Subchondral bone osteoblasts can induce chondrocyte mineralization during osteoarthritis and this process is relevant to cartilage degradation

Pathological mineralization of articular cartilage is a characteristic feature of osteoarthritis (OA); however, the underlying mechanisms, and their relevance to cartilage degeneration, are not clear. The involvement of subchondral bone changes in OA have been reported previously with the characterization of abnormal subchondral bone mineral density (BMD), osteiod volume, altered bone mechanical parameters and an increase in bone turnover markers. A number of osteoarthritic animal models have demonstrated that subchondral bone changes often precede cartilage degeneration. In this study site specific localization of mineralization markers were detected in the OA cartilage. Chondrocytes and osteoblasts derived from OA cartilage and subchondral bone showed a significant increase in the mRNA expressions of mineralization markers. Interestingly, osteoblasts from OA subchondral bone could significantly decrease cartilage matrix expression; whereas, increase mineralization of chondrocytes (Figure 1). Osteogenic factors, such as CBFA1, ALP, and type X collagen (Col-X), were detected in chondrocytes under mineralization conditions (Figure 2). Furthermore, chondrocyte mineralization was followed by increased mRNA and protein levels of MMP-2, MMP-9 and MMP-13, all of which are detrimental to cartilage integrity in vivo. The data reported here suggests that the upregulation of subchondral bone-mineralization, typical of OA progression, causes cartilage mineralization, and that the mineralization of chondrocytes induce increased MMP levels with a subsequent degradation of the articular cartilage.

Long-term effects of hydrogel properties on human chondrocyte behavior

Hydrogels provide a 3-dimensional network for embedded cells and offer promise for cartilage tissue engineering applications. Nature-derived hydrogels, including alginate, have been shown to enhance the chondrocyte phenotype but are variable and not entirely controllable. Synthetic hydrogels, including polyethylene glycol (PEG)-based matrices, have the advantage of repeatability and modularity; mechanical stiffness, cell adhesion, and degradability can be altered independently. In this study, we compared the long-term in vitro effects of different hydrogels (alginate and Factor XIIIa-cross-linked MMP-sensitive PEG at two stiffness levels) on the behavior of expanded human chondrocytes and the development of construct properties. Monolayer-expanded human chondrocytes remained viable throughout culture, but morphology varied greatly in different hydrogels. Chondrocytes were characteristically round in alginate but mostly spread in PEG gels at both concentrations. Chondrogenic gene (COL2A1, aggrecan) expression increased in all hydrogels, but alginate constructs had much higher expression levels of these genes (up to 90-fold for COL2A1), as well as proteoglycan 4, a functional marker of the superficial zone. Also, chondrocytes expressed COL1A1 and COL10A1, indicative of de-differentiation and hypertrophy. After 12 weeks, constructs with lower polymer content were stiffer than similar constructs with higher polymer content, with the highest compressive modulus measured in 2.5% PEG gels. Different materials and polymer concentrations have markedly different potency to affect chondrocyte behavior. While synthetic hydrogels offer many advantages over natural materials such as alginate, they must be further optimized to elicit desired chondrocyte responses for use as cartilage models and for development of functional tissue-engineered articular cartilage.

Tissue engineering of stratified articular cartilage from chondrocyte subpopulations

Objective: To test if subpopulations of chondrocytes from different cartilage zones could be used to engineer cartilage constructs with features of normal stratification. Design: Chondrocytes from the superficial and middle zones of immature bovine cartilage were cultured in alginate, released, and seeded either separately or sequentially to form cartilage constructs. Constructs were cultured for 1 or 2 weeks and were assessed for growth, compressive properties, and deposition, and localization of matrix molecules and superficial zone protein (SZP). Results: The cartilaginous constructs formed from superficial zone chondrocytes exhibited less matrix growth and lower compressive properties than constructs from middle zone chondrocytes, with the stratified superficial-middle constructs exhibiting intermediate properties. Expression of SZP was highest at the construct surfaces, with the localization of SZP in superficial-middle constructs being concentrated at the superficial surface. Conclusions: Manipulation of subpopulations of chondrocytes can be useful in engineering cartilage tissue with a biomimetic approach, and in fabricating constructs that exhibit stratified features of normal articular cartilage. (C) 2003 OsteoArthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

