the direction of joint motion. At 6 and 12?weeks after PTCD creation, histological changes were examined in the defect sites and surrounding cartilage. Immature cartilage showed a higher repair capability than mature cartilage. Although repaired immature cartilage had fibrocartilage, it exhibited better quality than any PTCD model, except for a fetus model and comparable quality to full-thickness cartilage defects (FTCD) after bone marrow stimulation. Elucidation of the underlining mechanisms that immature cartilage possesses for repairing PTCDs is necessary in order to aid the prevention or develop treatment for osteoarthritis.     

Advances and challenges in gene‐based approaches for osteoarthritis

Osteoarthritis (OA), a paramount cause of physical disability for which there is no definitive cure, is mainly characterized by the gradual loss of the articular cartilage. Current nonsurgical and reconstructive surgical therapies have not met success in reversing the OA phenotype so far. Gene transfer approaches allow for a long‐term and site‐specific presence of a therapeutic agent to re‐equilibrate the metabolic balance in OA cartilage and may consequently be suited to treat this slow and irreversible disorder. The distinct stages of OA need to be respected in individual gene therapy strategies. In this context, molecular therapy appears to be most effective for early OA. A critical step forward has been made by directly transferring candidate sequences into human articular chondrocytes embedded within their native extracellular matrix via recombinant adeno‐associated viral vectors. Although extensive studies in vitro attest to a growing interest in this approach, data from animal models of OA are sparse. A phase I dose‐escalating trial was recently performed in patients with advanced knee OA to examine the safety and activity of chondrocytes modified to produce the transforming growth factor β1 via intra‐articular injection, showing a dose‐dependent trend toward efficacy. Proof‐of‐concept studies in patients prior to undergoing total knee replacement may be privileged in the future to identify the best mode of translating this approach to clinical application, followed by randomized controlled trials. Copyright © 2013 John Wiley & Sons, Ltd.     

The minipig model for experimental chondral and osteochondral defect repair in tissue engineering: retrospective analysis of 180 defects

Articular cartilage repair is still a challenge in orthopaedic surgery. Although many treatment options have been developed in the last decade, true regeneration of hyaline articular cartilage is yet to be accomplished. In vitro experiments are useful for evaluating cell-matrix interactions under controlled parameters. When introducing new treatment options into clinical routine, adequate animal models are capable of closing the gap between in vitro experiments and the clinical use in human beings. We developed an animal model in the G?ttingen minipig (GMP) to evaluate the healing of osteochondral or full-thickness cartilage defects. The defects were located in the middle third of the medial portion of the patellofemoral joint at both distal femurs. Chondral defects were 6.3 mm, osteochondral defects either 5.4 or 6.3 mm in diameter and 8 or 10 mm deep. In both defects the endogenous repair response showed incomplete repair tissue formation up to 12 months postoperatively. Based on its limited capability for endogenous repair of chondral and osteochondral defects, the GMP is a useful model for critical assessment of new treatment strategies in articular cartilage tissue engineering.     

Compressive properties of mouse articular cartilage determined in a novel micro-indentation test method and biphasic finite element model

The mechanical properties of articular cartilage serve as important measures of tissue function or degeneration, and are known to change significantly with osteoarthritis. Interest in small animal and mouse models of osteoarthritis has increased as studies reveal the importance of genetic background in determining predisposition to osteoarthritis. While indentation testing provides a method of determining cartilage mechanical properties in situ, it has been of limited value in studying mouse joints due to the relatively small size of the joint and thickness of the cartilage layer. In this study, we developed a micro-indentation testing system to determine the compressive and biphasic mechanical properties of cartilage in the small joints of the mouse. A nonlinear optimization program employing a genetic algorithm for parameter estimation, combined with a biphasic finite element model of the micro-indentation test, was developed to obtain the biphasic, compressive material properties of articular cartilage. The creep response and material properties of lateral tibial plateau cartilage were obtained for wild-type mouse knee joints, by the micro-indentation testing and optimization algorithm. The newly developed genetic algorithm was found to be efficient and accurate when used with the finite element simulations for nonlinear optimization to the experimental creep data. The biphasic mechanical properties of mouse cartilage in compression (average values: Young's modulus, 2.0 MPa; Poisson's ratio, 0.20; and hydraulic permeability, 1.1 x 10(-16) m4/N-s) were found to be of similar orders of magnitude as previous findings for other animal cartilages, including human, bovine, rat, and rabbit and demonstrate the utility of the new test methods. This study provides the first available data for biphasic compressive properties in mouse cartilage and suggests a promising method for detecting altered cartilage mechanics in small animal models of osteoarthritis.     

