Articular cartilage regeneration with microfracture and hyaluronic acid

Microfracture used to treat articular cartilage injuries can facilitate access to stem cells in the bone marrow and stimulate cartilage regeneration. However, the regenerated cartilage is fibrocartilage as opposed to hyaline articular cartilage and is thinner than native cartilage. Following microfracture in rabbit knee cartilage defects, application of hyaluronic acid gel resulted in regeneration of a thicker, more hyaline-like cartilage. The addition of transforming growth factor-β3, an inducer of chondrogenic differentiation in mesenchymal stem cells, to the treatment with microfracture and hyaluronic acid did not significantly benefit cartilage regeneration.     

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.     

Depletion of chondrocyte primary cilia reduces the compressive modulus of articular cartilage

Primary cilia are slender, microtubule based structures found in the majority of cell types with one cilium per cell. In articular cartilage, primary cilia are required for chondrocyte mechanotransduction and the development of healthy tissue. Loss of primary cilia in Col2aCre;ift88^fl/fl transgenic mice results in up-regulation of osteoarthritic (OA) markers and development of OA like cartilage with greater thickness and reduced mechanical stiffness. However no previous studies have examined whether loss of primary cilia influences the intrinsic mechanical properties of articular cartilage matrix in the form of the modulus or just the structural properties of the tissue. The present study describes a modified analytical model to derive the viscoelastic moduli based on previous experimental indentation data. Results show that the increased thickness of the articular cartilage in the Col2aCre;ift88^fl/fl transgenic mice is associated with a reduction in both the instantaneous and equilibrium moduli at indentation strains of greater than 20%. This reveals that the loss of primary cilia causes a significant reduction in the mechanical properties of cartilage particularly in the deeper zones and possibly the underlying bone. This is consistent with histological analysis and confirms the importance of primary cilia in the development of a mechanically functional articular cartilage.     

Anatomically shaped osteochondral constructs for articular cartilage repair

Few successful treatment modalities exist for surface-wide, full-thickness lesions of articular cartilage. Functional tissue engineering offers a great potential for the clinical management of such lesions. Our long-term hypothesis is that anatomically shaped tissue constructs of entire articular layers can be engineered in vitro on a bony substrate, for subsequent implantation. To determine the feasibility, this study investigated the development of bilayered scaffolds of chondrocyte-seeded agarose on natural trabecular bone. In a series of three experiments, bovine chondrocytes were seeded in (1) cylindrical bilayered constructs of agarose and bovine trabecular bone, 0.53 cm² in surface area and 3.2 mm thick, and were cultured for up to 6 weeks; (2) chondrocyte-seeded anatomically shaped agarose constructs reproducing the human patellar articular layer (area=11.7 cm², mean THICKNESS=3.4 mm), cultured for up to 6 weeks; and (3) chondrocyte-seeded anatomically shaped agarose constructs of the patella (same as above) integrated into a corresponding anatomically shaped trabecular bone substrate, cultured for up to 2 weeks. Articular layer geometry, previously acquired from human cadaver joints, was used in conjunction with computer-aided design and manufacturing technology to create these anatomically accurate molds. In all experiments, chondrocytes remained viable over the entire culture period, with the agarose maintaining its shape while remaining firmly attached to the underlying bony substrate (when present). With culture time, the constructs exhibited positive type II collagen staining as well as increased matrix elaboration (Safranin O staining for glycosaminoglycans) and material properties (Young's modulus and aggregate modulus). Despite the use of relatively large agarose constructs partially integrated with trabecular bone, no adverse diffusion limitation effects were observed. Anatomically shaped constructs on a bony substrate may represent a new paradigm in the design of a functional articular cartilage tissue replacement.     