Synthesis of proteoglycan 4 by chondrocyte subpopulations in cartilage explants, monolayer cultures, and resurfaced cartilage cultures

Objective: To quantify the levels of proteoglycan 4 (PRG4) expression by subpopulations of chondrocytes from superficial, middle, and deep layers of normal bovine calf cartilage in various culture systems. Methods: Bovine calf articular cartilage discs or isolated cells were used in I of 3 systems of chondrocyte culture: explant, monolayer, or transplant, for 1-9 days. PRG4 expression was quantified by enzyme-linked immunosorbent assay of spent medium and localized by immunohistochemistry at the articular surface and within chondrocytes in explants and cultured cells. Results: Superficial chondrocytes secreted much more PRG4 than did middle and deep chondrocytes in all cultures. The pattern of PRG4 secretion into superficial culture medium varied with the duration of culture, decreasing with time in explant culture (from similar to25 mug/cm(2)/day on days 0-1 to similar to3 mug/cm(2)/day on days 5-9), while increasing in monolayer culture (from similar to1 pg/cell/day on days 0-1 to similar to7 pg/cell/day on days 7-9) and tending to increase in transplant culture (reaching similar to2 mug/cm(2)/day by days 7-9). In all of the culture systems, inclusion of ascorbic acid stimulated PRG4 secretion, and the source of PRG4 was immunolocalized to superficial cells. Conclusion: The results described here indicate that the phenotype of PRG4 secretion by chondrocytes in culture is generally maintained, in that PRG4 is expressed to a much greater degree by chondrocytes from the superficial zone than by those from the middle and deep zones. The marked up-regulation of PRG4 synthesis by ascorbic acid may have implications for cartilage homeostasis and prevention of osteoarthritic disease. Transplanting specialized cells that secrete PRG4 to a surface may impart functional lubrication and be generally applicable to many tissues in the body.

Cell density alters matrix accumulation in two distinct fractions and the mechanical integrity of alginate-chondrocyte constructs

Chondrocyte density in articular cartilage is known to change with the development and growth of the tissue and may play an important role in the formation of a functional extracellular matrix (ECM). The objective of this study was to determine how initial chondrocyte density in an alginate hydrogel affects the matrix composition, its distribution between the cell-associated (CM) and further removed matrix (FRM) fractions, and the tensile mechanical properties of the developing engineered cartilage. Alginate constructs containing primary bovine chondrocytes at densities of 0, 4, 16, and 64 million cells/ml were fabricated and cultured for 1 or 2 weeks, at which time structural, biochemical, and mechanical properties were analyzed. Both matrix content and distribution varied with the initial cell density. Increasing cell density resulted in an increasing content of collagen and sulfated-glycosaminoglycan (GAG) and an increasing proportion of these molecules localized in the CM. While the equilibrium tensile modulus of cell-free alginate did not change with time in culture, the constructs with highest cell density were 116% stiffer than cell-free controls after 2 weeks of culture. The equilibrium tensile modulus was positively correlated with total collagen (r2 = 0.47, p < 0.001) and GAG content (r2 = 0.68, p < 0.001), and these relationships were enhanced when analyzing only those matrix molecules in the CM fraction (r2 = 0.60 and 0.72 for collagen and GAG, respectively, each p < 0.001). Overall, the results of this study indicate that initial cell density has a considerable effect on the developing composition, structure, and function of alginate–chondrocyte constructs.

Short-term retention of labeled chondrocyte subpopulations in stratified tissue-engineered cartilaginous constructs implanted in vivo in mini-pigs