High-resolution measurements of the multilayer ultra-structure of articular cartilage and their translational potential

Current musculoskeletal imaging techniques usually target the macro-morphology of articular cartilage or use histological analysis. These techniques are able to reveal advanced osteoarthritic changes in articular cartilage but fail to give detailed information to distinguish early osteoarthritis from healthy cartilage, and this necessitates high-resolution imaging techniques measuring cells and the extracellular matrix within the multilayer structure of articular cartilage. This review provides a comprehensive exploration of the cellular components and extracellular matrix of articular cartilage as well as high-resolution imaging techniques, including magnetic resonance image, electron microscopy, confocal laser scanning microscopy, second harmonic generation microscopy, and laser scanning confocal arthroscopy, in the measurement of multilayer ultra-structures of articular cartilage. This review also provides an overview for micro-structural analysis of the main components of normal or osteoarthritic cartilage and discusses the potential and challenges associated with developing non-invasive high-resolution imaging techniques for both research and clinical diagnosis of early to late osteoarthritis.     

Mesenchymal stem cells in regenerative medicine: Opportunities and challenges for articular cartilage and intervertebral disc tissue engineering

Defects of load‐bearing connective tissues such as articular cartilage and intervertebral disc (IVD) can result from trauma, degenerative, endocrine, or age‐related disease. Current surgical and pharmacological options for the treatment of arthritic rheumatic conditions in the joints and spine are ineffective. Cell‐based surgical therapies such as autologous chondrocyte transplantation (ACT) have been in clinical use for cartilage repair for over a decade but this approach has shown mixed results. This review focuses on the potential of mesenchymal stem cells (MSCs) as an alternative to cells derived from patient tissues in autologous transplantation and tissue engineering. Here we discuss the prospects of using MSCs in regenerative medicine and summarize the advantages and disadvantages of these cells in articular cartilage and IVD tissue engineering. We discuss the conceptual and practical difficulties associated with differentiating and pre‐conditioning MSCs for subsequent survival in a physiologically harsh extracellular matrix, an environment that will be highly hypoxic, acidic, and nutrient deprived. Implanted MSCs will be exposed to traumatic physical loads and high levels of locally produced inflammatory mediators and catabolic cytokines. We also explore the potential of culture models of MSCs, fully differentiated cells and co‐cultures as “proof of principle” ethically acceptable “3Rs” models for engineering articular cartilage and IVD in vitro for the purpose of replacing the use of animals in arthritis research. J. Cell. Physiol. 222:23–32, 2010. © 2009 Wiley‐Liss, Inc.     

The mechanics of focal chondral defects in the hip

There is a mean incidence of osteoarthritis (OA) of the hip in 8% of the overall population. In the presence of focal chondral defects, defined as localized damage to the articular cartilage, there is an increased risk of symptomatic progression toward OA. This relationship between chondral defects and subsequent development of OA has led to substantial efforts to develop effective procedures for surgical cartilage repair. This study examined the effects of chondral defects and labral delamination on cartilage mechanics in the dysplastic hip during the gait cycle using subject-specific finite element analysis. Models were generated from volumetric CT data and analyzed with simulated chondral defects at the chondrolabral junction on the posterior acetabulum during five distinct points in the gait cycle. Focal chondral defects increased maximum shear stress on the osteochondral surface of the acetabular cartilage, when compared to the intact case. This effect was amplified with labral delamination. Additionally, chondral defects increased the first principal Lagrange strain on the articular surface of the acetabular cartilage and labrum. Labral delamination relieved some of this tensile strain. As defect size was increased, contact stress increased in the medial zone of the acetabulum, while it decreased anteriorly. The results suggest that in the presence of chondral defects and labral delamination the cartilage experiences elevated tensile strains and shear and contact stress, which could lead to further damage of the cartilage, and subsequent arthritic progression. The framework presented here will serve as the procedure for future finite element studies on cartilage mechanics in hips with varying disease states with simulated chondral defects and labral tears.     