A biphasic multiscale study of the mechanical microenvironment of chondrocytes within articular cartilage under unconfined compression

Computational analyses have been used to study the biomechanical microenvironment of the chondrocyte that cannot be assessed by in vitro experimental studies; yet all computational studies thus far have focused on the effect of zonal location (superficial, middle, and deep) on the mechanical microenvironment of chondrocytes. The aim of this paper was to study the effect of both zonal and radial locations on the biomechanical microenvironment of chondrocytes in inhomogeneous cartilage under unconfined stress relaxation. A biphasic multiscale approach was employed and nine chondrocytes in different locations were studied. Hyperelastic biphasic theory and depth-dependent aggregate modulus and permeability of articular cartilage were included in the models. It was found that both zonal and radial locations affected the biomechanical stresses and strains of the chondrocytes. Chondrocytes in the mid-radial location had increased volume during the early stage of the loading process. Maximum principal shear stress at the interface between the chondrocyte and the extracellular matrix (ECM) increased with depth, yet that at the ECM–pericellular matrix (PCM) interface had an inverse trend. Fluid pressure decreased with depth, while the fluid pressure difference between the top and bottom boundaries of the microscale model increased with depth. Regardless of location, fluid was exchanged between the chondrocyte, PCM, and ECM. These findings suggested that even under simple compressive loading conditions, the biomechanical microenvironment of the chondrocytes, PCM and ECM was spatially dependent. The current study provides new insight on chondrocyte biomechanics.     

Cryopreservation of articular cartilage. Part 1: conventional cryopreservation methods

There is increasing interest in the possibility of treating diseased or damaged areas of synovial joint surfaces by grafts of healthy allogeneic cartilage. Such grafts could be obtained from cadaver tissue donors or in the future they might be manufactured by 'tissue engineering' methods. Cartilage is an avascular tissue and hence is immunologically privileged but to take advantage of this is the graft must contain living cells. Preservation methods that achieve this are required to build up operational stocks of grafts, to provide a buffer between procurement and use, and to enable living grafts of a practical size to be provided at the right time for patient and surgeon. Review of the literature shows that it has been relatively straightforward to cryopreserve living isolated chondrocytes, but at the present time there is no satisfactory method to preserve cartilage between the time of procurement or manufacture and surgical use. In this paper, we review the relevant literature and we confirm that isolated ovine chondrocytes in suspension can be effectively cryopreserved by standard methods yet the survival of chondrocytes in situ in cartilage tissue is inadequate and extremely variable.     

Retinoic acid-induced cartilage degradation is caused by cartilage cells

Summary Cartilage cubes, prepared from the proximal epiphyses of neonatal rat humeri and consisting of cartilage tissue only, were cultured in the presence of retinoic acid. The retinoid induced the loss of metachromatic staining with toluidine blue, which correlates with the loss of proteoglycan, followed by tissue degradation processes resulting in a distinct reduction of the cartilage mass. Histologically, fibroblast-like cells appeared within chondrones, indicating a transformation of chondroblasts. Focal tissue degradation was observed after only 2 days. Electron microscopically, the clustered cells within the zone of tissue degradation were rich in various lysosomal structures indicating their lytic activity. Cycloheximide and EDTA completely blocked the retinoic acid effects suggesting that protein synthesis was required and that metalloproteinases may be involved in the degradation processes. In conclusion, with the new test system described here we demonstrated that cartilage cells themselves performed the tissue degradation induced by retinoic acid.     

Dynamic compression of cartilage constructs engineered from expanded human articular chondrocytes

Recent works have shown that mechanical loading can alter the metabolic activity of chondrocytes cultured in 3D scaffolds. In this study we determined whether the stage of development of engineered cartilaginous constructs (expanded adult human articular chondrocytes/Polyactive foams) regulates the effect of dynamic compression on glycosaminoglycan (GAG) metabolism. Construct maturation depended on the culture time (3-14 days) and the donor (4 individuals). When dynamic compression was subsequently applied for 3 days, changes in GAG synthesized, accumulated, and released were significantly positively correlated to the GAG content of the constructs prior to loading, and resulted in stimulation of GAG formation only in the most developed tissues. Conversely, none of these changes were correlated with the expression of collagen type II mRNA, indicating that the response of chondrocytes to dynamic compression does not depend directly upon the stage of cell differentiation, but rather on the extracellular matrix surrounding the cells.     