It is likely that effective application of cell-laden implants for cartilage defects depends on retention of implanted cells and interaction between implanted and host cells. The objectives of this study were to characterize stratified cartilaginous constructs seeded sequentially with superficial (S) and middle (M) chondrocyte subpopulations labelled with fluorescent cell tracking dye PKH26 (*) and determine the degree to which these stratified cartilaginous constructs maintain their architecture in vivo after implantation in mini-pigs for 1 week. Alginate-recovered cells were seeded sequentially to form stratified S*/M (only S cells labelled) and S*/M* (both S and M cells labelled) constructs. Full-thickness defects (4 mm diameter) were created in the patellofemoral groove of adult Yucatan mini-pigs and filled with portions of constructs or left empty. Constructs were characterized biochemically, histologically, and biomechanically, and stratification visualized and quantified, before and after implant. After 1 week, animals were sacrificed and implants retrieved. After 1 week in vivo, glycosaminoglycan and collagen content of constructs remained similar to that at implant, whereas DNA content increased. Histological analyses revealed features of an early repair response, with defects filled with tissues containing little matrix and abundant cells. Some implanted (PKH26-labeled) cells persisted in the defects, although constructs did not maintain a stratified organization. Of the labelled cells, 126 +/- 38% and 32 +/- 8% in S*/M and S*/M* constructs, respectively, were recovered. Distribution of labelled cells indicated interactions between implanted and host cells. Longer-term in vivo studies will be useful in determining whether implanted cells are sufficient to have a positive effect in repair.

Continuous passive motion applied to whole joints stimulates chondrocyte blosynthesis of PRG4

Continuous passive motion (CPM) is currently a part of patient rehabilitation regimens after a variety of orthopedic surgical procedures. While CPM can enhance the joint healing process, the direct effects of CPM on cartilage metabolism remain unknown. Recent in vivo and in vitro observations suggest that mechanical stimuli can regulate articular cartilage metabolism of proteoglycan 4 (PRG4), a putative lubricating and chondroprotective molecule found in synovial fluid and at the articular cartilage surface. ----- ----- Objectives: (1) Determine the topographical variation in intrinsic cartilage PRG4 secretion. (2) Apply a CPM device to whole joints in bioreactors and assess effects of CPM on PRG4 biosynthesis.----- ----- Methods: A bioreactor was developed to apply CPM to bovine stifle joints in vitro. Effects of 24 h of CPM on PRG4 biosynthesis were determined.----- ----- Results: PRG4 secretion rate varied markedly over the joint surface. Rehabilitative joint motion applied in the form of CPM regulated PRG4 biosynthesis, in a manner dependent on the duty cycle of cartilage sliding against opposing tissues. Specifically, in certain regions of the femoral condyle that were continuously or intermittently sliding against meniscus and tibial cartilage during CPM, chondrocyte PRG4 synthesis was higher with CPM than without.----- ----- Conclusions: Rehabilitative joint motion, applied in the form of CPM, stimulates chondrocyte PRG4 metabolism. The stimulation of PRG4 synthesis is one mechanism by which CPM may benefit cartilage and joint health in post-operative rehabilitation. (C) 2006 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

Zonal chondrocyte subpopulations reacquire zone-specific characteristics during in Vitro redifferentiation

Background: If chondrocytes from the superficial, middle, and deep zones of articular cartilage could maintain or regain their characteristic properties during in vitro culture, it would be feasible to create constructs comprising these distinctive zones. ----- ----- Hypothesis: Zone-specific characteristics of zonal cell populations will disappear during 2-dimensional expansion but will reappear after 3-dimensional redifferentiation, independent of the culture technique used (alginate beads versus pellet culture).----- ----- Study Design: Controlled laboratory study.----- ----- Methods: Equine articular chondrocytes from the 3 zones were expanded in monolayer culture (8 donors) and subsequently redifferentiated in pellet and alginate bead cultures for up to 4 weeks. Glycosaminoglycans and DNA were quantified, along with immunohistochemical assessment of the expression of various zonal markers, including cartilage oligomeric protein (marking cells from the deeper zones) and clusterin (specifically expressed by superficial chondrocytes).----- ----- Results: Cell yield varied between zones, but proliferation rates did not show significant differences. Expression of all evaluated zonal markers was lost during expansion. Compared to the alginate bead cultures, pellet cultures showed a higher amount of glycosaminoglycans produced per DNA after redifferentiation. In contrast to cells in pellet cultures, cells in alginate beads regained zonal differences, as evidenced by zone-specific reappearance of cartilage oligomeric protein and clusterin, as well as significantly higher glycosaminoglycans production by cells from the deep zone compared to the superficial zone.----- ----- Conclusion: Chondrocytes isolated from the 3 zones of equine cartilage can restore their zone-specific matrix expression when cultured in alginate after in vitro expansion.