Human model neurons in studies of brain cell damage and neural repair

Disorders of the central nervous system are a major concern in modern human societies. Studies of these disorders require the use of suitable model systems that accurately reproduce the human situation. In this article we focus on the possibilities of using the human NT-2 teratocarcinoma cell line for studies on neuronal differentiation, cellular function and neurodegeneration. Neurons generated from undifferentiated NT-2 precursor cells show neuronal morphology, express neuronal markers, exhibit action potentials and have the advantage of homogeneous cellular composition of clonally derived cells. They release a number of different neurotransmitters, respond to stimulation with glutamate, gamma-amino-butyric acid, and nitric oxide, and form functional synapses in culture. Depending on the differentiation protocol, NT-2 cells also have the capacity to develop into glial cells. Different neuronal differentiation procedures and biological properties of NT-2 neurons are described in the text. In transplantation experiments, differentiated NT-2 neurons integrated successfully into the nervous systems of both experimental animals and human patients without evidence for tumor formation, underlining their value for both basic research and clinical applications. We discuss some potential applications in the fields of basic research, drug discovery, and therapy of CNS damage with particular emphasis on neuronal transplantation and different cell death mechanisms in neuronal degeneration. Grafting of NT-2 neurons has been shown to effectively reverse functional defects in animal disease models. Moreover, an ongoing phase 2 randomized clinical trial indicates the safety and feasibility of NT-2 neuron transplantation for the treatment of human patients with cerebral stroke.     

Bone and cartilage in osteoarthritis: is what's best for one good or bad for the other?

The interest in the relationship between articular cartilage and the structural and functional properties of peri-articular bone relates to the intimate contact that exists between these tissues in joints that are susceptible to the development of osteoarthritis (OA). The demonstration in several animal models that osteoporosis and decreased bone tissue modulus leads to an increased propensity for the development of post-traumatic OA is paradoxical in light of the extensive epidemiological literature indicating that individuals with high systemic bone mass, assessed by bone mineral density, are at increased risk for OA. These observations underscore the need for further studies to define the pathophysiological mechanisms involved in the interaction between subchondral bone and articular cartilage and for applying this information to the development of therapeutic interventions to improve the outcomes in patients with OA.     

Development of a preclinical natural porcine knee simulation model for the tribological assessment of osteochondral grafts in vitro

In order to pre-clinically evaluate the performance and efficacy of novel osteochondral interventions, physiological and clinically relevant whole joint simulation models, capable of reproducing the complex loading and motions experienced in the natural knee environment are required. The aim of this study was to develop a method for the assessment of tribological performance of osteochondral grafts within an in vitro whole natural joint simulation model.The study assessed the effects of osteochondral allograft implantation (existing surgical intervention for the repair of osteochondral defects) on the wear, deformation and damage of the opposing articular surfaces. Tribological performance of osteochondral grafts was compared to the natural joint (negative control), an injury model (focal cartilage defects) and stainless steel pins (positive controls). A recently developed method using an optical profiler (Alicona Infinite Focus G5, Alicona Imaging GmbH, Austria) was used to quantify and characterise the wear, deformation and damage occurring on the opposing articular surfaces. Allografts inserted flush with the cartilage surface had the lowest levels of wear, deformation and damage following the 2 h test; increased levels of wear, deformation and damage were observed when allografts and stainless steel pins were inserted proud of the articular surface. The method developed will be applied in future studies to assess the tribological performance of novel early stage osteochondral interventions prior to in vivo studies, investigate variation in surgical precision and aid in the development of stratified interventions for the patient population.     

Direct transplantation of mesenchymal stem cells into the knee joints of Hartley strain guinea pigs with spontaneous osteoarthritis

Introduction

Mesenchymal stem cells (MSCs) can differentiate into various connective tissue cells. Several techniques have been used for the clinical application of MSCs in articular cartilage repair; however, there are many issues associated with the selection of the scaffold material, including its ability to support cell viability and differentiation and its retention and degradation in situ. The application of MSCs via a scaffold also requires a technically demanding surgical procedure. The aim of this study was to test the outcome of intra-articular transplantation of mesenchymal stem cells suspended in hyaluronic acid (HA) in the knee joints of Hartley strain guinea pigs with spontaneous osteoarthritis (OA).