Requirement of fibroblast growth factor signaling for regeneration of epiphyseal morphology in rabbit full-thickness defects of articular cartilage

The involvement of fibroblast growth factor-2 (FGF-2) during the repair process in rabbit full-thickness defects of articular cartilage was studied. Fibroblast growth factor-2 (50 pg/h) was administered for 2 weeks in a 5mm defect of articular cartilage, which is large enough not to repair spontaneously. The administration of FGF-2 resulted in the regeneration of the articular cartilage and the subchondral bone within 8 weeks. In these defects, undifferentiated mesenchymal cells initiated chondrogenic differentiation coupled with replacement by subchondral bone, resulting in the resurfacing of the defects with hyaline cartilage and the recovery of subchondral bone up to the original bone–articular cartilage junction. In rabbits, full-thickness defects are capable of regenerating articular cartilage as long as the defect size is limited to ≤3 mm in diameter. In the defects, strong immunoreactivity for FGF-2 was observed in the granulation tissue filling the defects in the early stage of repair, in association with the expression of FGF-2 mRNA shown by in situ hybridization. Once the undifferentiated mesenchymal cells had differentiated into chondrocytes, both the immunoreactivity and the in situ hybridization signal declined significantly. Upon the local administration of a monoclonal antibody against FGF-2 (bFM-1, 50ng/h), the defects were filled with fibrous tissue and no resurfacing hyaline cartilage was formed. Compared to the non-treated defects, there were marked increases in FGF-2 immunoreactivity and the overexpression of FGF-2 mRNA in the reparative tissue in the bFM-1 -treated defects. This rebound phenomenon indicates that the autocrine FGF-2 signaling is critically important for the regeneration of articular cartilage.     

Chondrogenesis,joint formation,and articular cartilage regeneration

The repair of joint surface defects remains a clinical challenge, as articular cartilage has a limited healing response. Despite this, articular cartilage does have the capacity to grow and remodel extensively during pre‐ and post‐natal development. As such, the elucidation of developmental mechanisms, particularly those in post‐natal animals, may shed valuable light on processes that could be harnessed to develop novel approaches for articular cartilage tissue engineering and/or regeneration to treat injuries or degeneration in adult joints. Much has been learned through mouse genetics regarding the embryonic development of joints. This knowledge, as well as the less extensive available information regarding post‐natal joint development is reviewed here and discussed in relation to their possible relevance to future directions in cartilage tissue repair and regeneration. J. Cell. Biochem. 107: 383–392, 2009. © 2009 Wiley‐Liss, Inc.     

Dielectric characterization of costal cartilage chondrocytes

Background

Chondrocytes respond to biomechanical and bioelectrochemical stimuli by secreting appropriate extracellular matrix proteins that enable the tissue to withstand the large forces it experiences. Although biomechanical aspects of cartilage are well described, little is known of the bioelectrochemical responses. The focus of this study is to identify bioelectrical characteristics of human costal cartilage cells using dielectric spectroscopy.

Methods

Dielectric spectroscopy allows non-invasive probing of biological cells. An in house computer program is developed to extract dielectric properties of human costal cartilage cells from raw cell suspension impedance data measured by a microfluidic device. The dielectric properties of chondrocytes are compared with other cell types in order to comparatively assess the electrical nature of chondrocytes.

Results

The results suggest that electrical cell membrane characteristics of chondrocyte cells are close to cardiomyoblast cells, cells known to possess an array of active ion channels. The blocking effect of the non-specific ion channel blocker gadolinium is tested on chondrocytes with a significant reduction in both membrane capacitance and conductance.

Conclusions

We have utilized a microfluidic chamber to mimic biomechanical events through changes in bioelectrochemistry and described the dielectric properties of chondrocytes to be closer to cells derived from electrically excitably tissues.

General significance

The study describes dielectric characterization of human costal chondrocyte cells using physical tools, where results and methodology can be used to identify potential anomalies in bioelectrochemical responses that may lead to cartilage disorders.     