Development of zonal cartilage constructs : effects of chondrocyte subpopulation, compressive stimulation, and culture models

Articular cartilage is a highly resilient tissue located at the ends of long bones. It has a zonal structure, which has functional significance in load-bearing. Cartilage does not spontaneously heal itself when damaged, and untreated cartilage lesions or age-related wear often lead to osteoarthritis (OA). OA is a degenerative condition that is highly prevalent, age-associated, and significantly affects patient mobility and quality of life. There is no cure for OA, and patients usually resort to replacing the biological joint with an artificial prosthesis. An alternative approach is to dynamically regenerate damaged or diseased cartilage through cartilage tissue engineering, where cells, materials, and stimuli are combined to form new cartilage. However, despite extensive research, major limitations remain that have prevented the wide-spread application of tissue-engineered cartilage. Critically, there is a dearth of information on whether autologous chondrocytes obtained from OA patients can be used to successfully generate cartilage tissues with structural hierarchy typically found in normal articular cartilage. I aim to address these limitations in this thesis by showing that chondrocyte subpopulations isolated from macroscopically normal areas of the cartilage can be used to engineer stratified cartilage tissues and that compressive loading plays an important role in zone-dependent biosynthesis of these chondrocytes. I first demonstrate that chondrocyte subpopulations from the superficial (S) and middle/deep (MD) zones of OA cartilage are responsive to compressive stimulation in vitro, and that the effect of compression on construct quality is zone-dependent. I also show that compressive stimulation can influence pericelluar matrix production, matrix metalloproteinase secretion, and cytokine expression in zonal chondrocytes in an alginate hydrogel model. Subsequently, I focus on recreating the zonal structure by forming layered constructs using the alginate-released chondrocyte (ARC) method either with or without polymeric scaffolds. Resulting zonal ARC constructs had hyaline morphology, and expressed cartilage matrix molecules such as proteoglycans and collagen type II in both scaffold-free and scaffold-based approaches. Overall, my findings demonstrate that chondrocyte subpopulations obtained from OA joints respond sensitively to compressive stimulation, and are able to form cartilaginous constructs with stratified organization similar to native cartilage using the scaffold-free and scaffold-based ARC technique. The ultimate goal in tissue engineering is to help provide improved treatment options for patients suffering from debilitating conditions such as OA. Further investigations in developing functional cartilage replacement tissues using autologous chondrocytes will bring us a step closer to improving the quality of life for millions of OA patients worldwide.

Non-invasive identification of proteoglycans and chondrocyte differentiation state by Raman microspectroscopy

Proteoglycans (PGs) are crucial extracellular matrix (ECM) components that are present in all tissues and organs. Pathological remodeling of these macromolecules can lead to severe diseases such as osteoarthritis or rheumatoid arthritis. To date, PG-associated ECM alterations are routinely diagnosed by invasive analytical methods. Here, we employed Raman microspectroscopy, a laser-based, marker-free and non-destructive technique that allows the generation of spectra with peaks originating from molecular vibrations within a sample, to identify specific Raman bands that can be assigned to PGs within human and porcine cartilage samples and chondrocytes. Based on the non-invasively acquired Raman spectra, we further revealed that a prolonged in vitro culture leads to phenotypic alterations of chondrocytes, resulting in a decreased PG synthesis rate and loss of lipid contents. Our results are the first to demonstrate the applicability of Raman microspectroscopy as an analytical and potential diagnostic tool for non-invasive cell and tissue state monitoring of cartilage in biomedical research. ((c) 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim).

Cartilage regeneration using zonal chondrocyte subpopulations : a promising approach or an overcomplicated strategy?

Cartilage defects heal imperfectly and osteoarthritic changes develop frequently as a result. Although the existence of specific behaviours of chondrocytes derived from various depth-related zones in vitro has been known for over 20 years, only a relatively small body of in vitro studies has been performed with zonal chondrocytes and current clinical treatment strategies do not reflect these native depth-dependent (zonal) differences. This is surprising since mimicking the zonal organization of articular cartilage in neo-tissue by the use of zonal chondrocyte subpopulations could enhance the functionality of the graft. Although some research groups including our own have made considerable progress in tailoring culture conditions using specific growth factors and biomechanical loading protocols, we conclude that an optimal regime has not yet been determined. Other unmet challenges include the lack of specific zonal cell sorting protocols and limited amounts of cells harvested per zone. As a result, the engineering of functional tissue has not yet been realized and no long-term in vivo studies using zonal chondrocytes have been described. This paper critically reviews the research performed to date and outlines our view of the potential future significance of zonal chondrocyte populations in regenerative approaches for the treatment of cartilage defects. Secondly, we briefly discuss the capabilities of additive manufacturing technologies that can not only create patient-specific grafts directly from medical imaging data sets but could also more accurately reproduce the complex 3D zonal extracellular matrix architecture using techniques such as hydrogel-based cell printing.