Methods

Commercially available human MSCs were cultured, labeled with carboxyfluorescein diacetate succinimidyl ester (CFDA-SE), suspended in either PBS or HA, and injected into the knee joints of 7-month-old animals. The control animals were injected with either PBS or HA alone. The animals were sacrificed at 1, 3, and 5 weeks post transplantation, the knee joints harvested, and fluorescent microscopic analysis was performed. Histological and immunohistochemical analysis were performed at 5 weeks post transplantation.

Results

At 5 weeks post transplantation, partial cartilage repair was noted in the HA-MSC group but not in the other groups. Examination of CFDA-SE-labeled cells demonstrated migration, differentiation, and proliferation of MSC in the HA-MSC group. There was strong immunostaining for type II collagen around both residual chondrocytes and transplanted MSCs in the OA cartilage.

Conclusion

This scaffold-free and technically undemanding technique appears to result in the regeneration of articular cartilage in the spontaneous OA animal model. Although further examination of the long-term effects of transplantation is necessary, the findings suggest that intra-articular injection of HA-MSC mixture is potentially beneficial for OA.     

Effects of shear stress on articular chondrocyte metabolism

The articular cartilage of diarthrodial joints experiences a variety of stresses, strains and pressures that result from normal activities of daily living. In normal cartilage, the extracellular matrix exists as a highly organized composite of specialized macromolecules that distributes loads at the bony ends. The chondrocyte response to mechanical loading is recognized as an integral component in the maintenance of articular cartilage matrix homeostasis. With inappropriate mechanical loading of the joint, as occurs with traumatic injury, ligament instability, bony malalignment or excessive weight bearing, the cartilage exhibits manifestations characteristic of osteoarthritis. Breakdown of cartilage in osteoarthritis involves degradation of the extracellular matrix macromolecules and decreased expression of chondrocyte proteins necessary for normal joint function. Osteoarthritic cartilage often exhibits increased amounts of type I collagen and synthesis of proteoglycans characteristic of immature cartilage. The shift in cartilage phenotype in response to altered load yields a matrix that fails to support normal joint function. Mathematical modeling and experimental studies in animal models confirm an association between altered loading of diarthrotic joints and arthritic changes. Both types of studies implicate shear forces as a critical component in the destructive profile. The severity of cartilage destruction in response to altered loads appears linked to expression of biological factors influencing matrix integrity and cellular metabolism. Determining how shear stress alters chondrocyte metabolism is fundamental to understanding how to limit matrix destruction and stimulate cartilage repair and regeneration. At present, the precise biochemical and molecular mechanisms by which shear forces alter chondrocyte metabolism from a normal to a degenerative phenotype remain unclear. The results presented here address the hypothesis that articular chondrocyte metabolism is modulated by direct effects of shear forces that act on the cell through mechanotransduction processes. The purpose of this work is to develop critical knowledge regarding the basic mechanisms by which mechanical loading modulates cartilage metabolism in health and disease. This presentation will describe the effects of using fluid induced shear stress as a model system for stimulation of articular chondrocytes in vitro. The fluid induced shear stress was applied using a cone viscometer system to stimulate all the cells uniformly under conditions of minimal turbulence. The experiments were carried using high-density primary monolayer cultures of normal and osteoarthritic human and normal bovine articular chondrocytes. The analysis of the cellular response included quantification of cytokine release, matrix metalloproteinase expression and activation of intracellular signaling pathways. The data presented here show that articular chondrocytes exhibit a dose- and time-dependent response to shear stress that results in the release of soluble mediators and extracellular matrix macromolecules. The data suggest that the chondrocyte response to mechanical stimulation contributes to the maintenance of articular cartilage homeostasis in vivo.     