Vitrification of porcine articular cartilage

The limited availability of fresh osteochondral allograft tissues necessitates the use of banking for long-term storage. A vitrification solution containing a 55% cryoprotectant formulation, VS55, previously studied using rabbit articular cartilage, was evaluated using porcine articular cartilage. Specimens ranging from 2 to 6 mm in thickness were obtained from 6 mm distal femoral cartilage cores and cryopreserved by vitrification or freezing. The results of post-rewarming viability assessments employing alamarBlue demonstrated a large decrease (p < 0.001) in viability in all three sizes of cartilage specimen vitrified with VS55. This is in marked contrast with prior experience with full thickness, 0.6 mm rabbit cartilage. Microscopic examination following cryosubstitution confirmed ice formation in the chondrocytes of porcine cartilage vitrified using VS55. Experiments using a more concentrated vitrification formulation (83%), VS83, showed a significant treatment benefit for larger segments of articular cartilage. Differences between the VS55 and the VS83 treatment groups were significant at p < 0.001 for 2 mm and 4 mm plugs, and at p < 0.01 for full thickness, 6 mm plugs. The percentage viability in fresh controls, compared to VS55 and VS83, was 24.7% and 80.7% in the 2 mm size group, 18.2% and 55.5% in the 4 mm size group, and 5.2% and 43.6% in the 6 mm group, respectively. The results of this study continue to indicate that vitrification is superior to conventional cryopreservation with low concentrations of dimethyl sulfoxide by freezing for cartilage. The vitrification technology presented here may, with further process development, enable the long-term storage and transportation of living cartilage for repair of human articular surfaces.     

Mesenchymal progenitor cells derived from chorionic villi of human placenta for cartilage tissue engineering

Human mesenchymal stem cells are currently being studied extensively because of their capability for self-renewal and differentiation to various connective tissues, which makes them attractive as cell sources for regenerative medicine. Herein we report the isolation of human placenta-derived mesenchymal cells (hPDMCs) that have the potential to differentiate into various lineages to explore the possibility of using these cells for regeneration of cartilage. We first evaluated the chondrogenesis of hPDMCs in vitro and then embedded the hPDMCs into an atelocollagen gel to make a cartilage-like tissue with chondrogenic induction media. For in vivo assay, preinduced hPDMCs embedded in collagen sponges were subcutaneously implanted into nude mice and also into nude rats with osteochondral defect. The results of these in vivo and in vitro studies suggested that hPDMCs can be one of the possible allogeneic cell sources for tissue engineering of cartilage.     

Age-related changes in the composition and mechanical properties of human nasal cartilage

Mutations in the tuberous sclerosis 2 (TSC2) gene product have been genetically linked to the pathology of both tuberous sclerosis (TSC) and the gender-specific lung disease, lymphangioleiomyomatosis (LAM). Both diseases are classified as disorders of cellular migration, proliferation, and differentiation. Earlier studies from our laboratory (1) linked TSC2 with steroid/nuclear receptor signaling. Studies presented here provide evidence for calmodulin (CaM) signaling in the propagation of this TSC2 activity. Far Western screening of a lambda phage human brain cDNA library to identify interacting proteins for the TSC2 gene product (tuberin) yielded multiple clones encoding human CaM. Direct binding with 32P-labeled tuberin demonstrated Ca2+-dependent binding to CaM-Sepharose which was lost upon deletion of the C-terminal 72 residues. The sequence (1740)WIARLRHIKRLRQRIC(1755) was identified as one capable of forming a basic amphipathic helix indicative of CaM binding domains in known calmodulin binding proteins. Studies with a synthetic peptide of this sequence demonstrated very tight Ca2+-dependent binding to CaM as judged by tryptophan fluorescence perturbation studies and phosphodiesterase activation by CaM. Deletion mutagenesis studies further suggested that this CaM binding domain is required for tuberin modulation of steroid receptor function and that mutations in this region may be involved in the pathology of TSC and LAM.     