The interplay between chondrocyte redifferentiation pellet size and oxygen concentration

Chondrocytes dedifferentiate during ex vivo expansion on 2-dimensional surfaces. Aggregation of the expanded cells into 3-dimensional pellets, in the presence of induction factors, facilitates their redifferentiation and restoration of the chondrogenic phenotype. Typically 1×105–5×105 chondrocytes are aggregated, resulting in “macro” pellets having diameters ranging from 1–2 mm. These macropellets are commonly used to study redifferentiation, and recently macropellets of autologous chondrocytes have been implanted directly into articular cartilage defects to facilitate their repair. However, diffusion of metabolites over the 1–2 mm pellet length-scales is inefficient, resulting in radial tissue heterogeneity. Herein we demonstrate that the aggregation of 2×105 human chondrocytes into micropellets of 166 cells each, rather than into larger single macropellets, enhances chondrogenic redifferentiation. In this study, we describe the development of a cost effective fabrication strategy to manufacture a microwell surface for the large-scale production of micropellets. The thousands of micropellets were manufactured using the microwell platform, which is an array of 360×360 µm microwells cast into polydimethylsiloxane (PDMS), that has been surface modified with an electrostatic multilayer of hyaluronic acid and chitosan to enhance micropellet formation. Such surface modification was essential to prevent chondrocyte spreading on the PDMS. Sulfated glycosaminoglycan (sGAG) production and collagen II gene expression in chondrocyte micropellets increased significantly relative to macropellet controls, and redifferentiation was enhanced in both macro and micropellets with the provision of a hypoxic atmosphere (2% O2). Once micropellet formation had been optimized, we demonstrated that micropellets could be assembled into larger cartilage tissues. Our results indicate that micropellet amalgamation efficiency is inversely related to the time cultured as discreet microtissues. In summary, we describe a micropellet production platform that represents an efficient tool for studying chondrocyte redifferentiation and demonstrate that the micropellets could be assembled into larger tissues, potentially useful in cartilage defect repair.

Porohyperelastic analysis of single chondrocyte using AFM and inverse FEA

The aim of this paper is to determine the creep and relaxation responses of single chondrocytes in vitro. Firstly, Atomic Force Microscopy (AFM) was used to obtain the force-indentation curves of single chondrocytes at the strain-rate of 7.05 s-1. This result was then employed in inverse finite element analysis (FEA) using porohyperelastic (PHE) idealization of the cells to determine their mechanical properties. The PHE model results agreed well with AFM experimental data. This PHE model was then utilized to study chondrocyte’s creep and relaxation behaviors. The results revealed that the effect of fluid was predominant for cell’s mechanical behaviors and that the PHE is a good model for biomechanics studies of chondrocytes.

Analysis of strain-rate dependent mechanical behavior of single chondrocyte : a finite element study

Various studies have been conducted to investigate the effects of impact loading on cartilage damage and chondrocyte death. These have shown that the rate and magnitude of the applied strain significantly influence chondrocyte death, and that cell death occurred mostly in the superficial zone of cartilage suggesting the need to further understand the fundamental mechanisms underlying the chondrocytes death induced at certain levels of strain-rate. To date there is no comprehensive study providing insight on this phenomenon. The aim of this study is to examine the strain-rate dependent behavior of a single chondrocyte using a computational approach based on Finite Element Method (FEM). An FEM model was developed using various mechanical models, which were Standard Neo-Hookean Solid (SnHS), porohyperelastic (PHE) and poroviscohyperelastic (PVHE) to simulate Atomic Force Microscopy (AFM) experiments of chondrocyte. The PVHE showed, it can capture both relaxation and loading rate dependent behaviors of chondrocytes, accurately compared to other models.