Pre-clinical studies of retrovirally transduced human chondrocytes expressing transforming growth factor-beta-1 (TG-C)

Background aimsThe aim was to evaluate cartilage regeneration in animal models involving induced knee joint damage. Through cell-mediated gene therapy methods, a cell mixture comprising a 3:1 ratio of genetically unmodified human chondrocytes and transforming growth factor beta-1 (TGF-β1)-secreting human chondrocytes (TG-C), generated via retroviral transduction, resulted in successful cartilage proliferation in damaged regions.MethodsNon-clinical toxicology assessments for efficacy, biodistribution and local/systemic toxicity of single intra-articular administration of the cell mixture in mice, rabbits and goats was conducted.ResultsAdministration of the mixture was tolerated well in all of the species. There was evidence of cartilage proliferation in rabbits and goats. As an additional precautionary step, the efficacy of TGF-β1 secretion in irradiated human chondrocytes was also demonstrated.ConclusionsFour studies in rabbits and goats demonstrated the safety and efficacy of TG-C following direct intra-articular administration in animal models involving induced knee joint damage. Based on these pre-clinical studies authorization has been received from the USA Food and Drug Administration (FDA) to proceed with an initial phase I clinical study of TG-C for degenerative arthritis.     

Cartilage repair: surgical techniques and tissue engineering using polysaccharide- and collagen-based biomaterials

Lesions of articular cartilage have a large variety of causes among which traumatic damage, osteoarthritis and osteochondritis dissecans are the most frequent. Replacement of articular defects in joints has assumed greater importance in recent years. This interest results in large part because cartilage defects cannot adequately heal themselves. Many techniques have been suggested over the last 30 years, but none allows the regeneration of the damaged cartilage, i.e. its replacement by a strictly identical tissue. In the first generation of techniques, relief of pain was the main concern, which could be provided by techniques in which cartilage was replaced by fibrocartilage. Disappointing results led investigators to focus on more appropriate bioregenerative approaches using transplantation of autologous cells into the lesion. Unfortunately, none of these approaches has provided a perfect final solution to the problem. The latest generation of techniques, currently in the developmental or preclinical stages, involve biomaterials for the repair of chondral or osteochondral lesions. Many of these scaffolds are designed to be seeded with chondrocytes or progenitor cells. Among natural and synthetic polymers, collagen- and polysaccharide-based biomaterials have been extensively used. For both these supports, studies have shown that chondrocytes maintain their phenotype when cultured in three dimensions. In both types of culture, a glycosaminoglycan-rich deposit is formed on the surface and in the inner region of the cultured cartilage, and type II collagen synthesis is also observed. Dynamic conditions can also improve the composition of such three-dimensional constructs. Many improvements are still required, however, in a number of key aspects that so far have received only scant attention. These aspects include: adhesion/integration of the graft with the adjacent native cartilage, cell-seeding with genetically-modified cell populations, biomaterials that can be implanted without open joint surgery and combined therapies, aimed at disease modification, pain relief and reduction of inflammation.     

The longitudinal relationship between body composition and patella cartilage in healthy adults

Background: Although obesity is a risk factor for patellofemoral osteoarthritis (OA), it is unclear whether the components of body composition, such as muscle and fat mass, are major determinants of articular cartilage properties at the patella. Objective: The aim of this study was to determine whether anthropometric and body composition measures, assessed over 10 years, were related to articular patella cartilage volume and defects in healthy adults with no clinical knee OA. Methods and Procedures: Two hundred and ninety‐seven healthy, community‐based adults aged 50–79 years with no clinical history of knee OA were recruited. Anthropometric and body composition (fat‐free mass and fat mass) data were measured at baseline (1990–1994) and follow‐up (2003–2004). Patella cartilage volume and defects were assessed at follow‐up (2003–2004) using magnetic resonance imaging (MRI). Results: After adjustment for potential confounders, increased measures of obesity (weight, BMI, waist circumference, and fat mass) at baseline and follow‐up were associated with an increased risk for the presence of patella cartilage defects at follow‐up for both men and women (all P ≤ 0.03). Increased baseline values for these variables tended to be associated with reduced patella cartilage volume at follow‐up for women (all P ≤ 0.11), but not men (all P ≤ 0.87). Discussion: We have demonstrated that increased anthropometric measures of obesity, as well as fat mass, are associated with an increased risk for the presence of patella cartilage defects in both men and women. Women, but not men, with greater baseline body mass, particularly adipose‐derived mass, appear to have an associated reduction in their patella cartilage volume. Interventions targeting a reduction in adipose tissue may help reduce the risk for the onset and progression of patellofemoral OA, particularly in women.