Applied osmotic loading for promoting development of engineered cartilage

This study investigated the potential use of static osmotic loading as a cartilage tissue engineering strategy for growing clinically relevant grafts from either synovium-derived stem cells (SDSCs) or chondrocytes. Bovine SDSCs and chondrocytes were individually encapsulated in 2% w/v agarose and divided into chondrogenic media of osmolarities 300 (hypotonic), 330 (isotonic), and 400 (hypertonic, physiologic) mOsM for up to 7 weeks. The application of hypertonic media to constructs comprised of SDSCs or chondrocytes led to increased mechanical properties as compared to hypotonic (300 mOsM) or isotonic (330 mOsM) media (p<0.05). Constant exposure of SDSC-seeded constructs to 400 mOsM media from day 0 to day 49 yielded a Young's modulus of 513±89 kPa and GAG content of 7.39±0.52%ww on day 49, well within the range of values of native, immature bovine cartilage. Primary chondrocyte-seeded constructs achieved almost as high a Young's modulus, reaching 487±187 kPa and 6.77±0.54%ww (GAG) for the 400 mOsM condition (day 42). These findings suggest hypertonic loading as a straightforward strategy for 3D cultivation with significant benefits for cartilage tissue engineering strategies. In an effort to understand potential mechanisms responsible for the observed response, cell volume measurements in response to varying osmotic conditions were evaluated in relation to the Boyle–van't Hoff (BVH) law. Results confirmed that chondrocytes behave as perfect osmometers; however SDSCs deviated from the BVH relation.     

Toward regeneration of articular cartilage

Articular cartilage is classified as permanent hyaline cartilage and has significant differences in structure, extracelluar matrix components, gene expression profile, and mechanical property from transient hyaline cartilage found in the epiphyseal growth plate. In the process of synovial joint development, articular cartilage originates from the interzone, developing at the edge of the cartilaginous anlagen, and establishes zonal structure over time and supports smooth movement of the synovial joint through life. The cascade actions of key regulators, such as Wnts, GDF5, Erg, and PTHLH, coordinate sequential steps of articular cartilage formation. Articular chondrocytes are restrictedly controlled not to differentiate into a hypertrophic stage by autocrine and paracrine factors and extracellular matrix microenvironment, but retain potential to undergo hypertrophy. The basal calcified zone of articular cartilage is connected with subchondral bone, but not invaded by blood vessels nor replaced by bone, which is highly contrasted with the growth plate. Articular cartilage has limited regenerative capacity, but likely possesses and potentially uses intrinsic stem cell source in the superficial layer, Ranvier's groove, the intra‐articular tissues such as synovium and fat pad, and marrow below the subchondral bone. Considering the biological views on articular cartilage, several important points are raised for regeneration of articular cartilage. We should evaluate the nature of regenerated cartilage as permanent hyaline cartilage and not just hyaline cartilage. We should study how a hypertrophic phenotype of transplanted cells can be lastingly suppressed in regenerating tissue. Furthermore, we should develop the methods and reagents to activate recruitment of intrinsic stem/progenitor cells into the damaged site. Birth Defects Research (Part C) 99:192–202, 2013 . © 2013 Wiley Periodicals, Inc .     

Nutrient channels and stirring enhanced the composition and stiffness of large cartilage constructs

A significant challenge in cartilage tissue engineering is to successfully culture functional tissues that are sufficiently large to treat osteoarthritic joints. Transport limitations due to nutrient consumption by peripheral cells produce heterogeneous constructs with matrix-deficient centers. Incorporation of nutrient channels into large constructs is a promising technique for alleviating transport limitations, in conjunction with simple yet effective methods for enhancing media flow through channels. Cultivation of cylindrical channeled constructs flat in culture dishes, with or without orbital shaking, produced asymmetric constructs with poor tissue properties. We therefore explored a method for exposing the entire construct surface to the culture media, while promoting flow through the channels. To this end, chondrocyte-seeded agarose constructs (∅10 mm, 2.34 mm thick), with zero or three nutrient channels (∅1 mm), were suspended on their sides in custom culture racks and subjected to three media stirring modes for 56 days: uniaxial rocking, orbital shaking, or static control. Orbital shaking led to the highest construct E_Y, sulfated glycosaminoglycan (sGAG), and collagen contents, whereas rocking had detrimental effects on sGAG and collagen versus static control. Nutrient channels increased E_Y as well as sGAG homogeneity, and the beneficial effects of channels were most marked in orbitally shaken samples. Under these conditions, the constructs developed symmetrically and reached or exceeded native levels of E_Y (~400 kPa) and sGAG (~9%/ww). These results suggest that the cultivation of channeled constructs in culture racks with orbital shaking is a promising method for engineering mechanically competent large cartilage constructs.